Li M, Lin Z, Johnson M E
Center for Pharmaceutical Biotechnology, University of Illinois at Chicago 60612-7230, USA.
Biochem J. 1995 May 15;308 ( Pt 1)(Pt 1):251-60. doi: 10.1042/bj3080251.
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.
色氨酸和5-溴色氨酸（5-BrTrp）是镰状血红蛋白聚合作用相对有效的抑制剂。通过使用自旋标记衍生物和/或计算机建模，已经提出了这些化合物与正常血红蛋白和镰状血红蛋白（HBA和HBS）的结合位点，但尚未得到确凿证实。在本研究中，我们利用光亲和标记技术来解决这个问题。分别用Nα-（4-叠氮基四氟苯甲酰基）色氨酸和Nα-（1-乙基-2-重氮丙二酰基）-5-溴色氨酸对HBA和HBS的氰化高铁形式进行光亲和标记。HBA和HBS的辐照样品均被变性，用胰蛋白酶消化，然后通过反相高效液相色谱进行分离。从用Nα-（1-乙基-2-重氮丙二酰基）-5-溴色氨酸对HBS进行光标记的产物中分离出一个标记的胰蛋白酶肽段。通过氨基酸分析、测序以及快原子轰击质谱，鉴定该肽段为Val1（α）-Lys7（α），标记附着在Val1（α）上。本文提出了Nα-（1-乙基-2-重氮丙二酰基）-5-溴色氨酸的结合模式，并讨论了其与基于5-BrTrp的抗镰状化剂效力的相关性。
