精氨酸对牛胰核糖核酸酶氧化复性的时相性抑制作用

Phase-specific Inhibition of Arginine on the Oxidative Regeneration of Bovine Pancreatic Ribonuclease A

精氨酸常在重组蛋白的体外复性中,作为一种小分子添加剂用于抑制蛋白聚集。精氨酸对蛋白复性折叠过程本身的影响尚不清楚。首次以不易聚集的牛胰核糖核酸酶为研究对象,通过飞行质谱检测氧化复性中间体的变化,通过酶学活性检测蛋白活性的恢复过程,观察了精氨酸对氧化复性的直接影响。发现不同浓度精氨酸对牛胰核糖核酸酶的氧化复性有直接的抑制作用。特别在0.5 mol/L浓度时,精氨酸对牛胰核糖核酸酶氧化复性的抑制作用具有独特的时相依赖性:早期复性可持续至4 h,其活性恢复达30%后,晚期复性基本终止。这种抑制特征与脲的抑制作用有明显的不同,精氨酸没有完全抑制关键结构中间体的形成。结果说明,牛胰核糖核酸酶的氧化复性,除经关键结构中间体的主要通路外,还可能存在一个潜在复性通路,它是其氧化复性后期的关键限速通路。该复性通路可以被0.5 mol/L精氨酸完全阻断。

Arginine is often used as a small molecule additive to suppress the aggregation of recombinant proteins during regeneration in vitro. It is not clear whether arginine has a direct impact on the refolding process. Here the effect of arginine on the oxidative regeneration of bovine pancreatic ribonuclease A which is free from aggregation, was studied for the first time, by detecting the folding intermediates through Mass Spectrometry and by monitoring its activity recovery through enzymatic assay during the refolding. Arginine at different concentrations inhibited the oxidative regeneration of bovine pancreatic ribonuclease A. At a concentration of 0.5 mol/L, arginine inhibited the refolding in a phase-specific manner that ribonuclease A continued to refold during the initial 4 h stage when the activity reached to about 30%, while it ceased to refold further at the later stage. It was different from the effect of urea which showed no phase-specific in- hibition. Arginine, unlike urea, did not abolish the formation of key structural intermediates. Thus, a poten- tial unknown folding pathway, other than the classic pathway with key structural intermediates, was proposed for the regeneration of bovine pancreatic ribonuclease A, which was a rate-limiting pathway at the late stage of its refolding. This unknown pathway was blocked completely by arginine at 0.5 mol/L.