小麦胆色素原脱氨酶的联合酶活法测定  

The Porphobilinogen Deaminase Assay Used δ-Aminolevulinate as Substrate by Coupled Enzyme Assay

作　　者：范军[1,2] 郭蔼光[1,2] 

机构地区：[1]安徽农业大学生物技术研究中心 [2]西北农业大学基础部植物生化室

出　　处：《安徽农业大学学报》1998年第1期76-80,共5页Journal of Anhui Agricultural University

基　　金：国家自然科学基金

摘　　要：本实验以小麦匀浆液中的δ－氨基酮戊酸脱水酶（ＡＬＡＤ）催化δ－氨基酮戊酸（ＡＬＡ）生成胆色素原（ＰＢＧ），并以ＰＢＧ作为底物测定胆色素原脱氨酶（ＰＢＧＤ）的酶活，结果表明：将ＡＬＡ缓冲液与粗ＡＬＡＤ酶液１∶２（ｖ／ｖ）混合，２５℃，黑暗反应４ｈ，可获得最大量的ＰＢＧ，加入部分纯化的ＰＢＧＤ，３７℃反应１ｈ后，测得底物ＰＢＧ减少，产物原尿卟啉原Ⅰ增加，该联合酶活测定法以ＰＢＧ减少来直接确定加热处理后的凝胶过滤和亲合层析纯化的ＰＢＧＤ酶活高峰管，具有经济、快速、方便等优点，可用于纯化小麦叶绿素突变体返白系中胆色素原脱氨酶和尿卟啉原Ⅲ合成酶。Porphobilinogen (PBG) was produced by δ-Aminolevulinate dehydratase (ALAD) from wheat leaf crude which catalyzed δ-Aminolevulinate (ALA) in vitro.Substrate was used to assay porphobilinogen deaminase (PBGD).The results showed the maximum production of PBG was obtained with ALA buffer and ALAD crude at the ratio of 1∶2 (v/v),after 4 h incubation in temperature of 25℃.1 hour incubation in temperature of 37℃ with addition of purified PBGD led to decrease of PBG and production of uroporphyrins which were measured spectrophotometrically at 553 nm and 405 5 nm,respectively.By decreasing PBG,this coupled enzyme assay can determine the peak frations in chromotography economically,quickly and easily.It can be used in purifing the PBGD and Uros from albino mutant of wheat.

关 键 词：Δ-氨基酮戊酸 小麦 胆色素原脱氨酶 酶活 

分 类 号：S512.101[农业科学—作物学]