Lello Z, Luca G, Maurizio B
Department of Biochemical Science, University La Sapienza, Rome, Italy.
Biochim Biophys Acta. 1990 Feb 9;1037(2):165-9. doi: 10.1016/0167-4838(90)90163-a.
In an attempt to isolate structural domains of arthropod hemocyanins and possibly to investigate their functional properties, we have undertaken proteolytic digestion experiments of isolated subunits from Panulirus interruptus and Limulus polyphemus oxy-hemocyanin. Satisfactory results have been obtained using trypsin at high concentration and short digestion times. Results show that, in the case of Panulirus hemocyanin, only subunit alpha is susceptible to trypsin digestion, but that proteolytic cleavage is associated with the loss of the copper-oxygen band; on the other hand, in the case of Limulus hemocyanin, four subunits (I, II, III and IV) show a significant susceptibility to trypsin, and their fragmentation takes place with preservation of the oxygen-binding capacity. A more detailed study of the digestion products of subunit IV from Limulus hemocyanin reveals that the proteolytic fragments keep together in a single non-covalent complex. Attempts to separate the native fragments result in the precipitation of the digestion products. Subunit IV of Limulus with proteolytic cuts binds O2 and CO with the same affinity as the native subunit, suggesting that the copper site is still preserved structurally and is functionally active in a 37 kDa trypsin-resistant domain.
为了分离节肢动物血蓝蛋白的结构域，并可能研究其功能特性，我们对来自断沟龙虾和美洲鲎氧合血蓝蛋白的分离亚基进行了蛋白水解消化实验。使用高浓度胰蛋白酶并缩短消化时间获得了满意的结果。结果表明，就断沟龙虾血蓝蛋白而言，只有α亚基易受胰蛋白酶消化，但蛋白水解切割与铜 - 氧带的丧失有关；另一方面，就美洲鲎血蓝蛋白而言，四个亚基（I、II、III和IV）对胰蛋白酶表现出显著的敏感性，并且它们的片段化在保持氧结合能力的情况下发生。对美洲鲎血蓝蛋白IV亚基消化产物的更详细研究表明，蛋白水解片段以单一非共价复合物的形式聚集在一起。分离天然片段的尝试导致消化产物沉淀。具有蛋白水解切割的美洲鲎IV亚基与天然亚基以相同的亲和力结合O2和CO，这表明铜位点在结构上仍然保留，并且在一个37 kDa的抗胰蛋白酶结构域中具有功能活性。
