Garlick R L, Mazer J S
J Biol Chem. 1983 May 25;258(10):6142-6.
We have determined the major site of nonenzymatic glycosylation of human serum albumin in vivo. This was accomplished by reacting freshly purified human serum albumin with sodium [3H]borohydride followed by aminoethylation and tryptic digestion. The tryptic peptides were separated into a soluble fraction which contained 88% of the total 3H radioactivity and an insoluble fraction. In order to isolate the 3H-labeled glycosylated peptides, the soluble tryptic peptide fraction was first subjected to boronic acid affinity chromatography. Cation exchange chromatography then separated the soluble glycosylated peptides into a major peak which contained 48% of the total recovered 3H radioactivity and a number of minor peptide fractions. The amino acid composition of the major peptide was: Thr, Glu2, Ala, Val2, Leu2, Lys, lysino-1-deoxysorbitol. In accord with the primary structure of human serum albumin, this amino acid composition corresponds precisely to residues 525-534. Glucitol-lysine, the NH2-terminal residue of this peptide, is totally resistant to cleavage by trypsin. Thus, lysine-525 is the predominant site of nonenzymatic glycosylation of human serum albumin in vivo. Chromatography on GlycoGel B boronic acid affinity gel indicates that 10-12% of normal serum albumin is glycosylated. The rate of nonenzymatic glycosylation of this protein in vivo is approximately 9 times that of human hemoglobin.
我们已经确定了人血清白蛋白在体内非酶糖基化的主要位点。这是通过将新鲜纯化的人血清白蛋白与[3H]硼氢化钠反应，然后进行氨乙基化和胰蛋白酶消化来实现的。胰蛋白酶肽段被分离成一个可溶性部分（其中包含总3H放射性的88%）和一个不溶性部分。为了分离出3H标记的糖基化肽段，首先将可溶性胰蛋白酶肽段进行硼酸亲和层析。阳离子交换层析随后将可溶性糖基化肽段分离成一个主峰（其中包含回收的总3H放射性的48%）和一些次要的肽段部分。主要肽段的氨基酸组成是：苏氨酸、2个谷氨酸、丙氨酸、2个缬氨酸、2个亮氨酸、赖氨酸、赖氨酸-1-脱氧山梨醇。根据人血清白蛋白的一级结构，这种氨基酸组成恰好对应于525-534位残基。该肽段的NH2末端残基葡糖醇-赖氨酸完全抗胰蛋白酶切割。因此，赖氨酸-525是人血清白蛋白在体内非酶糖基化的主要位点。在GlycoGel B硼酸亲和凝胶上进行层析表明，正常血清白蛋白中有10-12%被糖基化。这种蛋白质在体内的非酶糖基化速率约为人血红蛋白的9倍。
