Hunt J V, Bottoms M A, Mitchinson M J
Department of Pathology, University of Cambridge, U.K.
Biochem J. 1993 Apr 15;291 ( Pt 2)(Pt 2):529-35. doi: 10.1042/bj2910529.
Modification of human serum albumin (HSA) with formaldehyde resulted in a loss of 75% of available lysine residues, but there was no change in histidine content or susceptibility to free-radical-mediated fragmentation. The modified HSA appeared resistant to glycation and glucose-mediated fragmentation. Native HSA inhibited oxidant production by free glucose, as assessed by the hydroxylation of benzoic acid, but modified HSA had little effect. Thus the oxidation of free glucose appeared to be inhibited by glycatable protein, but not by unglycatable protein. Also, a close proximity of glucose to protein (decreased in the case of modified HSA) would seem to be a prerequisite for glucose-mediated protein fragmentation. This latter observation, in particular, led us to examine the role of oxidation of glucose attached to HSA in the production of reactive oxidants and subsequent molecular damage. Glycated HSA, washed free of unbound glucose, became fragmented and generated oxidants capable of hydroxylating benzoic acid and oxidizing cholesteryl linoleate-HSA complexes. Significant levels of benzoate hydroxylation and HSA fragmentation occurred with HSA (10 mg/ml) containing 3.3 mol of glucose bound/mol of HSA. This is equivalent to incubation of 10 mg/ml native HSA with 0.66 mM glucose, conditions which lead to little fragmentation or oxidant formation. The oxidative activity of glycated HSA was dependent on transition-metal concentration. The level of protein-bound glucose appeared to decrease during the oxidant production and protein fragmentation. Thus glucose can oxidize and generate reactive oxidants, whether in solution or attached to protein. We discuss which is the more likely mechanism of glucose oxidation under the near-physiological conditions used to study the effects of protein exposure to glucose in vitro.
用甲醛修饰人血清白蛋白（HSA）导致75%的可用赖氨酸残基丧失，但组氨酸含量或对自由基介导断裂的敏感性没有变化。修饰后的HSA似乎对糖基化和葡萄糖介导的断裂具有抗性。天然HSA可抑制游离葡萄糖产生氧化剂，通过苯甲酸羟基化评估，但修饰后的HSA几乎没有作用。因此，游离葡萄糖的氧化似乎受到可糖基化蛋白质的抑制，而不受不可糖基化蛋白质的抑制。此外，葡萄糖与蛋白质的紧密接近（在修饰后的HSA中减少）似乎是葡萄糖介导蛋白质断裂的先决条件。尤其是后一观察结果，促使我们研究与HSA结合的葡萄糖氧化在活性氧化剂产生及随后分子损伤中的作用。洗涤去除未结合葡萄糖的糖化HSA发生断裂，并产生能够使苯甲酸羟基化和氧化胆固醇亚油酸酯-HSA复合物的氧化剂。含有3.3摩尔葡萄糖/摩尔HSA的HSA（10毫克/毫升）出现显著水平的苯甲酸羟基化和HSA断裂。这相当于将10毫克/毫升天然HSA与0.66毫摩尔葡萄糖孵育，在此条件下几乎不发生断裂或氧化剂形成。糖化HSA的氧化活性取决于过渡金属浓度。在氧化剂产生和蛋白质断裂过程中，蛋白质结合的葡萄糖水平似乎降低。因此，葡萄糖无论是在溶液中还是与蛋白质结合，都能氧化并产生活性氧化剂。我们讨论了在用于体外研究蛋白质暴露于葡萄糖影响的近生理条件下，葡萄糖氧化更可能的机制。
