Watkins N G, Thorpe S R, Baynes J W
J Biol Chem. 1985 Sep 5;260(19):10629-36.
Ribonuclease A has been used as a model protein for studying the specificity of glycation of amino groups in protein under physiological conditions (phosphate buffer, pH 7.4, 37 degrees C). Incubation of RNase with glucose led to an enhanced rate of inactivation of the enzyme relative to the rate of modification of lysine residues, suggesting preferential modification of active site lysine residues. Sites of glycation of RNase were identified by amino acid analysis of tryptic peptides isolated by reverse-phase high pressure liquid chromatography and phenylboronate affinity chromatography. Schiff base adducts were trapped with Na-BH3CN and the alpha-amino group of Lys-1 was identified as the primary site (80-90%) of initial Schiff base formation on RNase. In contrast, Lys-41 and Lys-7 in the active site accounted for about 38 and 29%, respectively, of ketoamine adducts formed via the Amadori rearrangement. Other sites reactive in ketoamine formation included N alpha-Lys-1 (15%), N epsilon-Lys-1 (9%), and Lys-37 (9%) which are adjacent to acidic amino acids. The remaining six lysine residues in RNase, which are located on the surface of the protein, were relatively inactive in forming either the Schiff base or Amadori adduct. Both the equilibrium Schiff base concentration and the rate of the Amadori rearrangement at each site were found to be important in determining the specificity of glycation of RNase.
核糖核酸酶A已被用作模型蛋白，用于研究生理条件下（磷酸盐缓冲液，pH 7.4，37摄氏度）蛋白质中氨基糖基化的特异性。相对于赖氨酸残基的修饰速率，将核糖核酸酶与葡萄糖一起孵育会导致该酶的失活速率加快，这表明活性位点赖氨酸残基优先被修饰。通过对反相高压液相色谱和苯基硼酸亲和色谱分离得到的胰蛋白酶肽段进行氨基酸分析，确定了核糖核酸酶的糖基化位点。用Na-BH3CN捕获席夫碱加合物，并将Lys-1的α-氨基鉴定为核糖核酸酶上初始席夫碱形成的主要位点（80-90%）。相比之下，活性位点中的Lys-41和Lys-7分别占通过阿马多里重排形成的酮胺加合物的约38%和29%。参与酮胺形成的其他位点包括与酸性氨基酸相邻的Nα-Lys-1（15%）、Nε-Lys-1（9%）和Lys-37（9%）。核糖核酸酶中位于蛋白质表面的其余六个赖氨酸残基在形成席夫碱或阿马多里加合物方面相对不活跃。发现每个位点的平衡席夫碱浓度和阿马多里重排速率对于确定核糖核酸酶糖基化的特异性都很重要。
