Pankov Iu A, Karasev V S
Biokhimiia. 1984 Jan;49(1):111-26.
The luteinizing hormone isolated from sperm-whale pituitary was separated into two subunits, alpha- and beta-, by ion-exchange chromatography on sulfoethyl-Sephadex. The hormone subunits were reconstituted, carboxymethylated and cleaved by BrCN and proteolytic enzymes. In order to block tryptic hydrolysis at lysine residues the alpha-subunit was subjected to maleylation. Large-sized fragments of BrCN were cleaved by chymotrypsin and trypsin, while large-sized fragments of trypsin were split by chymotrypsin. The resulting peptides were separated by gel filtration on Sephadex, ion-exchange chromatography on Aminex A-5 and thin-layer partition chromatography on cellulose. The amino acid sequence of the peptides was determined by the Edman method, using identification of the N-terminal amino acids in a reaction with dansyl chloride or dimethylaminoazobenzene-4-isothiocyanate. It was shown that the alpha-subunit of the luteinizing hormone is a peptide chain consisting of 96 amino acid residues with covalently linked carbon chains at asparagine residues at positions 56 and 82. The N-terminal amino acid of the alpha-subunit is phenylalanine, the C-terminal amino acid is serine. The alpha-subunit is heterogeneous at the N-end, i. e. beside phenylalanine it contains threonine and trace amounts of proline, aspartate, glutamate and glycine.
从抹香鲸脑垂体中分离出的促黄体生成素，通过磺乙基-葡聚糖凝胶离子交换色谱法被分离成α和β两个亚基。对激素亚基进行重组、羧甲基化，并通过溴化氰和蛋白水解酶进行切割。为了阻断赖氨酸残基处的胰蛋白酶水解，对α亚基进行了马来酰化处理。溴化氰产生的大片段被胰凝乳蛋白酶和胰蛋白酶切割，而胰蛋白酶产生的大片段则被胰凝乳蛋白酶进一步裂解。通过葡聚糖凝胶过滤、Aminex A-5离子交换色谱以及纤维素薄层层析对所得肽段进行分离。采用丹磺酰氯或二甲基氨基偶氮苯-4-异硫氰酸酯反应鉴定N端氨基酸的方法，通过埃德曼法测定肽段的氨基酸序列。结果表明，促黄体生成素的α亚基是一条由96个氨基酸残基组成的肽链，在第56位和82位天冬酰胺残基处有共价连接的碳链。α亚基的N端氨基酸是苯丙氨酸，C端氨基酸是丝氨酸。α亚基在N端具有异质性，即除了苯丙氨酸外，还含有苏氨酸以及微量的脯氨酸、天冬氨酸、谷氨酸和甘氨酸。
