高效疏水色谱分离不同龋敏感者唾液α-淀粉酶  

Separation of Human Salivary α-amylase from Caries-free and Caries-active by High Performance Hydrophobic Interaction Chro-matography

作　　者：景娟[1] 侯铁舟[1] 赵东方[1] 王英[2] 阮梅生[1] 

机构地区：[1]西安交通大学口腔医学院口腔内科,西安710004 [2]第四军医大学口腔医学院口腔内科,西安710032

出　　处：《口腔医学》2004年第5期264-266,共3页Stomatology

基　　金：中华医学基金会科研经费资助项目(1992)

摘　　要：目的 采用疏水色谱技术分离提纯高龋者和无龋者腮腺液α-淀粉酶的方法,研究人唾液α-淀粉酶与龋病的关系。方法 样品为刺激性唾液,冻干样品配成20 g/L的浓度。以2.0 mol/L(NH4)2SO4和0.02 mol/L KH2PO4溶液分别为A、B流动相,过XDF-SGM型疏水柱,25 min梯度,A:100％-0％、B:0％-100％,于230 nm检测,收集所得6个组分峰,对硝基麦芽糖庚酰(PNPG7)限定底物法测定酶活性,SDS-PAGE电泳测定其分子质量对样品进行定性。结果 经酶活性检测,6号峰被证实为有高的淀粉酶活性,α-淀粉酶的平均出峰时间标准差小,个体间重复性好;高龋和无龋者峰面积百分比差异有显著性(P<0.05)。结论 α-淀粉酶与龋病有一定的相关性。Objective To separate and purify parotid salivary a-amylase from caries-free (CF) and caries-active (CA) by High Performance Hydrophobic Interaction Chromatography (HPHIC) to study the relationship between α-amylase and caries. Methods After acitric acid stimulation and lyophilized parotid were collected, salivary samples were prepared at the concentration of 20 mg/ml for further use. The separation was done by an XDF - SGM hydrophobic interaction column as analytical column and 2.0mol/L (NH4)SO4 as A mobile phase, 0.02 mol/L KH2PO4 as B mobile phase. Gradient separation was at 25 min when A mobile phase was 100%-0% and B mobile phase was 0%-100%. The detective wave length was 230 nm. Each peak collected was detected for amylase activated by the method of limited substrate to PNPG7, and qualitative analysis by SDS - PAGE for their molecular quantity. Results The peak 6 was verified to have highly active amylase and minimal standard error and good duplication of average retention time. There was considerable difference between the peak areas of a-amylase from CF and CA subjects (P<0.05) . Conclusion There was relationship between salivary a-amylase and caries.

关 键 词：龋敏感 高效疏水色谱 分离纯化 腮腺液 Α-淀粉酶 

分 类 号：R394.26[医药卫生—医学遗传学]