Cairns J R, Williamson M K, Price P A
Department of Biology, University of California, San Diego, La Jolla 92093.
Anal Biochem. 1991 Nov 15;199(1):93-7. doi: 10.1016/0003-2697(91)90274-w.
We report the first direct method for the identification of the vitamin K-dependent Ca2+ binding amino acid, gamma-carboxyglutamic acid (Gla), in the sequencing of proteins. The carboxyl groups on the protein are first converted to methyl esters with methanolic HCl, a procedure that reduces the polarity of the resulting ATZ derivative of dimethyl-Gla and so greatly improves its extraction from the polybrene-treated glass fiber filter. After conversion to the PTH derivative in methanolic HCl, the resulting dimethyl ester of PTH Gla can be identified directly by a simple modification of the standard HPLC program for the separation of PTH derivatives. This methylation procedure can be used to identify Gla residues in proteins bound to PVDF membranes, as we demonstrate for matrix Gla protein and prothrombin, and to evaluate directly the degree of partial gamma-carboxylation at given glutamic acid residues, as we demonstrate for the 50% gamma-carboxylation of residue 17 in human bone Gla protein.
我们报道了一种在蛋白质测序中鉴定维生素K依赖的Ca2+结合氨基酸——γ-羧基谷氨酸（Gla）的直接方法。首先用甲醇盐酸将蛋白质上的羧基转化为甲酯，这一过程降低了所得二甲基-Gla的ATZ衍生物的极性，从而极大地改善了其从经聚凝胺处理的玻璃纤维滤膜上的提取。在甲醇盐酸中转化为PTH衍生物后，所得PTH Gla的二甲基酯可通过对分离PTH衍生物的标准HPLC程序进行简单修改而直接鉴定。正如我们在基质Gla蛋白和凝血酶原中所证明的，这种甲基化程序可用于鉴定与PVDF膜结合的蛋白质中的Gla残基，并且正如我们在人骨Gla蛋白第17位残基50%的γ-羧化中所证明的，可直接评估给定谷氨酸残基处的部分γ-羧化程度。
