Rapid reverse phase-HPLC assay of HMG-CoA reductase activity.
Radioisotope-based and mass spectrometry coupled to chromatographic techniques are the conventional methods for monitoring HMG-CoA reductase (HMGR) activity. Irrespective of offering adequate sensitivity, these methods are often cumbersome and time-consuming, requiring the handling of radiolabeled chemicals or elaborate ad-hoc derivatizing procedures. We propose a rapid and versatile reverse phase-HPLC method for assaying HMGR activity capable of monitoring the levels of both substrates (HMG-CoA and NADPH) and products (CoA, mevalonate, and NADP(+)) in a single 20 min run with no pretreatment required. The linear dynamic range was 10-26 pmol for HMG-CoA, 7-27 nmol for NADPH, 0.5-40 pmol for CoA and mevalonate, and 2-27 nmol for NADP(+), and limit of detection values were 2.67 pmol, 2.77 nmol, 0.27 pmol, and 1.3 nmol, respectively.
Mozzicafreddo
Matteo
M
School of Biosciences and Biotechnology, University of Camerino, Camerino, MC, Italy. matteo.mozzicafreddo@unicam.it
Cuccioloni
Massimiliano
M
Eleuteri
Anna Maria
AM
Angeletti
Mauro
M
D055162:Biocatalysis; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D007700:Kinetics; D016014:Linear Models; D013997:Time Factors
D006903:Hydroxymethylglutaryl CoA Reductases