Photoaffinity labelling of cyanomethaemoglobin with derivatives of tryptophan and 5-bromotryptophan.
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.
Li
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M
Center for Pharmaceutical Biotechnology, University of Illinois at Chicago 60612-7230, USA.
Lin
Z
Z
Johnson
M E
ME
D000345:Affinity Labels; D000595:Amino Acid Sequence; D006801:Humans; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008706:Methemoglobin / Q000031:analogs & derivatives* / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000031:analogs & derivatives / Q000737:chemistry
D000345:Affinity Labels; D010455:Peptides; C007169:cyanomethemoglobin; C066614:5-bromotryptophan; D014364:Tryptophan; D008706:Methemoglobin