Hajós G, Delincée H
Int J Radiat Biol Relat Stud Phys Chem Med. 1983 Oct;44(4):333-42. doi: 10.1080/09553008314551271.
Following irradiation of bovine pancreatic ribonuclease in aqueous solution with 60Co gamma-rays protein aggregates are formed. The nature of the bonds linking these radiation-induced aggregates together has been investigated by chromatographic and electrophoretic methods. Thin-layer gel filtration and polyacrylamide gel electrophoresis, both in the presence of sodium dodecyl sulphate, demonstrated the existence of covalent crosslinks between the aggregates. However, non-covalent crosslinking also plays a role in the radiolysis of ribonuclease. Thin-layer gel filtration with and without 6 M urea and 2 per cent beta-mercaptoethanol added to the gel, revealed that only part of the covalent bonds between the aggregates consisted of disulphide linkages. By separation of the reduced aggregates by thin-layer gel filtration and electrophoresis, both with SDS, this finding was substantiated. Densitometric measurements indicated for example that the percentage of covalently linked dimers held together by disulphide bridges amounted to about 40-45 per cent, whereas the remaining 55-60 per cent of the dimers must be linked by other covalent bonds. The existence of covalent crosslinks other than disulphide bonds was also confirmed by isoelectric focusing. By this method definite differences were established between the proteolytic hydrolysates of the reduced aggregates and the reduced monomer of gamma-irradiated ribonuclease.
用60Coγ射线照射水溶液中的牛胰核糖核酸酶后，会形成蛋白质聚集体。已通过色谱和电泳方法研究了将这些辐射诱导的聚集体连接在一起的键的性质。在十二烷基硫酸钠存在下的薄层凝胶过滤和聚丙烯酰胺凝胶电泳表明聚集体之间存在共价交联。然而，非共价交联在核糖核酸酶的辐射分解中也起作用。在凝胶中添加和不添加6M尿素及2%β-巯基乙醇的薄层凝胶过滤显示，聚集体之间只有部分共价键是二硫键。通过用SDS进行薄层凝胶过滤和电泳分离还原后的聚集体，这一发现得到了证实。例如，光密度测量表明，由二硫桥连接在一起的共价连接二聚体的百分比约为40-45%，而其余55-60%的二聚体必须由其他共价键连接在一起。除二硫键以外的共价交联的存在也通过等电聚焦得到了证实。通过这种方法，确定了还原后的聚集体的蛋白水解产物与γ射线照射的核糖核酸酶的还原单体之间存在明显差异。
