Krungkrai J, Yuthavong Y, Webster H K
J Chromatogr. 1987 Jun 5;417(1):47-56. doi: 10.1016/0378-4347(87)80090-7.
A highly sensitive assay for pteroylpolyglutamate hydrolase is described employing high-performance liquid chromatography (HPLC) with ultraviolet detection at 280 nm. The method is based on the separation of pteroylpolyglutamates containing various glutamyl residues on a C18 muBondapak reversed-phase column. Individual pteroylpolyglutamates are eluted by a gradient of 2.5-8.5% acetonitrile in 0.1 M potassium phosphate buffer (pH 6.0) within 20 min. The polyglutamates with higher glutamyl residues were less well retained in the reversed-phase column. The relationship between the peak area and the amount of pteroylpolyglutamate was observed to be linear over the range 10 pmol to 2.5 nmol. Human serum pteroylpolyglutamate hydrolase was studied using pteroylpentaglutamate as substrate in 0.1 M sodium acetate buffer (pH 4.5). The enzyme appeared to function as an exopeptidase based on the detection of intermediates, pteroyltetra-, tri-, and -diglutamate, and the product, pteroylmonoglutamate. Using the HPLC assay, extracts of Plasmodium falciparum were found not to contain detectable enzyme activity.
描述了一种用于蝶酰多谷氨酸水解酶的高灵敏度检测方法，该方法采用在280nm处进行紫外检测的高效液相色谱（HPLC）。该方法基于在C18 μBondapak反相柱上分离含有不同谷氨酰基残基的蝶酰多谷氨酸。在20分钟内，通过在0.1M磷酸钾缓冲液（pH 6.0）中2.5 - 8.5%乙腈的梯度洗脱各个蝶酰多谷氨酸。谷氨酰基残基较多的多谷氨酸在反相柱中的保留效果较差。在10 pmol至2.5 nmol范围内，观察到峰面积与蝶酰多谷氨酸量之间呈线性关系。在0.1M醋酸钠缓冲液（pH 4.5）中，以蝶酰五谷氨酸为底物研究了人血清蝶酰多谷氨酸水解酶。基于中间体蝶酰四谷氨酸、三谷氨酸和二谷氨酸以及产物蝶酰单谷氨酸的检测，该酶似乎作为一种外肽酶发挥作用。使用HPLC检测法，发现恶性疟原虫提取物不含有可检测到的酶活性。
