来自美洲野牛的核糖核酸酶A的分离及部分特性鉴定
The isolation and partial characterization of ribonuclease A from Bison bison.
作者信息
Stewart G R, Stevenson K J
出版信息
Biochem J. 1973 Nov;135(3):427-41. doi: 10.1042/bj1350427.
Abstract
- Bison ribonuclease was isolated from pancreas glands of Bison bison by acid extraction, (NH(4))(2)SO(4) fractionation, affinity chromatography on Sepharose-5'-(4-aminophenylphosphoryl)uridine 2',3'-phosphate and ion-exchange chromatography on Bio-Rex-70. 2. The selectivity of the affinity column towards bison ribonuclease in heterogeneous protein solutions was greatly improved by employing piperazine buffers at pH5.3, which decreased non-specific interactions of other proteins. Rapid desorption from the affinity column was obtained with sodium phosphate buffer (pH3). 3. Bison ribonuclease has a total amino acid content very similar to ox ribonuclease. Inactivation of bison ribonuclease with iodoacetic acid leads to the formation of 0.62 residues of pi-carboxymethylhistidine and 0.36 residues of tau-carboxymethylhistidine. The amino acid composition of peptides isolated from diagonal peptide ;maps' and also of peptides isolated after pH1.6 and 2.4 two-dimensional high-voltage electrophoresis of a digest of bison ribonuclease labelled with pyridoxal 5-phosphate indicates that there is complete homology between ox and bison ribonucleases. 4. The Schiff-base attachment site of pyridoxal 5-phosphate was identified as lysine-41 by NaBH(4) reduction followed by peptide isolation.
摘要
- 美洲野牛核糖核酸酶是从美洲野牛的胰腺中通过酸提取、硫酸铵分级分离、在琼脂糖-5'-(4-氨基苯磷酰基)尿苷2',3'-磷酸上进行亲和层析以及在Bio-Rex-70上进行离子交换层析分离得到的。2. 通过使用pH5.3的哌嗪缓冲液，可大大提高亲和柱在异质蛋白质溶液中对美洲野牛核糖核酸酶的选择性，这减少了其他蛋白质的非特异性相互作用。用磷酸钠缓冲液（pH3）可实现从亲和柱上的快速解吸。3. 美洲野牛核糖核酸酶的总氨基酸含量与牛核糖核酸酶非常相似。用碘乙酸使美洲野牛核糖核酸酶失活会导致形成0.62个β-羧甲基组氨酸残基和0.36个τ-羧甲基组氨酸残基。从对角线肽“图谱”中分离出的肽段以及用磷酸吡哆醛标记的美洲野牛核糖核酸酶消化物在pH1.6和2.4二维高压电泳后分离出的肽段的氨基酸组成表明，牛和美洲野牛核糖核酸酶之间存在完全同源性。4. 通过硼氢化钠还原后分离肽段，确定磷酸吡哆醛的席夫碱连接位点为赖氨酸-41。
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