胶原酶的检测与巯基肽抑制其作用的研究  

A Spectrophotometric Collagenase Assay and Inhibition of Collagenase by a Thiol Peptide

作　　者：沈远平[1] 张泺[1] 李平[1] 

机构地区：[1]同济医科大学附属同济医院眼科,武汉430030

出　　处：《华中科技大学学报（医学版）》1997年第S1期63-65,共3页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

摘　　要：介绍一种考马斯亮蓝R250染色后微量光谱检测胶原酶含量方法。免角膜碱烧伤前后其泪液中胶原酶含量测定,并用抑制胶原酶药物巯基肽做体外实验研究。原理:①将定量的胶原铺在酶标用的聚苯乙烯塑料孔中,加入胶原酶则胶原会被溶解,且与胶原酶量成正比。②巯基肽抑制胶原酶主要利用螫合钙或形成不溶性钙化物,以减低Ca^(2+)浓度,另利用巯基(-SH)与酶的Zn^(2+)结合减低酶活性,在实验中测出IC50为4×10^(-6)g,此量在眼局部应用安全有效。A quantitative collagenase assay using comassie blue R250 staining and microtiter spectropho- tometry was described. Collagen was gelled and dried the bottom of microwells as substrated, incubated with samples, and stained. The residual collagen was then quantified using a spectrophotometer. The assay was safe, simple, fast, economical and sensitive. We used the method to detect collagenase in the tear of rabbits before and after alkali burn to their corneas. A thiol peptide was tested to inhibit collagenase in vitro. The mechanism by which the peptide inhibited collagenase was by the chelation of calcium, lowering the Ca^(2+) con- centration, and by the -SH binding active site Zn^(2+) in collagenase, reducing its activity. The IC50 of the thiol peptide by this assay was 4×10^(-6)g. The result of this study suggests that the thiol peptide might be a good candidate as a collagenase inhibitor for the treatment of the alkali-burned eyes.

关 键 词：胶原酶 巯基肽 角膜碱烧伤 泪液 

分 类 号：R779.1[医药卫生—眼科]