Critical sources of error in colorimetric assay for UDP- N-acetylglucosamine pyrophosphorylase

Abstract

UDP-N-acetylglucosamine pyrophosphorylase (UAP)1 is a key enzyme in prokaryotes and eukaryotes leading to synthesis of a ubiquitous metabolite, UDP-N-acetylglucosamine (UDP-N-GlcNAc), for polymerization. This enzyme catalyzes the reaction UTP+ N-acetylglucosamine-1-phosphate (GlcNAc-1-P)!UDP-GlcNAc+ pyrophosphate (PPi), and its activity was usually determined by a radioactive assay in which product from a radioactive substrate was identiWed by chromatography and measured by a scintillation counter [1–4]. However, this assay has major concerns about safety, complicated handling, and availability of radioactive substrates. Mio et al. [5] mentioned another strategy in which a coupling enzyme, pyrophosphatase, is included in the anabolic reaction to convert the pyrophosphate into inorganic phosphate that can be readily quantiWed by a malachite green colorimetric assay. Nevertheless, the precautions of this assay have never been discussed, and ignorance of proper controls was found to result in signiWcant artifacts. The current report describes the basic principle, reaction condition, and critical sources of error of this colorimetric assay to allow accurate and reliable measurement of UAP activity. The principle of the malachite green colorimetric assay is based on the reaction between phosphate and molybdate to form a phosphomolybdate complex that binds to the dye and induces the color changes [6]. It is