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. 1996 Apr;5(4):775–781. doi: 10.1002/pro.5560050423

Subunit dissociations in natural and recombinant hemoglobins.

L R Manning 1, W T Jenkins 1, J R Hess 1, K Vandegriff 1, R M Winslow 1, J M Manning 1
PMCID: PMC2143381  PMID: 8845768

Abstract

A precise and rapid procedure employing gel filtration on Superose-12 to measure the tetramer-dimer dissociation constants of some natural and recombinant hemoglobins in the oxy conformation is described. Natural sickle hemoglobin was chosen to verify the validity of the results by comparing the values with those reported using an independent method not based on gel filtration. Recombinant sickle hemoglobin, as well as a sickle double mutant with a substitution at the Val-6(beta) receptor site, had approximately the same dissociation constant as natural sickle hemoglobin. Of the two recombinant hemoglobins with amino acid replacements in the alpha 1 beta 2 subunit interface, one was found to be extensively dissociated and the other completely dissociated. In addition, the absence of an effect of the allosteric regulators DPG and IHP on the dissociation constant was demonstrated. Thus, a tetramer dissociation constant can now be determined readily and used together with other criteria for characterization of hemoglobins and their interaction with small regulatory molecules.

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Selected References

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  1. ACKERS G. K., THOMPSON T. E. DETERMINATION OF STOICHIOMETRY AND EQUILIBRIUM CONSTANTS FOR REVERSIBLY ASSOCIATING SYSTEMS BY MOLECULAR SIEVE CHROMATOGRAPHY. Proc Natl Acad Sci U S A. 1965 Feb;53:342–349. doi: 10.1073/pnas.53.2.342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andersen M. E., Moffat J. K., Gibson Q. H. The kinetics of ligand binding and of the association-dissociation reactions of human hemoglobin. Properties of deoxyhemoglobin dimers. J Biol Chem. 1971 May 10;246(9):2796–2807. [PubMed] [Google Scholar]
  3. Andrews P. Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem J. 1964 May;91(2):222–233. doi: 10.1042/bj0910222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baudin-Chich V., Marden M., Wajcman H. Investigation of the tetramer-dimer equilibrium in haemoglobin solutions by high-performance size-exclusion chromatography on a diol column. J Chromatogr. 1988 Mar 11;437(1):193–201. doi: 10.1016/s0021-9673(00)90382-0. [DOI] [PubMed] [Google Scholar]
  5. Benesch R. E., Kwong S. Coupled reactions in hemoglobin. Heme-globin and dimer-dimer association. J Biol Chem. 1995 Jun 9;270(23):13785–13786. doi: 10.1074/jbc.270.23.13785. [DOI] [PubMed] [Google Scholar]
  6. Bonaventura J., Riggs A. Hemoglobin Kansas, a human hemoglobin with a neutral amino acid substitution and an abnormal oxygen equilibrium. J Biol Chem. 1968 Mar 10;243(5):980–991. [PubMed] [Google Scholar]
  7. Bunn H. F., Jandl J. H. Exchange of heme among hemoglobins and between hemoglobin and albumin. J Biol Chem. 1968 Feb 10;243(3):465–475. [PubMed] [Google Scholar]
  8. Chatterjee R., Welty E. V., Walder R. Y., Pruitt S. L., Rogers P. H., Arnone A., Walder J. A. Isolation and characterization of a new hemoglobin derivative cross-linked between the alpha chains (lysine 99 alpha 1----lysine 99 alpha 2). J Biol Chem. 1986 Jul 25;261(21):9929–9937. [PubMed] [Google Scholar]
  9. Chiancone E. Dissociation of hemoglobin into subunits. II. Human oxyhemoglobin: gel filtration studies. J Biol Chem. 1968 Mar 25;243(6):1212–1219. [PubMed] [Google Scholar]
  10. Chu A. H., Ackers G. K. Mutual effects of protons, NaCl, and oxygen on the dimer-tetramer assembly of human hemoglobin. The dimer Bohr effect. J Biol Chem. 1981 Feb 10;256(3):1199–1205. [PubMed] [Google Scholar]
  11. DiDonato A., Fantl W. J., Acharya A. S., Manning J. M. Selective carboxymethylation of the alpha-amino groups of hemoglobin. Effect on functional properties. J Biol Chem. 1983 Oct 10;258(19):11890–11895. [PubMed] [Google Scholar]
  12. Fronticelli C., Gattoni M., Lu A. L., Brinigar W. S., Bucci J. L., Chiancone E. The dimer-tetramer equilibrium of recombinant hemoglobins. Stabilization of the alpha 1 beta 2 interface by the mutation beta(Cys112-->Gly) at the alpha 1 beta 1 interface. Biophys Chem. 1994 Jul;51(1):53–57. doi: 10.1016/0301-4622(94)00028-x. [DOI] [PubMed] [Google Scholar]
  13. GUIDOTTI G., KONIGSBERG W., CRAIG L. C. ON THE DISSOCIATION OF NORMAL ADULT HUMAN HEMOGLOBIN. Proc Natl Acad Sci U S A. 1963 Oct;50:774–782. doi: 10.1073/pnas.50.4.774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gacon G., Belkhodja O., Wajcman H., Labie D., Najman A. Structural and functional studies of Hb Rothschild beta (C3) Trp replaced by Arg. A new variant of the alpha1beta2 contact. FEBS Lett. 1977 Oct 15;82(2):243–246. doi: 10.1016/0014-5793(77)80593-0. [DOI] [PubMed] [Google Scholar]
  15. INGRAM V. M. A specific chemical difference between the globins of normal human and sickle-cell anaemia haemoglobin. Nature. 1956 Oct 13;178(4537):792–794. doi: 10.1038/178792a0. [DOI] [PubMed] [Google Scholar]
  16. Jack L. J., Mather I. H. Cloning and analysis of cDNA encoding bovine butyrophilin, an apical glycoprotein expressed in mammary tissue and secreted in association with the milk-fat globule membrane during lactation. J Biol Chem. 1990 Aug 25;265(24):14481–14486. [PubMed] [Google Scholar]
  17. Kellett G. L., Schachman H. K. Dissociation of hemoglobin into subunits. Monomer formation and the influence of ligands. J Mol Biol. 1971 Aug 14;59(3):387–399. doi: 10.1016/0022-2836(71)90306-8. [DOI] [PubMed] [Google Scholar]
  18. Manning L. R., Morgan S., Beavis R. C., Chait B. T., Manning J. M., Hess J. R., Cross M., Currell D. L., Marini M. A., Winslow R. M. Preparation, properties, and plasma retention of human hemoglobin derivatives: comparison of uncrosslinked carboxymethylated hemoglobin with crosslinked tetrameric hemoglobin. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3329–3333. doi: 10.1073/pnas.88.8.3329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Martin de Llano J. J., Jones W., Schneider K., Chait B. T., Manning J. M., Rodgers G., Benjamin L. J., Weksler B. Biochemical and functional properties of recombinant human sickle hemoglobin expressed in yeast. J Biol Chem. 1993 Dec 25;268(36):27004–27011. [PubMed] [Google Scholar]
  20. Martin de Llano J. J., Manning J. M. Properties of a recombinant human hemoglobin double mutant: sickle hemoglobin with Leu-88(beta) at the primary aggregation site substituted by Ala. Protein Sci. 1994 Aug;3(8):1206–1212. doi: 10.1002/pro.5560030806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Martin de Llano J. J., Schneewind O., Stetler G., Manning J. M. Recombinant human sickle hemoglobin expressed in yeast. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):918–922. doi: 10.1073/pnas.90.3.918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Perutz M. F. Mechanisms of cooperativity and allosteric regulation in proteins. Q Rev Biophys. 1989 May;22(2):139–237. doi: 10.1017/s0033583500003826. [DOI] [PubMed] [Google Scholar]
  23. Sharma V. S., Newton G. L., Ranney H. M., Ahmed F., Harris J. W., Danish E. H. Hemoglobin Rothschild (beta 37(C3)Trp replaced by Arg): A high/low affinity hemoglobin mutant. J Mol Biol. 1980 Dec 15;144(3):267–280. doi: 10.1016/0022-2836(80)90090-x. [DOI] [PubMed] [Google Scholar]
  24. Turner G. J., Galacteros F., Doyle M. L., Hedlund B., Pettigrew D. W., Turner B. W., Smith F. R., Moo-Penn W., Rucknagel D. L., Ackers G. K. Mutagenic dissection of hemoglobin cooperativity: effects of amino acid alteration on subunit assembly of oxy and deoxy tetramers. Proteins. 1992 Nov;14(3):333–350. doi: 10.1002/prot.340140303. [DOI] [PubMed] [Google Scholar]
  25. Vandegriff K. D., Medina F., Marini M. A., Winslow R. M. Equilibrium oxygen binding to human hemoglobin cross-linked between the alpha chains by bis(3,5-dibromosalicyl) fumarate. J Biol Chem. 1989 Oct 25;264(30):17824–17833. [PubMed] [Google Scholar]
  26. Williams R. C., Jr, Kim H. Carbamoylated hemoglobins A and S: physical properties. Biochemistry. 1976 May 18;15(10):2207–2211. doi: 10.1021/bi00655a028. [DOI] [PubMed] [Google Scholar]
  27. Yanase H., Cahill S., Martin de Llano J. J., Manning L. R., Schneider K., Chait B. T., Vandegriff K. D., Winslow R. M., Manning J. M. Properties of a recombinant human hemoglobin with aspartic acid 99(beta), an important intersubunit contact site, substituted by lysine. Protein Sci. 1994 Aug;3(8):1213–1223. doi: 10.1002/pro.5560030807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Yanase H., Manning L. R., Vandegriff K., Winslow R. M., Manning J. M. A recombinant human hemoglobin with asparagine-102(beta) substituted by alanine has a limiting low oxygen affinity, reduced marginally by chloride. Protein Sci. 1995 Jan;4(1):21–28. doi: 10.1002/pro.5560040104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zimmerman J. K., Ackers G. K. Molecular sieve studies of interacting protein systems. X. Behavior of small zone profiles for reversibly self-associating solutes. J Biol Chem. 1971 Dec 10;246(23):7289–7292. [PubMed] [Google Scholar]

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