Method for assaying components in an enzyme-protein substrate system

FIELD OF THE INVENTION
The present invention relates to rapid and sensitive fluorescence assays and assay reagents for measuring components involved in either protein substrate formation or protein substrate degradation. More particularly, the invention discloses assays and assay reagents for the activity of components known to those skilled in the art, such as protein substrates, enzymes, proenzymes, enzyme activators, enzyme inhibitors, and enzyme cofactors, which are active in an enzymeprotein substrate system.
BACKGROUND OF THE INVENTION
In biomedicine and industrial applications, there is a significant need for assays useful to determine the activity of components, particularly enzymes such as proteases, on their respective target substrates. Although chromogenic or fluorogenic substrates have been used in the past to assay the activity of enzymes, there is a need for a method or assay which is rapid, efficient, inexpensive, and highly sensitive.
For example, an illustration of the usefulness of such assays is the measurement of components in enzyme-protein substrate systems in biomedicine, such as involved in fibrinogen polymerization, fibrinolysis, or fibrinogenolysis. Fibrinogen is the key structural protein in blood clot formation. It is a fibrous protein having three pairs of polypeptide chains held together by disulfide bonds. In fibrinogen polymerization, as illustrated in FIG. 1A, fibrinogen is a substrate for thrombin proteolytic activity. Thrombin catalyzes the release of small peptides, fibrinopeptides A and B, from the chains of fibrinogen. The removal of the fibrinopeptides from the fibrinogen substrate results in molecules which polymerize spontaneously into fibers in forming fibrin. After serving its role as the protein matrix of a blood clot, and in tissue repair, fibrin is then removed or degraded in the process of fibrinolysis.
As illustrated in FIG. 1B, the inactive precursor, plasminogen, is converted into the active proteolytic enzyme, plasmin. In fibrinolysis, plasmin catalyzes the fibrin substrate into soluble degradation products. Further, since plasmin is enzymatically active against fibrinogen, fibrinogenolysis is a process by which fibrinogen substrate is degraded into soluble products.
Measurement of the various enzymes, inhibitors, activators, and protein substrates involved in clot formation, inhibition of clot formation, or dissolution of clots (thrombolysis) has medical applications. For example, thrombosis is an important cause of human illness and death. Thrombolytic agents are increasingly used in pharmacological prevention and/or dissolution of formed thrombi such as in acute myocardial infarction.
Blood components or other body fluid components active in an enzyme-protein substrate system, other than those involved in the clotting system, play a role in various clinical conditions in humans (for a review, see Gallimore and Friberger, 1991, Blood Rev. 5:117-127). Identification of each of such components has led to the development of assays to measure the component, and relation of its detectable activity to diagnosis and/or treatment one or more clinical conditions. Further, there are many industrial applications in which it is useful to measure a component in an enzyme-protein substrate system. The industrial applications include, but are not limited to, testing of enzymes or their protein substrates in detergent formulations, animal feeds, and food products (including dairy processing, infant formulas, beverages).
Typically, the assays currently used involve chromogenic assay techniques requiring enzyme labelling of the substrate, addition of the component(s) for which activity is to be measured, and subsequent detection of the amount of color formed. Similar assays have been described without the use of chromogens, but instead measure turbidity once the reaction has taken place. Disadvantages of these types of assays, particularly for the measurement of components which are normally in low concentrations in the sample to be measured, include: susceptibility to diffusion artifacts; relative time consumption to perform the assay and obtain the results; the concentration of substrate that is required for the assay; lack of kinetic information of the process being measured; and lack of sensitivity of detection to the picomolar or nanomolar level. Thus, for applications such as monitoring therapy in a clinical setting, or monitoring the degree of substrate degradation in an industrial process, previously described methods have been found to be time-consuming and insufficiently sensitive.
Therefore, there is interest and need in providing assays which are rapid, highly sensitive, provide measurement of a component involved in an enzyme-substrate system which reliably reflect that component's activity; and which can be used to detect and quantitate a wide variety of analytes including protein substrates, enzymes, proenzymes, enzyme activators, enzyme inhibitors, and enzyme cofactors which are active in an enzyme-protein substrate system.
SUMMARY OF THE INVENTION
A novel method is provided for detecting and quantitating components involved in either an enzyme-catalyzed protein polymer formation or an enzyme-catalyzed protein substrate degradation. The method involves tagging a substrate with a fluorescent label. The substrate is the protein template onto which molecular assembly with the respective enzyme occurs (i.e. the protein substrate and enzyme molecules are specifically bound with affinity and avidity), and is also the molecule, in a modified form as a result of its interaction with the enzyme, that generates a signal that is directly detected since it has incorporated therein, molecules of fluorescent label. Depending on whether the enzymatic reaction involves polymerization or degradation, quenching or dequenching of the fluorescence emission is measured which relates directly to the kinetics and quantitation of modification of the substrate catalyzed by the enzyme. Therefore, with a known amount of substrate, the activity of the respective enzyme is directly detected. The fluorescence emission also provides an indirect means for the quantitation of activity of an enzyme inhibitor or enzyme activator. The method avoids diffusion-limitation artifacts, and is useful for immediate (within minutes) determination of a component's activity.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a schematic representation of fibrinogen polymerization.
FIG. 1B is a schematic representation of fibrinolysis and fibrinogenolysis.
FIG. 2A is a graph illustrating dependence of fluorescence intensity of 30 nM fluorescent-labeled fibrinogen upon addition of 0.1 to 1.0 U/ml thrombin at 0.1M NaCl.
FIG. 2B is a graph illustrating dependence of fluorescence intensity of 30 nM fluorescent-labeled fibrinogen upon addition of 0.1 to 1.0 U/ml thrombin at 0.3M NaCl.
FIG. 3A is a graph showing the effects of addition of unlabeled fibrinogen to 35 nM of fluorescent-labeled fibrinogen prior to addition of 0.5 U/ml thrombin in 0.1M NaCl.
FIG. 3B is a graph showing the effects of addition of unlabeled fibrinogen to 35 nM of fluorescent-labeled fibrinogen prior to addition of 0.5 U/ml thrombin in 0.3M NaCl.
FIG. 4 is a graph showing fluorescence intensity of 250 nM fluorescent-labeled fibrinogen and 750 nM unlabeled fibrinogen upon addition of 10 nM plasmin.
FIG. 5 is a graph represents a reciprocal plot showing the effect of fluorescent-labeled fibrinogen concentration (1.25 to 20 nM) on plasmin-mediated fibrinogenolysis. The theoretical curve using Equations 5 and 6 is shown for k.sub.cat =0.479 sec.sup.-1 and K.sub.d =0.42 .mu.M.
FIG. 6 is a graph showing the effect of increasing plasmin concentration on the initial rate of fragment X generation at constant concentration of fibrinogen (10.0 nM). The theoretical curve using Equations 5 and 6 is shown for k.sub.cat =0.479 sec.sup.-1 and K.sub.d =0.42 .mu.M.
FIG. 7 is a graph showing the effect of dequenching by addition of .epsilon.-amino caproic acid-plasmin complex to fluorescent-labeled fibrinogen on dequenching compared to an equal amount of plasmin (1.0 nM) added to fluorescent-labeled fibrinogen.
FIG. 8 is a graph showing the effect on dequenching by addition of .epsilon.-amino caproic acid (0.1 to 10 nM) to fluorescent-labeled fibrinogen (20 nM) undergoing degradation by plasmin (1.0 nM).
FIG. 9 is a graph showing the effect of fibrin concentration on the initial rate of dequenching by plasmin (5.0 nM) using coarse fibrin fibers (formed in 0.1M NaCl) or fine fibrin fibers (formed in 0.3M NaCl).
FIG. 10 is a graph showing the effects of adding trypsin to a reaction mixture containing fluorescent-labeled C3.
FIG. 11 is a graph showing the effects of adding trypsin to a reaction mixture containing fluorescent-labeled C9.
FIG. 12 is a graph showing the effects of adding thrombin to a reaction mixture containing fluorescent-labeled C9.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Definitions
By the terms"protein substrate" or "substrate" is meant, for the purposes of the specification or claims, a protein molecule (or peptide molecule of greater than 100 amino acids) that is the substrate of an enzyme. More particularly, the protein substrate is a folded molecule. In one embodiment, a preferred substrate is naturally occurring as a body fluid component or is a synthetic substrate of a naturally occurring enzyme present in body fluids. The protein substrate: a) can be labeled with fluorescent molecules; b) is modifiable in either an enzyme-catalyzed polymerization reaction or an enzyme-catalyzed degradation (dissolution) reaction; c) where upon an enzyme-catalyzed modification of the fluorescent-labeled substrate results in a change in fluorescence emission; and d) measurement of the difference between the fluorescence emission as a result of the enzyme-substrate reaction, and the basal level of fluorescence emission (prior to substrate modification), directly relates to substrate concentration and/or enzyme activity and indirectly relates to the presence or absence of other components involved in the enzyme-substrate reaction. Exemplary protein substrates are shown in Table 1. While many of the protein substrates in Table 1 have clinical relevance, commercial preparations of such substrates can also be used to as a substrate in the method of the present invention to detect enzyme (e.g., proteolytic) activity in industrial processes.
By the term "components" is meant, for the purposes of the specification or claims, protein substrates, enzymes, proenzymes, enzyme activators, enzyme inhibitors, and enzyme cofactors, which: (a) are active in an enzyme-protein substrate system; and (b) are active in either a protein substrate polymerization reaction, or a protein substrate dissolution or degradation reaction.
By the term "enzyme inhibitor" is meant, for the purposes of the specification or claims, to refer to proteins or factors which have activity that directly or indirectly inhibits or inactivates an enzyme. The enzyme inhibitor may be naturally occurring or, alternatively, is a synthetic inhibitor of a naturally occurring enzyme. Although not found naturally occurring, a pharmaceutical having enzyme inhibitor activity is illustrative of an enzyme inhibitor which is synthetic. Exemplary enzyme inhibitors are shown in Table 1.
By the term "enzyme activator" is meant, for the purposes of the specification or claims, proteins or factors which interact with an enzyme in forming an active enzyme or active enzyme complex. The enzyme activator may be naturally occurring or, alternatively, is a synthetic activator of a naturally occurring enzyme. Although not found naturally occurring, a pharmaceutical having enzyme activator activity is illustrative of an enzyme activator which is synthetic. Exemplary enzyme activators are shown in Table 1.
By the term "analytes" is meant, for the purposes of the specification or claims, substances, including the protein substrate or other components in an enzyme-protein substrate system, that are detected and quantitated by the method of the present invention.
By the term "measurement directly" is meant, for the purposes of the specification or claims, that the fluorescence emission changes (as measured between a basal level of fluorescence emission, and the fluorescence emission following an enzyme-catalyzed reaction) reflect the direct effect an enzyme has on the protein substrate.
By the term "measurement indirectly" is meant, for the purposes of the specification or claims, an inhibitive, competitive, or enhancing secondary interaction on the direct reaction between enzyme and substrate.
By the terms "basal level of fluorescence emission" is meant, for the purposes of the specification or claims, the fluorescence emission from a control reference reaction. For example, when an enzyme's activity is being measured, the basal level of fluorescence would be that from a reaction containing the fluorescent-labeled protein substrate and reaction mixture alone.
By the terms "reference level of fluorescence emission" is meant, for the purposes of the specification or claims, the fluorescence emission from a reference reaction containing the fluorescent-labeled protein substrate, enzyme, and reaction mixture, for comparison with a reaction containing the fluorescent-labeled protein substrate, enzyme, reaction mixture, and enzyme inhibitor or enzyme activator. Thus, the difference between the reference level of fluorescence emission and the level of fluorescence from a reaction also containing an enzyme inhibitor or enzyme activator, may be a measure of the activity of the enzyme inhibitor or enzyme activator.
The method according to the invention for detecting and quantitating components involved in either enzyme-catalyzed protein polymer formation or enzyme-catalyzed protein polymer degradation is performed by using a protein substrate having been tagged with a fluorescent label. Because the substrate is the protein template with which molecular assembly with the respective enzyme occurs, and is also the molecule which in a modified form is directly detected since it has incorporated therein molecules of fluorescent label, enzymes that are dilute in solution achieve very high concentrations locally when assembled on the protein substrate. This gives the fluorescent-labeled substrate a very sensitive signal when used in low concentrations (e.g., 1 to 10 nM). This concentration is about 1000 fold lower than typical .mu.M levels for fluorogenic or chromogenic substrates used in other assays that have no ability to concentrate molecules locally on a template.
In measuring components involved in an enzyme-catalyzed reaction which leads to polymerization of the substrate (e.g., fibrinogen, collagen, C9, .beta.-amyloid protein, keratin, actin, tubulin, casein, and vimentin), the fluorescence of the label molecules of the polymerized substrate is quenched (reduced fluorescence emission), relative to the basal level of fluorescence prior to polymerization. The fluorescence quenching is due to enhanced neighbor-neighbor interactions between the fluorescent label molecules in the polymerized substrate. Measurement of quenching during the polymerization to an end point determination provides for information on the kinetics of the reaction. Measurement of fluorescence emission at the end point of quenching, relative to the basal level of fluorescence emission, can be used to quantitate enzyme activity, or enzyme activators or inhibitors (also relative to a reference level of fluorescence), depending on the components included in the reaction mixture.
In measuring components involved in an enzyme-catalyzed reaction which leads to degradation of the protein substrate or to degradation of a polymerized form of protein substrate, the fluorescence of the label molecules of the protein substrate or polymerized form of the protein substrate is dequenched (increased fluorescence emission), relative to the basal level of fluorescence measured in a reference reaction prior to degradation or dissolution. The fluorescence dequenching is due to an increase in the distance between neighboring molecules of fluorescent label as a result or unfolding or cleavage of the substrate or polymerized form of substrate; i.e., thereby being free from proximity-based quenching. Measurement of dequenching during degradation (dissolution) to an end point determination provides for information on the kinetics of the reaction. Measurement of fluorescence emission at the end point of dequenching, relative to the basal level of fluorescence emission, can be used to quantitate enzyme activity, or enzyme activators or enzyme inhibitors (also relative to a reference level of fluorescence), depending on the components included in the reaction mixture.
Various protocols may be employed for the method according to the present invention depending upon the protein substrate used, and the different analytes to be measured. Similarly, different reagents may be necessary, and various components may need be added, depending upon the protein substrate used and analyte(s) to be measured. However, common to all embodiments of the method of the present invention is that the protein substrate is a molecule that is folded in its native conformation; i.e., containing more than one domain or subunit (including dimers and oligomers). Preferably, the protein substrate is naturally occurring. However, as evident from Table 1, synthetic protein substrates may be used in the method of the present invention, wherein such substrates can be labeled with fluorescent molecules, modifiable in either an enzyme-catalyzed polymerization reaction or an enzyme-catalyzed degradation reaction, and wherein upon an enzyme-catalyzed modification of the fluorescent-labeled synthetic protein substrate results in a change in fluorescence emission. The difference between the fluorescence emission as a result of the enzyme-catalyzed reaction and the basal level of fluorescence emission (prior to substrate modification) directly relates to substrate concentration and/or enzyme activity and indirectly relates to the presence of absence of other components involved in that pathway. The protein substrate, being a folded molecule, facilitates the achievement of fluorescent labelling that is internally quenched.
Further common to all embodiments of the present invention, the various reagents or components of a reaction mixture are mixed in solution, and then incubated for a sufficient time to allow for a reaction to occur in solution. Thus, in another aspect the method of the present invention differs from methods using chromogenic or fluorogenic substrates bound to a solid support, by obviating the need for a wash phase in which the solid phase is washed to remove unbound or non-specifically bound label. It is important to note that the method according to the present invention is capable of detecting, and measuring in either a kinetic (if desired) or endpoint fashion, the direct or indirect action of an unknown amount of analyte on the fluorescent-labeled protein substrate.
In one illustrative embodiment of the present invention, one or more components required in the processes of blood coagulation or blood fibrinolysis is the analyte. Since fibrinogen and/or fibrin are the key reactive species in these processes, and since there are multiple components that affect the processes, numerous embodiments of assays using fluorescent-labeled fibrin or fibrinogen may be performed according to the method of the present invention. Further, in addition to blood components being tested as analytes in the method according to the present invention, synthetic materials such as pharmaceuticals which may activate or inhibit a blood component involved in the processes of blood coagulation or blood fibrinolysis, may be the analyte. The fluorescence emission from a reaction containing the pharmaceutical would then be compared to the reference level of fluorescence emission (from a reaction not containing the pharmaceutical) to determine whether the pharmaceutical inhibits or enhances the interaction between the enzyme and the protein substrate.
In using either fibrin or fibrinogen as the substrate, depending on the analyte to be measured, the method of the present invention is an assay reflective of both binding and activity. For example, according to the method of the present invention, plasmin (enzyme) must bind the fluorescent substrate in order for a signal change from the basal level of fluorescence to occur; i.e., to initiate detection of plasmin enzyme activity. Thus, the assay of the present invention can detect either the activity of components (e.g. .epsilon.-amino caproic acid) which inhibit the binding of the enzyme (plasmin), or the activity of components that affect or inhibit the active site (e.g., .alpha..sub.2 -Antiplasmin) of the enzyme (plasmin). In contrast, assays in the art which contain a peptide of fibrin or fibrinogen as a substrate typically measure only components that affect plasmin activity, but not plasmin binding. Therefore the substrate used with this embodiment of the present invention is a natural or natural-like (e.g., recombinant) substrate which more accurately represents the particular pathway in body fluids compared to the smaller synthesized (e.g. peptide) substrates.
This embodiment of the assay is particularly useful for the detection and quantitation of fibrinolytic components with accuracy, rapidity, and sensitivity (nanomolar to picomolar range) not achievable in current commercially available assays. The accuracy, rapidity and sensitivity provided by the method of the present invention will particularly be appreciated in a clinical setting in which thrombolytic therapy is being monitored. Further, the assay of the present invention utilizes fluorescent labeling, and thus avoids the use radioactive labels and the attendant problems with their use. Therefore in a clinical setting, the assay may be carried out simply in a cuvette or a 96-well plate and then measured using a fluorimeter.
In accordance with the method of the present invention, when an enzyme active in an enzyme-catalyzed reaction in body fluid is the analyte, it will be necessary to only include a known amount fluorescent-labeled protein substrate for that specific enzyme in a reaction mixture in solution such as diluted plasma. For enzymes present in the blood, a blood sample would be taken and plasma from the blood sample would be isolated, diluted, and then added to plasma deficient in the enzyme to be tested but containing a known amount of fluorescent-labeled substrate. Alternatively, serum or another body fluid (urine, synovial fluid, etc.) could be used in place of plasma, depending upon the specific enzyme-catalyzed reaction to be detected. Thus, an enzyme-catalyzed reaction with the protein substrate is a measurable effect of the activity of the enzyme contained in the sample.
If an enzyme activator or enzyme inhibitor is the analyte, then components (i.e. precursors to the enzyme, and/or enzyme) other than the analyte required in that particular pathway may need be added if they are not already contained within the reaction mixture or plasma, in addition to the fluorescent-labeled substrate. The measurable effect, when compared to a reference level of fluorescence emission, would then relate to the activity of the analyte in the sample. Knowing the concentration of the enzyme and fluorescent-labeled substrate in a reaction will allow for indirect quantitation of the activity of an enzyme inhibitor or activator present in the sample.
The following examples and tables are offered for the purposes of illustration, and not by way of limitation.
TABLE 1______________________________________ activator (A)/ relevantsubstrate enzyme inhibitor (I) utility______________________________________proendothelin endothelin phosphoramidon hypertension converting (I) enzymeangiotensin I angiotensin imidapril, cap- hypertension, converting topril, cila- heart failure, enzyme zapril, enala- sarcoidosis pril, benaze- pril, rami- pril (Is)angiotensino- renin pepstatin, CGP hypertensiongen 38 560 (Is)high molecular Factor XIIa, .alpha.-1-proteinase inflammation,weight kinino- human bronchi- inhibitor (I) asthmagen al kallikreinHIV HIV protease HIV protease AIDSpolyprotein inhibitors, N-acetylpep- statin (Is)latent trans- plasmin antiplasmin, wound healingforming growth macroglobulin restenosisfactor-.beta. (Is)trypsin sus- trypsin, amyloid .beta.- plateletceptible pro- chymotrypsin protein releaseteins precursor (I) productAla-Ala-Phe- pancreatic .alpha.-2-macro- inflammationMCA; collagen chymotrypsin A globulin, .alpha.-1-matrix proteo- (cathepsin G) proteinase in-glycan hibitor, .alpha.-1- antichymo- trypsin (Is)serotonin monoxidase A tricyclic N- depression/ arylamides (I) anxietyComplement neutral urinary tryp- preeclampsia,proteins (e.g. elastase sin inhibitor, rheumatoidC3 & C5); .alpha.-1-proteinase arthritis,fibrinogen; inhibitor (Is) inflammatorycollagen; IgG bowel diseaseSubstance P, neutral thiorphan, hypertensionbradykinin, metalloendo- candoxatril,neurotensin, peptidase phosphorami-chemotactic (endopepti- don, RB 38B,peptides dase) MDL 100.240, UK 73967 (Is)troponin T calpain calpain inhi- myocardialsubunit; bitor, AK295, ischemia,vitronectin calpastatin thrombotic (Is) thrombocyto- penic purpuraextracellular endonuclease- pepstatin A, tumor cellmatrix NX, cathepsins leukocyte invasionproteins D & L, LDH-.kappa. cysteine pro- teinase inhi- bitor, phenyl- butazone, chy- mostatin (Is)enkephalins aminopepti- Inhibitors 1- neurochemical dases 1-5, 3, bestatin, dypeptidyl- thiorphan, amminopepti- captopril (Is) dase, dypepti- dylcarboxypep- tidasethrombin thromboplastin anticoagulants blood (warfarin, coagulation heparin) (Is)fibrinogen thrombin anticoagulants blood (warfarin, coagulation heparin) (Is)fibrin; plasmin plasmin inhi- bloodfibrinogen bitors (see coagulation Table 3), .epsilon.- amino caproic acid (Is)Complement thrombin, inflammationC3; trypsin, C3Complement convertaseC9kallikrein; FX11a C1-esterase septic shockprekallikrein inhibitor, antithrombin III (Is)casein rennin, trans- food/dairy glutaminase, processing plasmin, trypsinproteins subtilisins detergentsinsulin metallo- pharmaceutical protease______________________________________
Other proteases (enzymes) and protein substrates are known to those skilled in the medical and industrial arts. For a review of substrate assays and the measurement of various clinically relevant proenzymes, enzyme activators, enzyme cofactors, and enzyme inhibitors in blood and other body fluids, and known in the art, see Gallimore et al. (1991, Blood Rev. 5:117-127, herein incorporated by reference).
EXAMPLE 1
Various fluorescent molecules are known in the art which are suitable for use to label a protein substrate for the method of the present invention. Because substrates for the method of he present invention are molecules which are folded in their native state, incorporation of fluorescent label into more than one chain of the substrate will lead to quenching due to molecular proximity. A partial, but representative list of the class of such fluorescent molecules is illustrated as Table 2, and includes the respective excitation wavelength (EX) and emission wavelength (EM) for detection (in nanometers, "nmo"). Common fluorescent labels for protein labeling include amine-reactive probes which are reactive to end terminal amines and the lysines of the protein substrate; isothiocyanates; succinimidyl esters; sulfonyl chlorides which are conjugated to the substrate through amine residues and tyrosine; sulfhydryl-reactive probes that react with thiols found in proteins; and the like. Depending on the fluorescent molecule used, methods of incorporating or tagging the substrate with the fluorescent molecule label include attachment by covalent or noncovalent means.
TABLE 2______________________________________Fluorescent molecule EX EM______________________________________Fluorescein isothiocyanate (FITC) 491 nm 515 nmTetramethylrhodamine isothiocyanate (TRITC) 540 nm 567 nmcarboxytetramethylrhodamine succinimidyl 540 nm 567 nmestercarboxyfluorescein succinimidyl ester 491 nm 515 nmTEXAS RED.sup.R sulfonyl chloride 589 nm 615 nm7-diethylamino-3-((4'iodoacetyl)amino) 382 nm 472 nmpheny10-4-methylcoumarin5-iodoacetamidofluorescein 491 nm 515 nmtetramethylrhodamine-5-(and 6)- 540 nm 567 nmiodoacetamideN-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a- 504 nm 510 nmdiaza-s-indacene-3-propionyl)-N'iodoacetyl-ethylenediamineCoumarin malemide 385 nm 471 nmFluorescein malemide 490 nm 515 nmRhodamine malemide 542 nm 566 nm______________________________________
The protocol for labeling of substrate with fluorescent molecule may vary depending upon the fluorescent molecule used and the particular protein substrate to be labelled. Such protocols are known in the art for the respective fluorescent molecule. For example, a general protocol for labeling the protein substrate with FITC or TRITC is as follows. The protein substrate, in a concentration of from 0.1 to 10 mg/ml, is dissolved in 0.1 to 0.2M bicarbonate buffer (pH 8.3 to 9) The substrate is then incubated with FITC or TRITC (1 mg/ml) with continuous stirring for 1 hour at 22.degree. C. in a labeling buffer of 0.1M sodium bicarbonate (pH 9.0). The reaction is stopped with hydroxylamine (0.15M final concentration, freshly prepared), incubated for 1 hour, after which the labeled substrate is dialyzed extensively in the dark at 4.degree. C., and then stored at -75.degree. C. until used (since only nanomolar levels of substrate are required per reaction). Alternatively, to remove unincorporated fluorescent label from the labelled substrate, the substrate can be subjected to molecular sieve chromatography.
Generally, the protocol for labeling substrate with amine-reactive probes is the same as that of labelling with FITC or TRITC except that the fluorescent label (e.g. TEXAS RED.RTM. sulfonyl chloride) is incubated with the protein substrate for 1 hour at room temperature or 4.degree. C. in 0.1 to 0.2M sodium bicarbonate solution at pH 8.3. The general protocol for labeling the substrate with a fluorescent molecule comprising a haloacetyl derivative (type of sulfhydryl-reactive label) is as follows. The protein substrate, in a concentration of 0.1 to 10 mg/ml, is incubated with iodoacetamide reagent at 0.1 to 10 mg/ml concentration for 1 hour at room temperature under neutral buffer conditions (pH<8.0) in the dark. The labeled protein can be separated from unincorporated label using molecular sieve chromatography or extensive dialysis in the dark. The resultant fluorescent-labeled substrate may then be stored frozen. For malemide derivatives (type of sulfhydryl-reactive label) the protein substrate, in a concentration of 0.1 to 10 mg/ml, is incubated with maleimide reagent at 0.1 to 10 mg/ml concentration for 1 hour at room temperature under neutral buffer conditions (pH<8.0) in the dark. The labeled protein substrate can be separated from unincorporated label using molecular sieve chromatography or extensive dialysis in the dark.
EXAMPLE 2
There are some general considerations in practicing the method of the present invention. The assay reaction is performed in the liquid phase contained within a cuvette, the well of a microtiter plate, or like container suitable for fluorimetry. Typically, the inner surface of the container is preincubated with an inert protein, such as serum albumin, to substantially reduce or eliminate nonspecific binding of the fluorescent-labelled substrate to the surface. Alternatively, transparent, low protein-binding materials may be used as incubation wells.
The fluorescent-labeled substrate is diluted in a reaction solution. The solution may comprise a buffer such as a physiological saline solution, or other compatible solution (for solubility and stability of substrate) depending on the substrate used. Likewise, if the sample to be analyzed requires dilution, the diluent should also be compatible with the substrate used. A general saline buffer using HEPES, Tris, or phosphate buffer is suitable. Salt concentrations in the buffer can range from 0.1M to 0.5M salt such as NaCl. Thus, an exemplary buffer comprises 0.1M NaCl, 5 mM CaCl.sub.2, and 10 to 50 mM Tris-HCl (pH 7.4). In general, a zinc salt (zinc ion, Zn.sup.2+) should be avoided as it may precipitate out of solution a substrate such as fibrin. Buffer components should also be chosen to include those which do not inhibit polymerization or dissolution/degradation processes. Additionally, it is known by those skilled in the art that certain enzymes, to be optimally active, are dependent on ion concentration. Thus, depending on the specific enzyme to be measured, the ion may need to be added to the reaction mixture. For example, calpain is Ca.sup.++ -dependent.
A fluorescence emission baseline is established before the sample containing the analyte is added to the reaction mixture. In that regard, the reaction should be performed to avoid the presence of any contaminating strongly fluorescent or quenching materials that overlap with the excitation or emission wavelength of the fluorescent-labeled substrate. Since the sample containing the analyte to be measured may be a body fluid or a solution of an industrial process, particulate matter (e.g., red blood cells) may be present. The inclusion of particulate matter in the reaction is not recommended, but may be tolerated in some conditions. The reaction buffer may also comprise platelet poor plasma into which the labeled-substrate is added.
The reaction is initiated by the addition of critical enzyme in the sample to the fluorescent substrate. The reaction mixture is then excited with light at a wavelength dependent on the fluorescent molecule used to label the substrate (see for example, Table 2). For example, using FITC-labeled substrate in the method according to the present invention, the optimal excitation is blue light (488 nm). Fluorescent emission is then measured with a light detector (i.e., photo-multiplier tube) in a fluorimeter. For example, for FITC-labeled substrate, green emission light (515 nm) is collected using a photomultiplier tube inside a fluorimeter. The intensity of emission of fluorescent light is monitored continuously by sampling the emission at 0.5 to 10 times per second. The development of signal is dependent on the initial state of the fluorescent-labeled substrate and enzyme-catalyzed reaction events occurring on the substrate (resulting in either polymerization or degradation/dissolution). However, a range of typical reaction times to an end-point determination is 1 to 5 minutes. The assay may be carried out at temperatures ranging from about 4.degree. C. to about 37.degree. C., and preferably at room temperature or 37.degree. C.
Other reagents (other proteins, enzyme inhibitor, enzyme activator, or enzyme cofactor, etc.) can be added to the fluorescent-labeled substrate prior to start of the reaction. Alternately, it is possible to preincubate components of the reaction mixture together prior to addition of fluorescentlabeled substrate to the incubation mixture. For example, a preincubation reaction can include an enzyme activator (e.g., urokinase) incubated with a proenzyme (e.g., plasminogen ) to generate an active form of the enzyme (e.g., plasmin). In another illustration of this embodiment, tPA (tissue plasminogen activator) can be incubated with unlabeled fibrin in the presence of plasminogen to generate plasmin. In either example, the preincubation reaction mixture can then be added to fluorescent-labeled substrate.
The use of a fluorescent-labeled substrate, as defined herein, to assay enzyme activities in a standard fluorimeter is novel. Because the substrate is a molecule which is folded in its native conformation, and onto which molecular assemblies with enzyme are recreated, the molecular size of the substrate to be used in the method of the present invention is preferably greater than about 100,000 daltons (e.g. the molecular size of fibrinogen is 340,000 daltons). Thus, the method of the present invention of following the fluorescence signal of such substrate during polymerization (with quenching of the signal) or proteolysis (with dequenching of the signal) is novel and very distinct from fluorescence polarization anisotropy measurements which are not useful for substrates having a molecular size above approximately 65,000 daltons.
Concerning assay reagents comprising components, other than the analyte, to be added to facilitate process of modification of the fluorescent-labeled substrate, in many instances it will not be necessary to use such components from the same species as the species of the analyte. For example, it is known that non-human components involved in fibrinolysis display cross-activity with the respective components found in humans. Thus, while one embodiment of the method of the present invention is preferably performed to measure analytes involved in human processes, assay reagents may include components from other species, if desirable. Further, various assay reagents useful for the particular process to be measured, can be included in a kit form to facilitate practice of the method of the present invention. In this regard, fluorescent-labeled substrate is typically stable for at least 9 months, and may be stable up to two years, with proper storage conditions.
Reference levels for many body fluid components are known to those skilled in the art from using the various assay methods currently available. However, as known in the art, reference levels may be different between the various methods utilized for assaying the same body fluid component. Further, when a new method is implemented in a clinical laboratory, a body fluid component is measured from samples of a (clinically) normal population to establish a reference level. Likewise, to implement the method according to the present invention in a clinical facility, reference levels of the various body fluid components to be measured may need to be established for comparison to levels detected by the method of the present invention in clinical specimens.
EXAMPLE 3
Fibrinogen Polymerization
An assay for fibrinogen polymerization according to the method of the present invention was performed in accordance with materials and methods outlined in Examples 1 and 2. Fibrinogen was labeled with FITC using a 0.1M sodium bicarbonate buffer. After stopping the reaction with hydroxylamine (0.15M final concentration), the FITC-fibrinogen was dialyzed extensively before storage. The buffer for the polymerization reaction comprised either 0.1M NaCl, 0.05M Tris-HCl, 5 mM CaCl.sub.2 ("buffer 1") or 0.3M NaCl, 0.05M Tris-HC1, 5 mM CaCl.sub.2 ("buffer 2"). Cuvettes were incubated with 10 .mu.M bovine serum albumin for 30 minutes to minimize adsorption of substrate to the cuvette walls.
In a series of reactions to produce fibrinogen polymerization into fibrin fibers, thrombin (final concentrations ranging from 0.1 U/ml, 0.25 U/ml, 0.5 U/ml and 1 U/ml) was mixed for about 5 seconds in either buffer 1 or buffer 2 containing a dilute FITC-fibrinogen (<40 nM) solution. Polymerization was monitored by exciting the reaction mixture with blue light (488 nm) and monitoring the fluorescence emission at 515 nm using a fluorimeter. The addition of thrombin to FITC-fibrinogen led to rapid and dose-dependent reduction of fluorescence emission due to quenching. At these extremely low concentration of fluorescent-labeled substrate (<40 nM), the solutions were optically clear and the drop in fluorescence was not due to turbidity or scattering effects during polymerization. Release of fibrinopeptides A and B from fibrinogen, mediated by thrombin action, caused no detectable dequenching of the FITC moieties on fibrin monomers.
The initial rate of quenching was dependent on the initial concentration of thrombin, as illustrated in
FIG. 2A (30 mM FITC-fibrinogen polymerized in buffer 1) and in FIG. 2B (30 mM FITC-fibrinogen polymerized in buffer 2). At the lowest concentrations of thrombin used, a comparatively significant lag time was observed before dequenching proceeded. After generation of sufficient monomer and protofibril extension, lateral aggregation of fibrils proceeded. Once dequenching began, the maximum rate of dequenching with time appeared to be largely independent of thrombin concentration, as seen by the similar maximum slopes on curves in FIGS. 2A or 2B. Also, the final extent of quenching appeared to be modulated by the thickness of the fiber. At low thrombin concentration and low salt (buffer 1) the thicker fibers that formed (taking longer to form) resulted in a greater final extent of quenching compared to the thinner fibers formed at higher thrombin concentrations (FIG. 2A). In high salt, favoring the formation of thinner fibers, the modulation of the final extent of quenching by adjusting the thrombin concentration was qualitatively similar to reactions with low salt, but the final differences were much smaller. As seen in FIGS. 2A and 2B, at all thrombin concentrations, the lag phase was considerably longer in buffer 2 as compared to in buffer 1, suggesting the importance of lateral aggregation during dequenching (i.e., protofibril extension is fast at higher salt concentrations). However, when quenching achieved its maximal rate at a given stage of the polymerization, there was little difference between that rate for low (buffer 1) and high (buffer 2) salt concentrations.
Addition of unlabeled fibrinogen to the reaction mixture attenuated the thrombin-induced quenching in a dose-dependent manner (FIGS. 3A and 3B). This result would not be seen for a scattering-based mechanism of loss of fluorescence signal. Increasing the concentration of unlabeled fibrinogen reduces the probability of two FITC-fibrin monomers either being incorporated sequentially in a protofibril or interacting within a given cross-section of fiber. Increasing the unlabeled fibrinogen in the reaction altered the initial quenching rate slightly (at constant thrombin concentration) and reduced the final extent of quenching in a dose-dependent manner. At a 5:1 ratio of unlabeled fibrinogen to FITC-fibrinogen, the quenching during polymerization was completely eliminated in fine fibers (FIG. 3A) while a small amount of quenching occurred at this ratio during polymerization of coarse fibers (FIG. 3B). This is consistent with an increased probability of two or more labeled FITC-fibrin monomers quenching each other in a thick fiber cross-section as compared to the quenching probability in a thin fiber-cross section which contains less fibrils. Thus, the quenching during polymerization is due to a mechanism involving molecular proximity which is achieved by two processes: fibril extension and fibril aggregation.
It was also observed that polymerization of FITC-fibrinogen by 1 U/ml of thrombin in the presence 0.1, 10, and 100 .mu.M Gly-Pro-Arg-Pro ("GPRP", a peptide that prevents polymerization; Laudano et al., 1980 Biochem. 19:1013-1019) reduced in a dose-dependent manner the rate and extent of quenching associated with polymerization. Over 80% of the thrombin-induced quenching was prevented when 100 .mu.M GPRP was added before polymerization. Similar to the findings given for unlabeled fibrinogen, this finding with GPRP verifies and validates the novel mechanism by which the assay works.
EXAMPLE 4
Thrombin Activity Assay
An assay for thrombin activity in fibrinogen polymerization according to the method of the present invention can be performed in accordance with materials and methods outlined in Examples 1, 2, and 3. The amount of biological sample, comprising body fluid to be tested for thrombin activity, to be added to the reaction mixture can range from about 1 .mu.l to 100 .mu.l, depending on the body fluid and if dilution of the sample is desirable. Likewise, one skilled in the art will appreciate that the amount of reaction buffer to which the sample is added (e.g., 0.1 ml to 2.5 ml) and the amount of FITC-fibrinogen in the reaction (e.g., 1 nM to 50 nM) will depend on the sample to be analyzed, and the mode (cuvette, microtiter well, etc.) in which the reaction takes place.
The sample is added to the reaction buffer containing FITCfibrinogen. Polymerization is then monitored by exciting the reaction mixture with blue light (488 nm) and monitoring the fluorescence emission at 515 nm using a fluorimeter. In a parallel set of reactions, one or more thrombin standards (e.g., between 0.1 U/ml and 10 U/ml) may be used to calibrate the assay of the sample. The extent of quenching at the end of desired time interval (e.g., 5-25 minutes) is related to the thrombin activity, and hence the thrombin concentration, present in the sample. Since plasmin, if present in the sample, may cause fibrinogenolysis in an assay intended to measure thrombin activity in fibrinogen polymerization, a plasmin inhibitor may be added when measuring thrombin activity. For example, added to the reaction buffer containing the thrombin and FITC-fibrinogen may be a plasmin inhibitor, such as .epsilon.-amino caproic acid, to eliminate interference by plasmin contained in the sample.
If desired, the specificity of the reaction for thrombin activity may be validated by the addition of specific thrombin inhibitors, into the reaction mixture containing the sample and FITC-fibrinogen. Such thrombin inhibitors include natural inhibitors like antithrombin III, and synthetic inhibitors like benzamidine and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone. Addition of a known amount of thrombin inhibitor will increase the amount of sample required (i.e., increase the amount of thrombin) to be added to obtain equivalent fluorescence emission. From the percent inhibition and from the thrombin concentration in the absence of inhibitor, can be calculated the increased thrombin activity (concentration) required to obtain equivalent activity.
EXAMPLE 5
Heparin Activity Assay (reverse thrombin assay)
Heparin and its variants are used in anticoagulation therapy, wherein it prevents fibrinogen polymerization by accelerating complexation of thrombin with antithrombin. An assay for heparin activity in fibrinogen polymerization according to the method of the present invention can be performed in accordance with materials and methods outlined in Examples 1, 2, 3, and 4.
The sample containing an unknown amount of heparin activity is added to the reaction buffer simultaneously with a known amount of thrombin (e.g., 1 U/ml), antithrombin (e.g., 0 to 5000 U/ml), and containing FITC-fibrinogen (e.g., 10 nM). Polymerization is then monitored by exciting the reaction mixture with blue light (488 nm) and monitoring the fluorescence emission at 515 nm using a fluorimeter. A parallel set of standard reactions containing thrombin, antithrombin, FITC-fibrinogen may be used to determine the maximum amount of fluorescence emission (quenching) during the desired time interval. The extent of interference of fibrinogen polymerization (i.e., inhibition of quenching) at the end of desired time interval in the reaction containing the sample, is related to the heparin activity, and hence the heparin concentration, present in the sample. Thus, from the percent inhibition of fibrinogen polymerization (as observed by a reduction in quenching) as compared to the standard(s), can be calculated the heparin activity (concentration) in the sample. Further, if desired, a parallel set of standard reactions may be performed containing thrombin, antithrombin, a known concentration of heparin, and FITC-fibrinogen for use to calculate the heparin activity (concentration) in the sample.
EXAMPLE 6
Antithrombin Activity Assay (reverse thrombin assay)
Antithrombin is a component that may be found in body fluid, wherein it prevents fibrinogen polymerization by direct suicide complexation with thrombin. An assay for antithrombin activity in fibrinogen polymerization according to the method of the present invention can be performed in accordance with materials and methods outlined in Examples 1, 2, 3, and 4.
The sample containing an unknown amount of antithrombin activity is added to the reaction buffer simultaneously with a known amount of thrombin (e.g., 1 U/ml), heparin (e.g., 0 to 5000 U/ml), and containing FITC-fibrinogen (e.g., 10 nM). Polymerization is then monitored by exciting the reaction mixture with blue light (488 nm) and monitoring the fluorescence emission at 515 nm using a fluorimeter. A parallel set of standard reactions containing thrombin, antithrombin, FITC-fibrinogen may be used to determine the maximum amount of fluorescence emission (quenching) during the desired time interval. The extent of interference of fibrinogen polymerization (i.e., inhibition of quenching) at the end of desired time interval in the reaction containing the sample, is related to the antithrombin activity, and hence the antithrombin concentration, present in the sample. Thus, from the percent inhibition of fibrinogen polymerization (as observed by a reduction in quenching) as compared to the standard(s), can be calculated the antithrombin activity (concentration) in the sample. Further, if desired, a parallel set of standard reactions may be performed containing thrombin, heparin, a known concentration of antithrombin, and FITC-fibrinogen for use to calculate the antithrombin activity (concentration) in the sample.
EXAMPLE 7
Fibrinogenolysis
An assay for fibrinogenolysis according to the method of the present invention was performed in accordance with materials and methods outlined in Examples 1 and 2. Fibrinogen was labeled with FITC using a 0.1M sodium bicarbonate buffer. After stopping the reaction with hydroxylamine (0.15M final concentration), the FITC-fibrinogen was dialyzed extensively before storage. The buffer for the lysis reaction comprised either 0.1M NaCl, 0.05M Tris-HCl, 5 mM CaCl.sub.2 ("buffer 1") or 0.3M NaCl, 0.05M Tris-HCl, 5 mM CaCl.sub.2 ("buffer 2".) Cuvettes were incubated with 10.mu.M bovine serum albumin for 30 minutes to minimize adsorption of substrate to the cuvette walls.
To illustrate plasmin-mediated fibrinogenolysis into degradation products, 10 nM of plasmin was mixed for about 5 seconds in the reaction buffer containing 250 nM FITC-fibrinogen. Substrate degradation was monitored by exciting the reaction mixture with blue light (488 nm) and then simultaneously monitoring the fluorescence emission at 515 nm using a fluorimeter. The action of plasmin on FITC-fibrinogen produced an immediate large increase in fluorescence intensity (dequenching) in a plasmin-dependent manner (FIG. 4). The detection limit has been found under routine conditions to be at least 10 picomolar plasmin.
Degradation events were also monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as indicated by the arrows representing sampling times (FIG. 4). Four fragments (X, Y, D and E) were detected in the degradation process. The major extent and most rapid rate of dequenching occurred at the earliest reaction times which correlated temporally with the degradation of fibrinogen to a fragment of about 200 kD (X) during the first three minutes of the reaction. A second fragment of about 140 kD (Y) first became visible by silver-stained SDS-PAGE after 3 minutes when over 50% of the dequenching had already occurred. Fragment Y reached its maximal concentration between 10 and 30 minutes after digestion was initiated when the dequenching rate had already slowed considerably. The obtainment of the final extent of dequenching was seen at 60 minutes and occurred concomitantly with the presence of the last remaining fragment X in the digestion. A third fragment (D) of about 85 kD appeared at about 10 minutes after digestion was initiated, and remained the prominent fragment from 30 to 300 minutes after the digestion was initiated. A fourth fragment (E) of about 45 kD appeared about 30 minutes after digestion was initiated, and again at 300 minutes, but not at 60 minutes and 120 minutes possibly due to its known, unreliable detection by silver staining.
During fibrinogenolysis, the total fluorescence emission at any time, F(t), is related to the amount of emission from fluorescent-labeled fibrinogen, .sigma.�fbg(t)!, and the amount of emission from individual fluorescent fibrinogen degradation products in the sample, .SIGMA..beta..sub.i .multidot.�FDP.sub.i (t)!. The intensity of fluorescence emission F(t) is described by Equation 1:
F(t)=.sigma.�fbg(t)!+.SIGMA..beta..sub.i .multidot.�FDP.sub.i (t)!
where i=X, Y, D, E; and .sigma.=F(0)/�fbg(0)! at t=0
The majority of dequenching occurs temporally during the generation of fragment X. Thus, an initial reaction rate analysis would relate the rate of dequenching with the first reaction step- the rate of fragment X generation. All degradation products appear to have the same fluorescence intensity per molecule, .beta., which is justified by the fact that the dequenching signal reaches steady state long before lysis is complete; i.e., conversion of fragment Y to fragment D produced little additional dequenching. Plasmin degradation of one fibrinogen molecule can produce at least three dequenched fragments that include a single X fragment and at least two small fragments from the carboxy terminus of the .alpha.chains. For the reaction where plasmin converts one quenched fibrinogen molecule to n dequenched fragments (of different size, but the same .beta.) with n.gtoreq.3, the Equation 1 becomes either:
F(t)=.sigma.�fbg(t)!+.beta..multidot.�n dequenched fragment(t)!Equation 2a
or
F(t)=.sigma.�fbg(t)!+.beta.'.multidot.�X(t)! Equation 2b
where .beta.'=.beta.n and .beta.'=F.sub.max /�fbg(0)! as all fibrinogen .fwdarw.>X
Differentiating Eqn. 2(b) where �fbg(0)! =�fbg(t)! +�X(t)!, the rate of dequenching is related to X generation by Eqn. 3. One measure of the consistency of this approach is that the expression �fbg(0)!/(F(0)-F.sub.max) which is equal to 1/(.alpha.-.beta.') was measured experimentally and found to remain constant as expected, regardless of reaction conditions.
1/(.sigma.-.beta.') dF/dt=d�fbg!/dt=-d�X!/dt Equation 3
For kinetic analysis where reversible binding and dissociation occurred by distinct binding sites separate from the catalytic site, the rate of complex formation is described by:
d�plm.sup.b !/dt=k.sub.f �plm.sup.f ! �.theta..sub.plm -plm.sup.b !-k.sub.r �plm.sup.b ! Equation 4
(Diamond et al., 1993, Biophysical J. 65:2622-2643)
where the total available plasmin binding site concentration, .theta..sub.plm, is equal to q.multidot.�fbg! and q=2 initially for two plasmin binding sites per fibrinogen molecule, and k.sub.f is the forward association rate, and k.sub.r is the reverse dissociation rate (Lucas et al., 1983, J. Biol. Chem. 258:4249-4256).
At all times, the free plasmin concentration �plm.sup.f ! plus the bound plasmin concentration �plm.sup.b ! is equal to the initial plasmin concentration �plm.sup.o !. For rapid binding that equilibrates quickly (d�plm.sup.b !/dt =0), the concentration of bound plasmin is given by (Equation 5):
�plm.sup.b !=(K.sub.d +�plm.sup.o !-(K.sub.d +�plm.sup.o !+q�fbg.sup.o !) .sup.2 -4q�fbg.sup.o !�plm.sup.o !2
and the initial reaction rate is given as:
v=k.sub.cat .multidot.�plm.sup.b ! Equation 6
Using this model, as the initial concentration of fibrinogen increases to very large values (at constant plasmin concentration), the maximum rate goes to v.sub.max =k.sub.cat .multidot.�plm.sup.o !. As the initial plasmin concentration increases (at constant fibrinogen), the maximum reaction rate goes to v.sub.max =k.sub.cat .multidot.q�fbg.sup.o !. Using 1 nM plasmin and varying concentrations of fibrinogen from 1.25 to 20 nM in the method according to the present invention, a kinetic analysis for the generation of fragment X was conducted. A reciprocal plot is given for the initial rate of fragment X generation (FIG. 5) calculated from Equation 3. A linear regression of the experimental data allowed direct calculation of v.sub.max and a determination of k.sub.cat =0.479 sec.sup.-1. Using Equations 5 and 6, the K.sub.d (dissociation constant) of active plasmin binding to fibrinogen was then calculated to be 0.42 .mu.M.
Although enzyme assays are typically conducted under conditions of excess substrate, the sensitivity of the fluorescence-based method of the present invention allows determination of excess plasmin on nanomolar concentrations of fibrinogen. The fibrinogen substrate has two binding sites for plasmin, each with fairly high affinity for plasmin. Thus, it is expected that under the conditions of excess plasmin, these binding sites can be saturated with plasmin. This was indeed observed experimentally for the initial stage of the reaction when the ratio of plasmin to fibrinogen exceeded twice that of the initial concentration of fibrinogen (FIG. 6). Using values of k.sub.cat =0.479 sec.sup.-1 and K.sub.d =0.42 .mu.M, the kinetic rates for reactions where the plasmin/fibrinogen ratio was less than one were accurately predicted with Equations 5 and 6. A large deviation from the model was observed when the plasmin was in excess of the initial fibrinogen, a regime where kinetic formulations typically breakdown. Given the number of lysine binding sites per fibrinogen molecule, the measured reaction rates reached saturation at the expected plasmin concentrations, but the value of that maximum rate was considerably less than predicted. This may be due to the very short linear regime of the initial rate and the rapid destruction of fibrinogen such that the assumption of local binding equilibrium does not hold or the initial concentration of fibrinogen cannot be used in Equation 5 since it drops so rapidly at high plasmin concentration.
It was found that preincubation of plasmin with .epsilon.-amino caproic acid (10 mM) prior to addition of fluorescent-labeled fibrinogen greatly reduced the dequenching rate which indicates that plasmin must bind fibrinogen in order to initiate lysis (FIG. 7). Addition of excess .epsilon.-amino caproic acid, after plasmin-mediated fibrinogenolysis was initiated, resulted in a dose-dependent inhibition of dequenching (FIG. 8). Addition of .epsilon.-amino caproic acid (10 mM) immediately inhibited the progression of fibrinogenolysis as indicated by the cessation of dequenching. This indicated that plasmin can be removed from fibrinogen either by .epsilon.-amino caproic acid disruption of fibrinogen-plasma complex or by .epsilon.-amino caproic acid capture of desorbing plasmin. Addition of 10 nM plasmin to a mixture of 10 mM .epsilon.-amino caproic acid and 20 nM FITC-fibrinogen resulted in a competition between free plasmin binding with fibrinogen and plasmin binding with .epsilon.-amino caproic acid. In that competition, some plasmin was able to bind fibrinogen as indicated by the initial rate of dequenching.
EXAMPLE 8
Fibrinolysis
An assay for fibrinolysis according to the method of the present invention was performed in accordance with materials and methods outlined in Examples 1 and 2. To produce suspensions of fluorescent-labeled fibrin fibers, thrombin (final concentration of 1 U/ml) was mixed for 5 seconds with FITC-fibrinogen (80 nM) in either buffer 1 or buffer 2 after which the polymerization was monitored until fluorescence quenching was complete and stable for 1 hour. The daily-fiber suspensions yielded a highly repeatable extent of quenching and were stable for several hours as indicated by the stability of the fluorescence signal. Prior to reactions for fibrinolysis, cuvettes were incubated with 10 .mu.M bovine serum albumin for 30 minutes to minimize adsorption of substrate to the cuvette walls. Small volumes of the FITC-fibrin were then pipetted into 2.4 ml of the reaction buffer and monitored for 200 to 400 seconds to establish the fluorescence baseline before addition of plasmin or other reagents.
To illustrate plasmin-mediated fibrinolysis, plasmin (5 nM) was mixed for about 5 seconds in the reaction buffer containing between 0.5 to 30 nM FITC-.fibrin. Polymerization was monitored by exciting the reaction mixture with blue light (488 nm) and monitoring the fluorescence emission at 515 nm using a fluorimeter. The action of plasmin on FITC-fibrinogen produced a large increase in fluorescence intensity (dequenching) in a plasmin-dependent manner. During fibrinolysis, the monomers in the fibrils are degraded with subsequent loss of fibril/fiber structure. The early dequenching during fibrinolysis is due to the loss of quenching in the fibril/fiber due to loss of structure, and the generation of dequenched degradation products. The initial rate of dequenching was very similar for lysis of coarse and fine fibers (FIG. 9) and was dependent on the concentration of fibrin used at constant plasmin concentration. It was noted that the final dequenched state of lysed fibrin (non-crosslinked) achieved a final fluorescence emission identical to the final dequenched state of lysed fibrinogen. Also, the initial rate of fluorescence change for FITC-fibrin in fibrinolysis appeared similar to that seen for FITC-fibrinogen in fibrinogenolysis.
EXAMPLE 9
Assays for Inhibitors of Fibrinolysis and Fibrinogenolysis
There are various plasmin inhibitors (also known as "antiplasmins") which may be present in body fluid and can inhibit fibrinolysis or fibrinogenolysis. Table 3 illustrates several of the physiologically important plasmin inhibitors.
TABLE 3______________________________________ Molecular size Approximate normalplasmin inhibitor (kilodaltons, kD) plasma/serum conc.______________________________________antiplasmin 70 kD 115-150 mg/dl.sup.-1.alpha..sub.1 -antitrypsin 51 kD 100-160 mg/dl.sup.-1.alpha..sub.2 -macroglobulin 820 kD 130-334 mg/dl.sup.-1C.sub.1 inhibitor 90 kD >11 mg/dl.sup.-1inter-.alpha.-trypsin 170 kD 20-70; mg/dl.sup.-1inhibitorantithrombin III 62 kD 200-400 mg/dl.sup.-1______________________________________
An assay for a plasmin inhibitor's activity in fibrinolysis or fibrinogenolysis according to the method of the present invention can be performed in accordance with materials and methods outlined in Examples 1, 2, 7, and 8.
The sample containing an unknown amount of plasmin inhibitor activity is added to the reaction buffer simultaneously with a known amount of plasmin (e.g., 5 nM) and FITC-fibrinogen or FITC-fibrin (e.g., 10 nM; "FITC-substrate"). Lysis of the FITC-substrate is then monitored by exciting the reaction mixture with blue light (488 nm) and monitoring the fluorescence emission at 515 nm using a fluorimeter. A parallel set of standard reactions containing a known amount of the plasmin inhibitor may be used to determine the amount of fluorescence emission (dequenching) during the desired time interval. The extent of interference of lysis of the FITC-substrate (i.e., prevention of dequenching) at the end of desired time interval in the reaction containing the sample, is related to the plasmin inhibitor activity, and hence the plasmin inhibitor concentration, present in the sample. Thus, from the percent inhibition of lysis of the FITC-substrate (as observed by a decrease in dequenching) as compared to the standard(s), can be calculated the plasmin inhibitor activity (concentration) in the sample. Further, if desired, a specific antithrombin agent can be added to prevent thrombin interference.
EXAMPLE 10
Assays for Plasminogen Activators
There are various plasminogen activators which may be present in body fluid and play an indirect role in fibrinolysis or fibrinogenolysis. Plasminogen is the inactive precursor of plasmin. Plasminogen activators are proteases that cleave plasminogen into plasmin. There are at least two biochemically distinct types: tissue-type (t-PA), approximately 70 kD in size, and urokinase-type or urinary (u-PA), approximately 33-53 kD. tPA activating activity is enhanced by fibrin. Other substances, such as streptokinase produced by certain strains of streptococci, can function to activate plasminogen. Thus, the method of the present invention can be used to detect and measure plasminogen activator activity. Measuring plasminogen activator activity in body fluid is of clinical importance for several reasons. Plasminogen activators are used in therapeutic thrombolysis; i.e., therapy designed to degrade inappropriate fibrin deposition in blood vessels such as can occur in myocardial infarction. Also, some tumors have been shown to produce uPA, resulting in plasmin-mediated tumor-associated proteolysis that is related to the invasive and metastatic potential of malignant cells in a patient having such a tumor.
10.1 tPA Activity Assay
An assay for tPA activity in fibrinolysis or fibrinogenolysis according to the method of the present invention can be performed in accordance with materials and methods outlined in Examples 1, 2, 7, and 8.
A biological sample containing an unknown amount of tPA activity is added to the reaction buffer simultaneously with a known amount of plasminogen (e.g., 5 nM) and FITC-fibrin or FITCfibrinogen (e.g., 10 nM; "FITC-substrate"). In this instance, it is noted that FITC-fibrin works better than FITC-fibrinogen because it contains tPA binding site that is not present prior to polymerization of fibrinogen. Lysis of the FITC-substrate is then monitored by exciting the reaction mixture with blue light (488 nm) and monitoring the fluorescence emission at 515 nm using a fluorimeter. A parallel set of standard reactions containing a known amount of tPA may be used to determine the amount of fluorescence emission (dequenching) during the desired time interval (i.e., in calibrating the assay). The extent of lysis of the FITC-substrate (i.e., dequenching) at the end of desired time interval in the reaction containing the sample, is related to the tPA activity, and hence the tPA concentration, present in the sample. Thus, the amount of lysis of the FITC-substrate (as observed by the amount of dequenching) as compared to the standard(s), can be calculated the tPA activity (concentration) in the sample. Further, if desired, to increase the sensitivity, tPA may be pre-incubated with plasminogen in the presence of unlabeled fibrin for a fixed period of time (e.g., 15 to 90 minutes) before addition of FITC-substrate. The detection limit is at least 20 picomolar tPA (single chain, recombinant) under routine conditions.
10.2 uPA Activity Assay
An assay for uPA activity in fibrinolysis or fibrinogenolysis according to the method of the present invention can be performed in accordance with materials and methods outlined in Examples 1, 2, 7, and 8.
A biological sample containing an unknown amount of uPA activity is added to the reaction buffer simultaneously with a known amount of plasminogen (e.g., 5 nM) and FITC-fibrin or FITCfibrinogen (e.g., 10 nM; "FITC-substrate"). Lysis of the FITC-substrate is then monitored by exciting the reaction mixture with blue light (488 nm) and monitoring the fluorescence emission at 515 nm using a fluorimeter. A parallel set of standard reactions containing a known amount of uPA may be used to determine the amount of fluorescence emission (dequenching) during the desired time interval (i.e., in calibrating the assay). The extent of lysis of the FITC-substrate (i.e., dequenching) at the end of desired time interval in the reaction containing the sample, is related to the uPA activity, and hence the uPA concentration, present in the sample. Thus, the amount of lysis of the FITC-substrate (as observed by the amount of dequenching) as compared to the standard(s), can be calculated the uPA activity (concentration) in the sample. To increase the sensitivity, uPA may be preincubated with plasminogen in the presence of unlabeled fibrin for a fixed period of time (e.g., 15 to 90 minutes) before addition of FITC-substrate. The detection limit is at least 200 picomolar uPA under routine conditions.
EXAMPLE 11
In another embodiment:, using the method of the present invention, and in accordance with materials and methods outlined in Examples 1-10, naturally-occurring molecular abnormalities fibrinogen ("dysfibrinogens") can be detected clinically. A sample suspected of containing a dysfibrinogen can be isolated from an individual. The apparent dysfibrinogen can be fluorescently labeled, and used as a fluorescent-labeled substrate in one or more of the assays measuring enzyme activity. For example, a dysfibrinogen has been described wherein there is a mutation in the thrombin-cleavage site (position 16 of the A.alpha. chain) involved in fibrinogen polymerization to fibrin. Therefore, in an assay including the sample and a known amount of thrombin, interference with quenching as compared to a standard reaction containing a known amount of fibrinogen may be used to determine if the suspected dysfibrinogen is present in the sample.
EXAMPLE 12
While some of the preceding Examples disclose embodiments that illustrate the fluorescence-based method of the present invention in detecting and measuring activity of components involved or active in clot formation or dissolution, it will be apparent to those skilled in the art that the fluorescence-based method of the present invention can equally be applied to the detection and measurement of components in industrial processes employing enzyme-substrate systems for quality assurance or quantitative purposes. The substrate in such systems is a folded molecule which a) can be labeled with fluorescent molecules; b) is modifiable in either an enzyme-catalyzed polymerization reaction or an enzyme-catalyzed degradation or dissolution reaction; c) where upon enzyme-catalyzed modification of the fluorescent-labeled substrate, a change in fluorescence emission results; and d) measurement of the difference between the fluorescence emission as a result of the enzyme-catalyzed reaction and the basal level of fluorescence emission (prior to substrate modification) directly relates to substrate concentration and/or enzyme activity and indirectly relates to the presence of absence of other components involved in that process.
In one embodiment, an assay for proteolytic activity of a sample containing an unknown amount of a proteolytic enzyme using the method of the present invention can be performed in accordance with materials and methods outlined in Examples 1, 2, 7, and 8. FITC-fibrin or FITC-fibrinogen can be used as a substrate to be degraded in detecting and measuring a sample containing an unknown amount of activity of a specific protease, including such proteases as trypsin, elastase, and papain. The extent of lysis of the FITC-substrate (i.e., dequenching) at the end of desired time interval in the reaction containing the sample, is related to the protease activity, and hence the protease concentration, present in the sample. Standard reactions containing a known amount of the protease may be run in parallel to calibrate the assay.
In another embodiment FITC-casein can be used as a substrate to be degraded in detecting and measuring a sample containing an unknown amount of activity of rennin before use of the enzyme in dairy processing. The extent of lysis of the FITC-substrate (i.e., dequenching) at the end of desired time interval in the reaction containing the sample, is related to the protease activity rennin, and hence the protease concentration, present in the sample. Standard reactions containing a known amount of the rennin may be run in parallel to calibrate the assay.
In another embodiment of the method of the present invention, and according to methods and materials outlined in Examples 1-10, procollagen may serve as the substrate which is fluorescently labeled. Fluorescent-labeled procollagen is a substrate which can be addled as a monomer or in a polymerized state in a reaction mixture along with a sample containing an unknown amount of activity of a component such as collagenase, gelatinase, and/or collagenase inhibitor to detect and measure the component in that sample using the method according to the present invention. Such components are used frequently in the cosmetics industry.
This embodiment is a further illustration of using the fluorescence-based method according to the present invention to detect and measure blood components active in other processes, such as the complement cascade. Complement protein C3 is a 195 kD protein found in serum at a concentration of 550-1200 .mu.g/ml. C3 is a disulfide-linked heterodimer composed of an .alpha. and .beta. polypeptide chain. After activation of the complement system, and during complement assembly on foreign surfaces, C3 becomes cleaved with C3 and its cleavage products becoming part of the C3 convertase complex and the C5 convertase complex. Complement protein C9 is a 79 kD protein found in serum at a concentration of about 60 .mu.g/ml. C9 is a single chain, folded protein which is inserted in bacterial membranes to form a lytic pore as the final step of complement-based killing of bacteria. In the terminal complement complex, C9 polymerizes. Vitronectin has been shown to inhibit C9 polymerization.
An assay for the cleavage of complement components, which may be useful as indicia of inflammation, was performed according to the method of the present invention in accordance with materials and methods outlined in Examples 1 and 2. Briefly, commercially available preparations of C3 and C9 were each fluorescently labelled by combining 100 .mu.g of the respective protein with FITC, incubating the mixture for 0.5 to 1.5 hours, and stopping the reaction by the addition of 1.5M hydroxylamine. Free FITC was removed from the fluorescent-labeled substrate by either dialysis or by gel permeation chromatography.
For measurement of C3 cleavage, the FITC-C3 substrate was diluted in a reaction buffer containing 0.1M NaCl, 5 mM CaCl.sub.2, 50 mM Tris-HCl (pH 7.4), and monitored for 200 to 400 seconds to establish the fluorescence baseline before addition of enzyme or other reagents. Trypsin was then added (50 .mu.l of 0.25% by weight trypsin) to the reaction mixture. As illustrated in FIG. 10, when trypsin was added to the reaction mixture containing FITC-C3 (as indicated by the arrow), the sample underwent sustained dequenching. Thus, an enzyme such as neutral elastase may be measured in a clinical sample by adding to the sample a known amount of FITC-C3. A parallel set of standard reactions containing known and various amounts of neutral elastase and a known amount of FITC-C3 may be used to determine the maximum amount of fluorescence emission (dequenching) during the desired time interval in an elastase-dependent manner. A standard curve is then provided by which to calculate neutral elastase concentration present in the sample.
For measurement of C9 cleavage, the FITC-C9 substrate was diluted in a reaction buffer containing 0.1M NaCl, 5 mM CaCl.sub.2, 50 mM Tris-HCl (pH 7.4), and monitored for 200 to 400 seconds to establish the fluorescence baseline before addition of enzyme or other reagents. In one embodiment, trypsin was then added (50 .mu.l of 0.25% by weight trypsin) to the reaction mixture. As illustrated in FIG. 11, when trypsin was added to the reaction mixture containing FITC-C9 (as indicated by the arrow), the sample underwent sustained dequenching. In a separate embodiment, thrombin was then added (1 U/ml final concentration) to the reaction mixture. As illustrated in FIG. 12, when thrombin was added to the reaction mixture containing FITC-C9 (as indicated by the arrow), the sample underwent sustained dequenching. Thus, fluorescent-labeled C9 may be used as a substrate to measure proteolytic activity of an enzyme present in a clinical sample by adding to the sample a known amount of fluorescent-labeled C9. A parallel set of standard reactions containing known and various amounts of the enzyme and a known amount of fluorescent-labeled C9 may be used to determine the maximum amount of fluorescence emission (dequenching) during the desired time interval in an enzyme-dependent manner. A standard curve is then provided by which to calculate the concentration of the enzyme present in the sample. Since C3 and C9 are available as commercial preparations, they could also be used to assay for activity of industrial enzymes.
Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, various modifications will become apparent to those skilled in the related arts from the foregoing description and figures. Such modifications are intended to be included within the spirit of this application and within the scope of the appended claims.

Number	Name	Date
4046635	Moroz	Sep 1977
4668623	Kinnunen et al.	May 1987
5071745	Triscott et al.	Dec 1991
5567596	Diamond et al.	Oct 1996

Method for assaying components in an enzyme-protein substrate system

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

Parent Case Info

US Referenced Citations (4)

Non-Patent Literature Citations (1)

Continuation in Parts (1)