Muszynska G, Riordan J F
Biochemistry. 1976 Jan 13;15(1):46-51. doi: 10.1021/bi00646a008.
Nitration of bovine carboxypeptidase A crystals with tetranitromethane increases esterase activity, decreases peptidase activity, and modifies about one tyrosyl residue. Modification of enzyme crystals avoids the polymerization that occurs when the enzyme is nitrated in solution. Two procedures have been employed to identify the tyrosyl residues nitrated. The first involves cyanogen bromide cleavage and isolation of the fragment containing residues 104-301. After solubilization by succinylation, this fragment is digested with chymotrypsin, the peptides are fractionated by gel filtration, and the nitrotyrosyl peptides are purified by affinity chromatography on an antinitrotyrosyl antibody-Sepharose conjugate followed by ion-exchange chromatography. In the second, the nitroenzyme is heat denatured, digested by chymotrypsin, and fractionated on the affinity and ion-exchange columns. By both methods, the major mitropeptides, representing between 60 and 80% of the nitrotyrosyl label, are uniquely compatible with that segment of the sequence of carboxypeptidase containing Tyr-248. A nearby cation, either the active site zinc ion or Arg-145, would seem to be an important factor in determining the selective nitration of this residue.
用四硝基甲烷对牛羧肽酶A晶体进行硝化，可提高酯酶活性，降低肽酶活性，并修饰大约一个酪氨酸残基。对酶晶体进行修饰可避免在溶液中对酶进行硝化时发生的聚合反应。已采用两种方法来鉴定被硝化的酪氨酸残基。第一种方法是用溴化氰裂解并分离含有104 - 301位残基的片段。通过琥珀酰化使其溶解后，用胰凝乳蛋白酶消化该片段，通过凝胶过滤对肽进行分级分离，然后通过在抗硝基酪氨酸抗体 - 琼脂糖偶联物上进行亲和色谱，再进行离子交换色谱来纯化硝基酪氨酸肽。第二种方法是将硝基酶加热变性，用胰凝乳蛋白酶消化，然后在亲和柱和离子交换柱上进行分级分离。通过这两种方法，占硝基酪氨酸标记60%至80%的主要硝基肽与羧肽酶序列中含有Tyr - 248的片段唯一匹配。附近的阳离子，无论是活性位点锌离子还是Arg - 145，似乎都是决定该残基选择性硝化的重要因素。
