沙棘籽乙醇脱氢酶激活肽的激活机理及稳定性

Activation Mechanism and Stability of Alcohol Dehydrogenase Activating Peptide from Sea Buckthorn Seed

摘要:

该研究通过测定不同质量浓度沙棘籽乙醇脱氢酶（Alcohol Dehydrogenase, ADH）激活肽的ADH激活率、抗氧化性、稳定ADH作用、内源荧光光谱、氨基酸组成，探索其作用机理及不同条件下的稳定性。结果表明，沙棘籽ADH激活肽的ADH激活活性呈浓度依赖性，质量浓度为10 mg/mL时ADH激活率为48.92%。沙棘籽ADH激活肽具有较强自由基清除力，1 mg/mL该肽的DPPH自由基清除率达87.17%、ABTS自由基清除率达99.76%，·OH清除率则在4 mg/mL时达87.98%，这使得ADH免受自由基攻击。沙棘籽ADH激活肽能与ADH相互作用，在5 mg/mL及10 mg/mL下使ADH结构伸展，这种结构调整更利于ADH与底物结合，这也是ADH活性及稳定性提高的原因。该肽的氨基酸构成较为均衡，亮氨酸、丙氨酸等促酒精代谢氨基酸含量较为丰富。沙棘籽ADH激活肽在热、酸碱处理、部分金属离子（Na+, Ca2+, K+）处理和胃消化中稳定，Fe2+和Fe3+对其活性有抑制作用且呈浓度依赖性，经肠道消化后仍可保持50.70%左右的ADH激活活性。综上，沙棘籽ADH激活肽具较强的ADH激活作用，能为ADH创造稳定有利环境，且具有较好稳定性。

Abstract:

In this study, the activation rate of alcohol dehydrogenase (ADH), antioxidant properties, stabilizing effects on ADH, endogenous fluorescence spectra, and amino acid composition of ADH-activating peptides from sea buckthorn seeds at various concentrations were assessed in order to investigate their mechanism of action and stability in diverse conditions. It was demonstrated by the results that the ADH activation activity of sea buckthorn seed peptides is concentration-dependent, with an activation rate of 48.92% observed at a concentration of 10 mg/mL. Strong free radical scavenging capabilities were exhibited by the peptides, with a DPPH free radical scavenging rate of 87.17% and an ABTS free radical scavenging rate of 99.76% achieved at a concentration of 1 mg/mL, while an ·OH scavenging rate of 87.98% was reached at 4 mg/mL. ADH is protected from free radical attacks by this activity. The sea buckthorn seed ADH activating peptide can be interacted with by ADH, resulting in structural extension of ADH at concentrations of 5 mg/mL and 10 mg/mL. This structural adjustment facilitates the binding of ADH to the substrate, which is also the reason for the increased activity and stability of ADH. The amino acid composition of the peptide is considered relatively balanced, with a rich content of alcohol-metabolizing amino acids such as leucine and alanine. The sea buckthorn seed ADH activating peptide is stable under heat, acidic and alkaline treatments, exposure to certain metal ions (Na?, Ca2?, K?), and gastric digestion. An inhibitory effect is exerted on its activity by Fe2+ and Fe3+ in a concentration-dependent manner, and approximately 50.70% of its ADH activating activity can still be maintained after intestinal digestion. In summary, a strong ADH activating effect is possessed by the sea buckthorn seed ADH activating peptide, a stable and favorable environment for ADH is created, and good stability is demonstrated.