Publication: High-performance liquid chromatographic assay for pteroylpolyglutamate hydrolase
Issued Date
1987-01-01
Resource Type
ISSN
03784347
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2-s2.0-0023204060
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Mahidol University
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SCOPUS
Bibliographic Citation
Journal of Chromatography B: Biomedical Sciences and Applications. Vol.417, No.C (1987), 47-56
Suggested Citation
Jerapan Krungkrai, Yongyuth Yuthavong, H. Kyle Webster High-performance liquid chromatographic assay for pteroylpolyglutamate hydrolase. Journal of Chromatography B: Biomedical Sciences and Applications. Vol.417, No.C (1987), 47-56. doi:10.1016/0378-4347(87)80090-7 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/15330
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Title
High-performance liquid chromatographic assay for pteroylpolyglutamate hydrolase
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Abstract
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 C 18 μBondapak 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. © 1987.