Sacchetta P, Battista P, Santarone S, Di Cola D
Institute of Scienze Biochimiche, Faculty of Medicine, University of Chieti, Italy.
Biochim Biophys Acta. 1990 Mar 1;1037(3):337-43. doi: 10.1016/0167-4838(90)90035-e.
Exposure of human red cells to oxidants such as phenylhydrazine, 2,4-dimethylphenylhydrazine and 4-hydrazinobenzoic acid stimulates the proteolysis of hemoglobin as evidenced by the increase in the rate of the free alanine and acid soluble amino groups released. An enzyme responsible for proteolytic degradation of oxidized hemoglobin, was purified from cytosolic fraction of erythrocytes by a DEAE-batch procedure followed by gel-filtration and ion-exchange chromatography. The final enzyme preparation produces a single band in non-denaturing polyacrylamide gel electrophoresis, and eight different bands of 23-32 kDa when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The native enzyme has a molecular mass of about 700 kDa as estimated by gel filtration. The enzyme, unable to hydrolyze native hemoglobin, cleaves phenylhydrazine-treated hemoglobin into small peptides without free amino acid release. In addition, the enzyme shows an endopeptidase activity towards synthetic peptides having a tyrosine or an arginine in the P1 position, whereas it does not hydrolyze shorter peptides and those with a proline in the P1 or P2 position. The proteolytic activity of the enzyme against oxidized hemoglobin is inhibited by chymostatin and p-chloromercuribenzoate, while it is stimulated by N-ethylmaleimide and epoxysuccinylleucylamido-(4-guanidino)butane (E-64). The peptidase activity assayed on succinyl-Leu-Leu-Val-Tyr-MCA is inhibited by chymostatin, hemin, N-ethylmaleimide and p-chloromercuribenzoate. The results obtained show that in human erythrocytes oxidized hemoglobin is cleaved into peptides by a high molecular mass proteinase identified as a member of the multicatalytic proteinase family. It is also suggested that the complete degradation of oxidized hemoglobin to free amino acids requires the involvement of a further proteolytic enzyme(s) which remain(s) to be identified.
将人类红细胞暴露于苯肼、2,4-二甲基苯肼和4-肼基苯甲酸等氧化剂中，会刺激血红蛋白的蛋白水解，这可通过游离丙氨酸和酸溶性氨基释放速率的增加得到证明。一种负责氧化血红蛋白蛋白水解降解的酶，通过DEAE分批法，随后进行凝胶过滤和离子交换色谱，从红细胞的胞质部分中纯化出来。最终的酶制剂在非变性聚丙烯酰胺凝胶电泳中产生一条带，而在变性条件下进行聚丙烯酰胺凝胶电泳时产生八条23 - 32 kDa的不同条带。通过凝胶过滤估计，天然酶的分子量约为700 kDa。该酶无法水解天然血红蛋白，能将经苯肼处理的血红蛋白切割成小肽，且不释放游离氨基酸。此外，该酶对在P1位置含有酪氨酸或精氨酸的合成肽表现出内肽酶活性，而不水解较短的肽以及在P1或P2位置含有脯氨酸的肽。该酶对氧化血红蛋白的蛋白水解活性受到抑肽酶和对氯汞苯甲酸的抑制，而受到N-乙基马来酰亚胺和环氧琥珀酰亮氨酰胺基-(4-胍基)丁烷（E-64）的刺激。对琥珀酰-Leu-Leu-Val-Tyr-MCA测定的肽酶活性受到抑肽酶、血红素、N-乙基马来酰亚胺和对氯汞苯甲酸的抑制。所得结果表明，在人类红细胞中，氧化血红蛋白被一种高分子量蛋白酶切割成肽，该蛋白酶被鉴定为多催化蛋白酶家族的成员。还表明，氧化血红蛋白完全降解为游离氨基酸需要进一步的蛋白水解酶参与，而这些酶仍有待确定。
