Question

1.   How would you assay for HMG-CoA Reductase activity? Be specific

a) What is the chemical reaction?

b)   How do you quantify the reaction?

c)    What kind of instrumentation is/are required to perform this enzyme assay?

Answer 1

1.a) The chemical reaction for HMG-CoA reductase activity is-HMG-CoA reductase reduces HMG-CoA to mevalonate and CoA,in an NADPH-dependent reaction, an intermediate step in cholesterol biosynthesis.

b) Quantification of the reaction is based on the consumption of NADPH by the enzyme HMG-CoA reductase. For this test samples are preparedreaction mixtures are addedabsorbance is measured at 340 nm in a kinetic mode at 37⁰ C for 10 minutes. Then the decrease in absorbance is measured which is a measure of NADPH consumption i.e the enzyme activity.

c) To perform this enzyme assay,HMG-CoA reductase activity assay kit(colorimetric)-ab204701 is required,which contains all the components like the enzyme HMG-CoA reductase,the substrate HMG-CoA,the enzyme assay buffer,NADPH and inhibitor. Multi-well spectrophotometer is also required.