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从13天龄鸡胚胎提取原胶原脯氨酰羟化酶,在FeSO_4、2-酮戊二酸、抗坏血酸、过氧化氢酶等辅助因子存在下,将底物(Pro-Pro-Gly)10·9H_2O中的Pro羟化成羟脯氨酰(HOPro)的羟化反应体系,观察IH764-3对胶原生物合成中脯氨酰羟化作用的影响。结果表明,脯氨酰羟化酶催化的原胶原脯氨酰底物(Pro-Pro-Gly)10·9H_2O的羟基化作用可被IH764-3所抑制。在酶促反应标准条件下,IH764-3为0.24mmol/L时,对脯氨酰羟化的抑制率为50.78±16.03％。本实验还证明,IH764-3不能与脯氨酰羟化酶结合,提示IH764-3对酶促脯氨酰羟化的抑制是由于其同酶反应所需的Fe~(2+)形成络合物所致。平衡移动法表明,IH764-3与Fe~(2+)形成的稳定络合物的Fe(2+):IH764-3摩尔比为1:3,表现稳定常数Ks=1.04×10~(12)。
Prolactyl hydroxylase was extracted from a 13-day-old chicken embryo and the substrate (Pro-Pro-Gly) 10 · (2-hydroxyproline) was hydrolyzed in the presence of cofactors such as FeSO 4, 2- ketoglutarate, ascorbate and catalase Hydroxyproline (HOPro) hydroxylation reaction of Pro hydroxylation in 9H 2 O was carried out to observe the effect of IH764-3 on prolyl hydroxylation in collagen biosynthesis. The results showed that prolyl hydroxylase catalyzed hydroxylation of Pro-Pro-Gly 10 · 9H 2 O could be inhibited by IH764-3. Under the standard conditions of enzymatic reaction, the inhibition rate of prolyl hydroxylation was 50.78 ± 16.03% when IH764-3 was 0.24mmol / L. This experiment also demonstrated that IH764-3 can not bind to prolyl hydroxylase, suggesting that the inhibition of enzymatic prolyl hydroxylation by IH764-3 is due to the complexation of Fe2 + required for its enzymatic reaction Cause caused. The equilibrium shift method showed that the molar ratio of Fe (2 +): IH764-3 to stable complex formed by IH764-3 and Fe ~ (2+) was 1: 3 and the stability constant Ks was 1.04 × 10 ~ (12) .