Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay |
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| Authors: | Vashist Sandeep Kumar Zhang BinBin Zheng Dan Al-Rubeaan Khalid Luong John H T Sheu Fwu-Shan |
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| Affiliation: | Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA |
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| Abstract: | A high-performance liquid chromatography (HPLC)-based fluorometric method for measuring serine hydroxymethyltransferase (SHMT) activity toward formation of serine and (6S)-H4PteGlun has been developed. In this method, serine formed by SHMT activity is reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to form the fluorescent adduct NBD–serine. The fluorescent assay components are then separated by reversed-phase chromatography, and NBD–serine is quantified by comparison with standards. This method was used to determine the Km and kcat values for 5,10-CH2–H4PteGlu5 of an SHMT from Arabidopsis thaliana. These data represent the first determination of kinetic parameters for (6S)-5,10-CH2–H4PteGlu5 for an SHMT from any organism. |
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