Milton E. Bailey

A Method for Determining Binding of Hexanal by Myosin and Actin Using Equilibrium Headspace Sampling Gas Chromatography

SUMMARY An equilibrium headspace sampling method was developed and used to investigate the binding of hexanal to muscle myofibrillar proteins in aqueous solution. Utilizing a diphasic system (lipid/water), headspace concentrations were measured by headspace sampling gas chromatography over a range of ligand concentrations and binding constants calculated. Coefficient of variation between duplicate headspace measurements averaged 4% and less than 10% between replications; however, calculated Klotz constants varied significantly. In an attempt to reduce Klotz plot variation, linear regression was applied to headspace measurements prior to the calculation of BOUND verses FREE variables used to plot Klotz plots. Headspace data modification of hexanal standard curves (with and without added protein) did not reduce variation in calculated Klotz constants N, the number of binding sites (number of molecules of hexanal per protein molecule) or in the Klotz constant (K). In a second attempt to reduce Klotz plot variation, two calculation methods were compared. A nonlinear calculation method should be used on nonlinear Klotz plots while the traditional linear calculation method should be used on linear Klotz plots to calculate binding constants. There were significant (P