磷酸化处理对蚕蛹多肽结合钙能力的影响

Effect of Phosphorylation on Calcium-Binding Ability of Silkworm Pupa Polypeptide

为探索磷酸化处理对多肽结合钙能力的促进作用,以蚕蛹多肽为研究对象,采用不同种类和添加量的磷酸化试剂对其进行磷酸化改性,再与CaCl_(2)反应制备蚕蛹多肽结合钙复合物,并对样品进行钙和磷含量测定、磷酸化位点检测、红外光谱分析和扫描电镜分析。研究结果表明:Na_(3)PO_(4)、NaH_(2)PO_(4)、(NaPO_(3))_(6)3种试剂可与蚕蛹多肽发生磷酸化反应,改性后蚕蛹多肽结合钙含量可从145.12 mg/g提高至202.32 mg/g;不同种类磷酸化试剂处理对多肽的结合钙能力无显著性差异,但磷酸化程度对肽结合钙能力有较大影响;多肽结合钙复合物中钙含量随磷酸化试剂的增加先升高后下降,当采用质量分数为0.50%的NaH_(2)PO_(4)时,改性后蚕蛹多肽结合钙含量达到最大值。红外光谱和磷酸化位点检测表明:NaH_(2)PO_(4)处理可使蚕蛹酶解产物中的17种肽段被磷酸化,增加磷酸化位点22个;肽段主要通过与丝氨酸和苏氨酸上的—OH反应接入磷酸基团。研究表明,磷酸化改性方法可显著提高多肽结合钙能力,希望为高效的补钙制品开发提供新的思路和方法。

To study the improvement of phosphorylation on calcium-binding ability of polypeptide,silkworm pupa polypeptide was phosphorylated by different kinds of phosphorylation reagents with diverse additive volume,then reacted with CaCl_(2)to prepare calcium-peptide complex.The content of calcium and phosphorus,phosphorylation site,FTIR and SEM of samples were analyzed.The results showed that phosphorylation occurred between polypeptide and Na_(3)PO_(4),NaH_(2)PO_(4),and(NaPO_(3))_(6),which led to the calcium content of the calcium-peptide complex enhanced from 145.12 mg/g to 202.32 mg/g.The calcium-binding ability was irrelated to the kinds of phosphorylation reagents,but related to the level of phosphorylation.The calcium content first increased then decreased with the increment of phosphorylation reagents.When the mass fraction of NaH_(2)PO_(4)was 0.50%,the binding calcium content reached maximum.Twenty-two increased phosphorylation sites were identified in 17 peptides after phosphorylation treatment by NaH_(2)PO_(4).FTIR and phosphorylation site detection confirmed that phosphate groups were successfully grafted onto the peptide backbone through hydroxyl groups on serine and threonine.Phosphorylation could improve the calcium-binding ability of polypeptide,and provide new ideas and methods for development of effective calcium supplements.