Kinetics of the inhibition of calmodulin-dependent nitric oxide synthase by flaxseed protein-derived peptides

Calmodulin-dependent Nitric Oxide Synthase (NOS) catalyses the production of Nitric Oxide (NO) by converting L-arginine to L-citrulline. Although NO mediates several physiological functions, excessive levels of NO have been found to contribute to the pathology of many diseases. Therefore, inhibition of the activities of NOS may be used in the treatment of pathological conditions due to uncontrolled production of NO.; We studied the inhibition of NOS through the inhibition of calmodulin (CaM) activity by peptides obtained from enzymatic hydrolysis of flaxseed proteins. Low molecular weight peptides (molecular weight <1000) with CaM-binding activity were obtained by enzymatic hydrolysis of flaxseed protein using alcalase. Positively charged peptides were isolated using cation exchange chromatography. Two peptide fractions with different levels of positive charge were obtained, and both were used for inhibition of CaM-dependent neuronal and endothelial NOS activity. At fixed CaM concentration, enzyme activity decreased moderately as peptide concentration increased. Enzyme inhibition kinetics showed that both peptides inhibited nNOS in a non-competitive manner, while eNOS inhibition was competitive. Furthermore, as CaM concentration increased at fixed peptide concentration, enzyme activity increased, suggesting that inhibition can be overcome by increasing the concentration of CaM.; Fluorescence spectroscopy and circular dichroism were used to determine the structural changes of CaM in the presence of inhibitory peptides. Fluorescence studies showed that flaxseed protein derived peptides are capable of inducing structural changes in CaM, while CD studies demonstrated changes in secondary and tertiary structures of CaM upon peptide binding.; Our results show that flaxseed protein derived peptides have CaM-binding activity and that interaction of the peptides with CaM is detrimental to optimum NOS catalytic activity. This could provide an effective means of treatment of diseases associated with over production of NO, and contribute to exploring the potential of flaxseed proteins as a functional food source.