Publication: A kinetic spectrophotometric method for the determination of pyridoxal-5′-phosphate based on coenzyme activation of apo-D-phenylglycine aminotransferase
Issued Date
2018-10-01
Resource Type
ISSN
18790909
01410229
01410229
Other identifier(s)
2-s2.0-85048895900
Rights
Mahidol University
Rights Holder(s)
SCOPUS
Bibliographic Citation
Enzyme and Microbial Technology. Vol.117, (2018), 64-71
Suggested Citation
Juntratip Jomrit, Duangnate Isarangkul, Pijug Summpunn, Suthep Wiyakrutta A kinetic spectrophotometric method for the determination of pyridoxal-5′-phosphate based on coenzyme activation of apo-D-phenylglycine aminotransferase. Enzyme and Microbial Technology. Vol.117, (2018), 64-71. doi:10.1016/j.enzmictec.2018.06.003 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/45034
Research Projects
Organizational Units
Authors
Journal Issue
Thesis
Title
A kinetic spectrophotometric method for the determination of pyridoxal-5′-phosphate based on coenzyme activation of apo-D-phenylglycine aminotransferase
Other Contributor(s)
Abstract
© 2018 Elsevier Inc. A new PLP assay method based on the coenzyme activation of apo-D-phenylglycine aminotransferase (apo-D-PhgAT) is reported. The assay process is comprised of two steps. First, PLP present in plasma samples is allowed to reconstitute apo-D-PhgAT, forming active holo-D-PhgAT. In the second step, the enzymatic activity of reconstituted D-PhgAT is determined using D-4-OH-phenylglycine as the amino donor substrate with 4-OH-benzoylformate (OH-BZF) as the reaction product. OH-BZF absorbs UV light strongly at 334 nm (molar absorption coefficient = 25.4 × 103 M−1cm−1) and its rate of formation is monitored spectrophotometrically. The rate of the transamination reaction catalyzed by the reconstituted D-PhgAT is directly proportional to the amount of PLP in the sample. The method is applicable for determining PLP in the concentration range from 5.2 to 250 nM and requires 50 μL of plasma sample. The mean within- and between-run coefficient of variations (CVs) were 8.1% and 12.4%, respectively. Analytical recoveries ranged from 98 to 108%. The assay was specific and showed good correlation with the established method (CDC, Method No: 4002.05). The assay requires one reaction catalyzed by a single enzyme, does not require a radioactive substrate, and a derivatization reagent is not needed. This PLP determination process is relatively simple to perform and can be completed using common laboratory equipment.