Zisapel N
Eur J Biochem. 1978 Sep 15;90(1):199-203. doi: 10.1111/j.1432-1033.1978.tb12591.x.
Cadmium-carboxypeptidase B was nitrated with tetranitromethane. The enzyme polymerized extensively during nitration. In the monomer nitrated Cd-carboxypeptidase B, 70% of the activity of Cd-carboxypeptidase B was retained. In order to identify the tyrosyl residues nitrated, the enzyme was digested with chymotrypsin and subtilisin and the nitrotyrosyl peptides were purified by affinity chromatography on antityrosyl-antibody-Sepharose conjugate followed by two-dimensional thin-layer chromatography. The major nitropeptides, representing 65% of the nitrotyrosyl label, were compatible with the segment of the sequence containing Tyr-240 and Tyr-259. Only 10% of the nitrotyrosyl label was found in the segment of Tyr-248. This indicates that the state of Tyr-248 in Cd-carboxypeptidase B differs from that in zinc-carboxypeptidase B where it shows chemical hyperreactivity due to its proximity to the metal ion.
羧肽酶B用四硝基甲烷进行硝化。该酶在硝化过程中广泛聚合。在单体硝化的镉羧肽酶B中，保留了70%的镉羧肽酶B活性。为了鉴定被硝化的酪氨酸残基，用胰凝乳蛋白酶和枯草杆菌蛋白酶消化该酶，并用抗酪氨酸抗体-琼脂糖凝胶偶联物进行亲和层析，随后进行二维薄层色谱法纯化硝基酪氨酸肽。占硝基酪氨酸标记65%的主要硝基肽与包含Tyr-240和Tyr-259的序列片段相符。在Tyr-248片段中仅发现10%的硝基酪氨酸标记。这表明镉羧肽酶B中Tyr-248的状态与锌羧肽酶B中不同，在锌羧肽酶B中由于其靠近金属离子而表现出化学超反应性。
