Assay of the enzymatic hydrolysis of pantetheine

✍ Scribed by Carl Wittwer; Bonita Wyse; R.Gaurth Hansen

• Publisher: Elsevier Science
• Year: 1982
• Tongue: English
• Weight: 747 KB
• Volume: 122
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(82)90273-1

✦ Synopsis

Four rapid, independent assays of enzymatic pantetheine hydrolysis are described and compared using an enzyme partially purified from pig kidney. Two assays detect specifically the hydrolysis products: cysteamine (2-aminoethanethiol)

is measured by the absorbance of its fluoropyruvate adduct at 300 nm and pantothenate is measured by radioimmunoassay. Methods of [ '%]pantethine synthesis are discussed and the labeled substrate employed in a third enzymatic assay. A fourth assay continuously monitors the absorbance of mercaptide ion at 240 nm. The mercaptide ion concentration increases proportionally with hydrolysis at a buffered pH because of a difference in pK(-SH) between pantetheine (9.9) and cysteamine (8.1) at 37'C. The enzyme shows a pH optimum of ca. 9 and an apparent K,,, of 20 pM.