Claims
- 1. Method for treating or preventing a disease or medical disorder which can be ameliorated by delivery of NO or its biological equivalent to tissues affected by the disease or medical disorder, in an animal or human, comprising administering to the animal or human nitrosyl-heme-containing donors of NO.
- 2. The method of claim 1 wherein the nitrosyl-heme-containing donor of NO is nitrosylhemoglobin.
- 3. Method for making stable nitrosyl-deoxyhemoglobin comprising adding NO to deoxyhemoglobin in an aqueous solution such that the ratio of added NO: heme is less than about 1:100 or greater than about 0.75.
- 4. Method for making SNO-oxyhemoglobin, comprising adding NO to an aqueous solution of oxyhemoglobin and buffer having a pK of at least about 9.4, at a concentration of approximately 10 mM to 200 mM, at pH 7.4.
- 5. Method for making nitrosyl-oxyhemoglobin comprising adding NO to oxyhemoglobin in an aqueous solution such that the ratio of NO: hemoglobin is less than about 1:30.
- 6. Hemoglobin conjugated to an NO-donor.
- 7. Hemoglobin of claim 6, wherein the NO-donor is selected from the group consisting of:
diazeniumdiolates, nitroprusside, nitroglycerin and nitrosothiol.
- 8. A composition comprising hemoglobin and one or more NO-donors.
- 9. A method for treating or preventing a disease or medical disorder which can be ameliorated by delivery of NO or its biological equivalent to tissues affected by the disease or medical disorder, in an animal or human, comprising administering a heme-based blood substitute and inhaled NO to the animal or human.
- 10. A method for delivering CO to the tissues in an animal or human, comprising administering CO-derivatized hemoglobin to the animal or human.
- 11. A method of treating or preventing a disease or medical disorder which can be ameliorated by delivery of NO or its biological equivalent to tissues affected by the disease or medical disorder in an animal or human, comprising administering CO-derivatized hemoglobin and a nitrosated hemoglobin to the animal or human.
- 12. Nitrosylhemoglobin conjugated to one or more electron acceptors.
- 13. Nitrosylhemoglobin of claim 12, wherein the electron acceptor is selected from the group consisting of:
superoxide dismutase, stable nitroxide radicals, and oxidized forms of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, ascorbate and dehydroascorbate.
- 14. A composition comprising nitrosylhemoglobin and one or more electron acceptors.
- 15. Hemoglobin conjugated to nitric oxide synthase.
- 16. Hemoglobin of claim 15, wherein the nitric oxide synthase is nitric oxide synthase of neurons.
- 17. A composition comprising hemoglobin and nitric oxide synthase.
- 18. A method for making isolated erythrocytes comprising nitrosylhemoglobin, comprising incubating deoxygenated erythrocytes in a solution comprising NO.
- 19. Isolated erythrocytes comprising nitrosylhemoglobin.
- 20. A method for treating shock in an animal or human comprising administering hemoglobin α-chains to the animal or human.
- 21. A method for treating or preventing a disease or medical disorder which can be ameliorated by delivery of NO or its biological equivalent to tissues affected by the disease or medical disorder, in an animal or human, comprising administering hemoglobin β-chains to the animal or human.
- 22. A method for measuring NO equivalents in S-nitrosohemoglobin and nitrosyl(FeII)-hemoglobin in blood comprising red blood cells, comprising the steps of:
a) lysing the red blood cells of a blood sample; b) preparing a desalted protein fraction of the lysed red blood cells; c) subjecting a protein fraction to photolysis, thereby liberating nitric oxide from S-nitrosohemoglobin and nitrosyl (FeII)-hemoglobin; and d) quantitating the nitric oxide in the protein fraction by measuring a chemiluminescence signal generated by a chemical reaction between nitric oxide and ozone, thereby measuring NO equivalents in S-nitrosohemoglobin and nitrosyl(FeII)-hemoglobin in blood.
- 23. A method for assaying nitric oxide production in disease states, comprising the steps of:
a) lysing the red blood cells of a blood sample; b) preparing a protein fraction of the lysed red blood cells; c) subjecting a protein fraction to photolysis, thereby liberating nitric oxide from S-nitrosohemoglobin and nitrosyl(FeII)-hemoglobin; and d) quantitating [the amount of] nitric oxide in the protein fraction by measuring a chemiluminescence signal generated by a chemical reaction between nitric oxide and ozone.
- 24. A method for assaying thiol-bound NO in S-nitrosohemoglobin in red blood cells, comprising:
a) isolating washed red blood cells from blood; b) lysing the red blood cells, thereby obtaining a lysate; c) desalting the lysate; d) dividing the lysate into an aliquot contacted with mercury ions in excess over protein concentration in the lysate, and leaving another aliquot untreated by mercury ions, thereby obtaining a mercury-treated aliquot and an untreated aliquot; e) exposing the mercury-treated aliquot and untreated aliquot to oxygen; f) isolating a mercury-treated low molecular weight fraction and an untreated low molecular weight fraction from the respective aliquots of e); g) contacting the low molecular weight fractions of f) with excess low molecular weight thiol under acidic conditions, thereby producing S-nitrosothiol in each fraction; h) measuring NO liberated from S-nitrosothiol in the fractions of g) by photolysis-chemiluminescence; and i) determining a quantity of thiol-bound NO in S-nitrosohemoglobin from a difference in measurements in h).
- 25. A method for assaying NO equivalents in S-nitrosohemoglobin and nitrosyl(FeII)-hemoglobin in purified hemoglobin, comprising measuring NO equivalents in the purified hemoglobin by photolysis-chemiluminescence, thereby measuring NO equivalents in S-nitrosohemoglobin and nitrosyl(FeII)-hemoglobin.
- 26. A method for measuring NO equivalents in S-nitrosohemoglobin and nitrosyl(FeII)-hemoglobin in red blood cells, comprising:
a) isolating washed red blood cells from blood; b) lysing the red blood cells, thereby obtaining a lysate; c) desalting the lysate; and d) measuring NO equivalents in the lysate of c) by photolysis-chemiluminescence, thereby measuring NO equivalents in S-nitrosohemoglobin and nitrosyl(FeII)-hemoglobin in red blood cells.
- 27. A method for measuring NO bound to nitrosyl(FeII)-hemoglobin in red blood cells, comprising:
a) making a protein fraction from the red blood cells; b) treating the protein fraction with excess HgCl2, followed by exposure to air; and c) subjecting the protein fraction of b) to photolysis of the NO ligand of nitrosyl(FeII)-hemoglobin followed by detection of NO by chemiluminescence, thereby measuring NO bound to nitrosyl(FeII)-hemoglobin.
- 28. A method for assaying S-nitrosohemoglobin comprising:
a) isolating washed red blood cells from blood; b) lysing the red blood cells, thereby obtaining a lysate; c) desalting the lysate; d) contacting an aliquot of the lysate of c) with mercury ions in excess over protein concentration, thereby obtaining a mercury-treated aliquot and an untreated aliquot; e) exposing the mercury-treated aliquot and the untreated aliquot to oxygen; f) measuring NO equivalents in the mercury-treated aliquot of e) and NO equivalents in the untreated aliquot of e) by photolysis-chemiluminescence; and g) determining a quantity of S-nitrosohemoglobin from the NO equivalents measured in f).
- 29. A method for measuring S-nitrosohemoglobin and nitrosyl(FeII)-hemoglobin in red blood cells, comprising:
a) isolating washed red blood cells from blood; b) lysing the red blood cells, thereby obtaining a lysate; c) desalting the lysate; and d) measuring NO equivalents from the lysate of c) by photolysis-chemiluminescence.
- 30. A method for assaying nitrosyl(Fe)-hemoglobin comprising:
a) isolating washed red blood cells from blood; b) lysing the red blood cells, thereby obtaining a lysate; c) desalting the lysate; d) contacting an aliquot of the lysate with mercury ions in excess over protein concentration, thereby obtaining a mercury-treated aliquot and an untreated aliquot; e) exposing the mercury-treated aliquot and untreated aliquot to oxygen; f) measuring NO equivalents in the aliquots of e) by photolysis-chemiluminescence, thereby obtaining 1) a quantity for S-nitrosohemoglobin and 2) a quantity for nitrosyl(FeII)-hemoglobin and S-nitrosohemoglobin combined, respectively; and g) determining a quantity for nitrosyl(FeII)-hemoglobin by subtracting 1) from 2).
- 31. A method for assessing oxygen delivery to a site in the body, comprising determining S-nitrosohemoglobin and nitrosylhemoglobin values in a blood sample from the site, and using said values to assess oxygen delivery.
- 32. The method of claim 31, further comprising determining an oxygen value in the blood sample and using said value to assess oxygen delivery.
GOVERNMENT SUPPORT
[0001] This invention was made with government support under Grant Nos. HL52529 and HR59130 awarded by the National Institutes of Health. The government has certain rights in the invention.