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. 1995 May 15;308(Pt 1):251–260. doi: 10.1042/bj3080251

Photoaffinity labelling of cyanomethaemoglobin with derivatives of tryptophan and 5-bromotryptophan.

M Li 1, Z Lin 1, M E Johnson 1
PMCID: PMC1136870  PMID: 7755572

Abstract

Tryptophan and 5-bromotryptophan (5-BrTrp) are relatively potent inhibitors of sickle-haemoglobin polymerization. The binding sites of these compounds to normal and sickle haemoglobin (HBA and HBS) have been suggested, but not firmly established, through the use of spin-labelled derivatives and/or computer modeling. In the present study we approached the problem by utilizing the technique of photoaffinity labelling. The cyanomet forms of HBA and HBS were subjected to photoaffinity labelling with N alpha-(4-azidotetrafluorobenzoyl)tryptophan and N alpha-(1-ethyl-2-diazomalonyl)-5-bromotryptophan respectively. Both irradiated samples of HBA and HBS were denatured, digested with trypsin, and then separated by reversed-phase HPLC. A labelled tryptic peptide was isolated from the photolabelling of HBS with N alpha-(1-ethyl-2-diazomalonyl)-5-bromotryptophan. The peptide was identified to be Val1(alpha)-Lys7(alpha), with the label attached to Val1(alpha), by virtue of amino acid analysis and sequencing, in conjunction with fast-atom-bombardment MS. The binding mode of N alpha-(1-ethyl-2-diazomalonyl)-5-bromotryptophan is proposed and its relevance to the potency of the 5-BrTrp-based anti-sickling agents is discussed.

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Selected References

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  1. Abraham D. J., Gazze D. M., Kennedy P. E., Mokotoff M. Design, synthesis, and testing of potential antisickling agents. 5. Disubstituted benzoic acids designed for the donor site and proline salicylates designed for the acceptor site. J Med Chem. 1984 Dec;27(12):1549–1559. doi: 10.1021/jm00378a005. [DOI] [PubMed] [Google Scholar]
  2. Abraham D. J., Perutz M. F., Phillips S. E. Physiological and x-ray studies of potential antisickling agents. Proc Natl Acad Sci U S A. 1983 Jan;80(2):324–328. doi: 10.1073/pnas.80.2.324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Belleau B., Malek G. A new convenient reagent for peptide syntheses. J Am Chem Soc. 1968 Mar 13;90(6):1651–1652. doi: 10.1021/ja01008a045. [DOI] [PubMed] [Google Scholar]
  4. Bernstein F. C., Koetzle T. F., Williams G. J., Meyer E. F., Jr, Brice M. D., Rodgers J. R., Kennard O., Shimanouchi T., Tasumi M. The Protein Data Bank. A computer-based archival file for macromolecular structures. Eur J Biochem. 1977 Nov 1;80(2):319–324. doi: 10.1111/j.1432-1033.1977.tb11885.x. [DOI] [PubMed] [Google Scholar]
  5. De Croos P. Z., Sangdee P., Stockwell B. L., Kar L., Thompson E. B., Johnson M. E., Currie B. L. Hemoglobin S antigelation agents based on 5-bromotryptophan with potential for sickle cell anemia. J Med Chem. 1990 Dec;33(12):3138–3142. doi: 10.1021/jm00174a008. [DOI] [PubMed] [Google Scholar]
  6. Di Iorio E. E. Preparation of derivatives of ferrous and ferric hemoglobin. Methods Enzymol. 1981;76:57–72. doi: 10.1016/0076-6879(81)76114-7. [DOI] [PubMed] [Google Scholar]
  7. Hexter C. S., Westheimer F. H. S-carboxymethylcysteine from the photolysis of diazoacyl trypsin and chymotrypsin. J Biol Chem. 1971 Jun 25;246(12):3934–3938. [PubMed] [Google Scholar]
  8. Johnson C. S., Schroeder W. A., Shelton J. B., Shelton J. R. The first example of a deletion in the human alpha chain: hemoglobin Boyle Heights or alpha 2 6 (A4) Asp----to O beta 2. Hemoglobin. 1983;7(2):125–140. doi: 10.3109/03630268309048642. [DOI] [PubMed] [Google Scholar]
  9. Klotz I. M., Haney D. N., King L. C. Rational approaches to chemotherapy: antisickling agents. Science. 1981 Aug 14;213(4509):724–731. doi: 10.1126/science.7256275. [DOI] [PubMed] [Google Scholar]
  10. Lee Y. H., Currie B. L., Johnson M. E. Interaction of a spin-labeled phenylalanine analogue with normal and sickle hemoglobins: detection of site-specific interactions through spin-label-induced 1H NMR relaxation. Biochemistry. 1986 Sep 23;25(19):5647–5654. doi: 10.1021/bi00367a045. [DOI] [PubMed] [Google Scholar]
  11. Manavalan P., Prabhakaran M., Johnson M. E. Location of potential binding sites on deoxy hemoglobin for the design of antigelling agents. J Mol Biol. 1992 Feb 5;223(3):791–800. doi: 10.1016/0022-2836(92)90990-2. [DOI] [PubMed] [Google Scholar]
  12. Mazhani L., Kim B. C., Poillon W. N. Noncovalent inhibitors of sickle hemoglobin gelation: effects of tetrasubstituted ammonium salts. Hemoglobin. 1984;8(2):129–136. doi: 10.3109/03630268408991706. [DOI] [PubMed] [Google Scholar]
  13. Noguchi C. T., Schechter A. N. Effects of amino acids on gelation kinetics and solubility of sickle hemoglobin. Biochem Biophys Res Commun. 1977 Jan 24;74(2):637–642. doi: 10.1016/0006-291x(77)90350-3. [DOI] [PubMed] [Google Scholar]
  14. Noguchi C. T., Schechter A. N. Inhibition of sickle hemoglobin gelation by amino acids and related compounds. Biochemistry. 1978 Dec 12;17(25):5455–5459. doi: 10.1021/bi00618a020. [DOI] [PubMed] [Google Scholar]
  15. Padlan E. A., Love W. E. Refined crystal structure of deoxyhemoglobin S. I. Restrained least-squares refinement at 3.0-A resolution. J Biol Chem. 1985 Jul 15;260(14):8272–8279. doi: 10.2210/pdb1hbs/pdb. [DOI] [PubMed] [Google Scholar]
  16. Padlan E. A., Love W. E. Refined crystal structure of deoxyhemoglobin S. II. Molecular interactions in the crystal. J Biol Chem. 1985 Jul 15;260(14):8280–8291. [PubMed] [Google Scholar]
  17. Poillon W. N. Noncovalent inhibitors of sickle hemoglobin gelation: effects of aryl-substituted alanines. Biochemistry. 1982 Mar 16;21(6):1400–1406. doi: 10.1021/bi00535a046. [DOI] [PubMed] [Google Scholar]
  18. ROSSI-FANELLI A., ANTONINI E., CAPUTO A. Studies on the relations between molecular and functional properties of hemoglobin. I. The effect of salts on the molecular weight of human hemoglobin. J Biol Chem. 1961 Feb;236:391–396. [PubMed] [Google Scholar]
  19. Schroeder W. A., Shelton J. B., Shelton J. R., Powars D. Hemoglobin Sunshine Seth - alpha 2 (94 (G1) Asp replaced by His) beta 2. Hemoglobin. 1979;3(2-3):145–159. doi: 10.3109/03630267908998910. [DOI] [PubMed] [Google Scholar]
  20. Shelton J. B., Shelton J. R., Schroeder W. A., Powars D. R. Hb Aztec or alpha 2 76 (EF5) Met----Thr beta 2 detection of a silent mutant by high performance liquid chromatography. Hemoglobin. 1985;9(4):325–332. doi: 10.3109/03630268508997008. [DOI] [PubMed] [Google Scholar]
  21. Vaughan R. J., Westheimer F. H. A method for marking the hydrophobic binding sites of enzymes. An insertion into the methyl group of an alanine residue of trypsin. J Am Chem Soc. 1969 Jan 1;91(1):217–218. doi: 10.1021/ja01029a055. [DOI] [PubMed] [Google Scholar]
  22. Votano J. R., Altman J., Wilchek M., Gorecki M., Rich A. Potential use of biaromatic L-phenylalanyl derivatives as therapeutic agents in the treatment of sickle cell disease. Proc Natl Acad Sci U S A. 1984 May;81(10):3190–3194. doi: 10.1073/pnas.81.10.3190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Votano J. R., Gorecki M., Rich A. Sickle hemoglobin aggregation: a new class of inhibitors. Science. 1977 Jun 10;196(4295):1216–1219. doi: 10.1126/science.870976. [DOI] [PubMed] [Google Scholar]
  24. Votano J. R., Rich A. Inhibition of deoxyhemoglobin S polymerization by biaromatic peptides found to associate with the hemoglobin molecule at a preferred site. Biochemistry. 1985 Apr 9;24(8):1966–1970. doi: 10.1021/bi00329a025. [DOI] [PubMed] [Google Scholar]
  25. Wireko F. C., Kellogg G. E., Abraham D. J. Allosteric modifiers of hemoglobin. 2. Crystallographically determined binding sites and hydrophobic binding/interaction analysis of novel hemoglobin oxygen effectors. J Med Chem. 1991 Feb;34(2):758–767. doi: 10.1021/jm00106a042. [DOI] [PubMed] [Google Scholar]

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