CHARACTERIZATION OF GLYCOSYLATED HEMOGLOBINS - RELEVANCE TO MONITORING OF DIABETIC CONTROL AND ANALYSIS OF OTHER PROTEINS
CHARACTERIZATION OF GLYCOSYLATED HEMOGLOBINS - RELEVANCE TO MONITORING OF DIABETIC CONTROL AND ANALYSIS OF OTHER PROTEINS
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DOI：
10.1172/jci110856
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发表时间：
1983-01-01
影响因子：
15.9
通讯作者：
BUNN, HF
中科院分区：
文献类型：
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作者：
GARLICK, RL;MAZER, JS;BUNN, HF
使用硼酸盐亲和层析和离子交换层析来测量[人]正常和糖尿病溶血物中糖基化Hb的水平，以及葡萄糖加合物在α-NH2-缬氨酸和ε-NH2-赖氨酸残基上的分布。当通过BioRex 70树脂上的离子交换色谱分析时，Hb A1c峰包含4.4±。 15 种正常溶血产物的 0.6% 和 9.1 .+-. 15 种糖尿病溶血物中的 2.1%。 Hb A1c在GlycoGel B硼酸酯亲和树脂上重新色谱分析，该树脂结合共价连接的糖的邻位羟基。只有 70 .+-。 5% 的 Hb 粘附在树脂上。硫代巴比妥酸比色试验分析证实，亲和树脂可以有效分离糖基化和非糖基化的 Hb。当针对非糖基化污染物进行校正时，正常溶血产物中 Hb A1c 的平均水平为 2.9.+-。 0.4%，远低于之前报道的数值。除 Hb AIc 外，还有 5.2 .+-。剩余 Hb (Hb Ao) 的 0.5% 被糖基化。在糖尿病患者中，糖基化 Ao 与 Hb Alc 平行增加。用[3H]硼氢化物还原并酸水解后，首先在 Affi-Gel 硼酸亲和树脂上纯化糖基化氨基酸，然后通过离子交换色谱法进行分析。 Hb Ao 上的葡萄糖加合物分布如下： α-链 N-末端缬氨酸，14%； α-链赖氨酸，40%，β-链赖氨酸，46%。揭示了非酶糖基化蛋白质分析中的几个缺陷。通过离子交换色谱或电泳分离的峰可能被非糖基化蛋白质污染。硫代巴比妥酸测试和[3H]硼氢化物还原都显示出不同的反应性，具体取决于酮胺连接的葡萄糖的位点。
Boronate affinity chromatography and ion exchange chromatography were used to measure the levels of glycosylated Hb in [human] normal and diabetic hemolysates, as well as the distribution of glucose adducts on .alpha.-NH2-valine and .epsilon.-NH2-lysine residues. When analyzed by ion exchange chromatography on BioRex 70 resin, the Hb AIc peak comprised 4.4 .+-. 0.6% of 15 normal hemolysates and 9.1 .+-. 2.1% of 15 diabetic hemolysates. The Hb AIc was rechromatographed on GlycoGel B boronate affinity resin that binds vicinal hydroxyl groups of covalently linked sugars. Only 70 .+-. 5% of the Hb adhered to the resin. Analysis by the thiobarbituric acid colorimetric test confirmed that the affinity resin effectively separated glycosylated from nonglycosylated Hb. When corrected for nonglycosylated contaminants, the mean level of Hb AIc in normal hemolysates was 2.9 .+-. 0.4%, a value considerably lower than those previously reported. In addition to Hb AIc, 5.2 .+-. 0.5% of the remaining Hb (Hb Ao) was glycosylated. In diabetics, glycosylated Ao was increased in parallel with Hb AIc. After reduction with [3H]borohydride and acid hydrolysis, glycosylated amino acids were first purified on Affi-Gel boronate affinity resin and then analyzed by ion exchange chromatography. The glucose adducts on Hb Ao were distributed as follows: .alpha.-chain N-terminal valine, 14%; .alpha.-chain lysines, 40%, .beta.-chain lysines, 46%. Several pitfalls in the analysis of nonenzymatically glycosylated proteins were revealed. Peaks isolated by ion exchange chromatography or electrophoresis are likely to be contaminated by nonglycosylated proteins. Both the thiobarbituric acid test and [3H]borohydride reduction show variable reactivity depending upon the site of the ketoamine-linked glucose.