摘要
目的优化碱水解-光度法测定胶原中羟脯氨酸含量的水解条件,建立一种简化且准确的羟脯氨酸含量测定方法。方法设计两因素五水平的正交试验,优化了胶原水解时碱浓度及水解时间对水解的影响。并对比碱水解-光度法与酸水解-光度法以及氨基酸分析仪法的检测结果,验证碱水解-光度法的稳定性和准确性。结果分光光度法检测羟脯氨酸含量为0 mg/ml^0.15 mg/ml时线性良好,线性回归方程为y=0.008x+0.014 2,相关系数r=0.999。碱水解的适宜条件:0.1 k Pa、121℃,2 mol/L Na OH,水解时间为20 min。在优化后的操作条件下,该法的平均回收率为101.3%,且与酸水解-光度法以及氨基酸分析仪法的检测结果差异无统计学意义。结论在高温碱条件下水解,无需充氮,无需中和或去除盐酸,实验操作简便,缩短用时,水解速度大幅提高,且灵敏度高、重现性好,提升了检测效率,可快速测定羟脯氨酸含量,应用于动物蛋白的检测。
Objective To optimize the hydrolysis conditions for alkaline hydrolysis- spectrophotometric determination of hydroxyproline in collagen,and establish a simplified and accurate method for the determination of hydroxyproline in collagen.Methods Two factors and five levels of orthogonal test was designed for optimizing the collagen hydrolyzed with alkali concentration and hydrolysis time on the influence of the hydrolysis. The detection results byalkaline hydrolysis- spectrophotometry,acid hydrolysis- spectrophotometry and amino acid analyzer were contrasted to evaluate the stability and accuracy of alkaline hydrolysis- spectrophotometry. Results The linear of spectrophotometry within the range 0 mg / ml- 0. 15 mg / ml was good. The linear regression equation was y = 0. 008 x ＋ 0. 014 2,with correlation coefficient r = 0. 999. The optimal condition of the method based on alkaline hydrolysis： 0. 1 kPa,121 ℃,2 mol / L NaOH and 20 min for hydrolysis. After the optimization of operating conditions,the average recovery was 101. 3%. This method had no significant differences in the test results with acid hydrolysis-spectrophotometry and amino acid analyzer. Conclusion This assay method under the condition of high temperature and alkali hydrolysis need no nitrogen,neutralization or removing hydrochloric acid. This method simplifies experimental procedure,reduces hydrolysis time,increases hydrolysis speed greatly,improves detection efficiency,which is highly sensitive and good reproducible,and can be applied to measure the hydroxyproline in collagen rapidly.
出处
《中国卫生检验杂志》
CAS
2015年第6期783-785,788,共4页
Chinese Journal of Health Laboratory Technology
基金
广东省海洋经济创新发展区域示范专项项目(GD2012-B03-002)
广东省科技计划项目(2012B070800007)
关键词
胶原
碱水解
羟脯氨酸
Collagen
Alkaline hydrolysis
Hydroxyproline
