3.2.4. HMG-CoA Reductase Enzyme Assay

The HMG-CoA reductase inhibitory activities of the cowpea peptide fractions and synthesised peptides were assayed using the HMG-CoA reductase kit (Sigma–Aldrich^®, St. Louis, MO, USA). The TPH sample and its fractions, containing peptides > 30 kDa, peptides of 30 to 10 kDa, peptides of 10 to 3 kDa, and peptides smaller than 3 kDa, were tested and diluted in aqueous buffer at the final concentration of 5000 µg/mL. QGF, IAF, and QDF peptides were diluted in the same buffer at the final concentration of 500 µM, following the manufacturer’s instructions. The peptide concentration was calculated based on the measured molecular mass of each one of them (351.2 g/mol, 350.2 g/mol, and 408.2 g/mol, respectively). No aggregation was observed in the hydrolysate, peptide fractions, or synthesized peptides. Enzyme activity was measured by monitoring the decrease in absorbance at 340 nm and 37 °C, following the supplier’s instructions. Specific enzyme activity was defined as number of micromoles of oxidised NADPH/min/mg protein (mgP). The results are expressed as the percentage of the control-specific activity of the enzyme in the absence of pravastatin and the compounds under investigation. Activity was calculated using the following equation: Units/mgP = (∆A₃₄₀/min_sample − ∆A₃₄₀/min_blank) × TV/12.44 × V × 0.6 × LP, where 12.44 = ε^mM; the extinction coefficient for NADPH at 340 nm is 6.22 mM⁻¹ cm⁻¹, hence 12.44 represents 2 NADPH units consumed in the reaction; TV = total volume of the reaction (mL); V = volume of the enzyme used in the assay (mL); 0.6 = enzyme concentration in mgP/mL; and LP = light path in cm.