A novel method for total protein analysis of protein mixtures using enzyme hydrolysis and derivatisation with o-phthaldialdehyde – Application to dairy products

A novel approach to analysis of proteins in dairy products was demonstrated using enzyme hydrolysis followed by derivatisation with o-phthaldialdehyde (OPA) and fluorescence detection. Unlike the response of other dye-binding methods, enzyme hydrolysis significantly eliminated dependence of the fluorescence intensity upon protein primary sequence and, compared with chemical hydrolysis, was achieved under relatively mild conditions. Enzyme hydrolysis and OPA-derivatisation steps were successfully automated using a flow-injection manifold, with a method coefficient of repeatability <6%. The enzyme-OPA (eOPA) method was applied to analysis of total (TP) and serum-soluble proteins (SSP, proteins soluble at pH 4.6) in reference standards spanning two decades of protein concentration. Method accuracy was very good for analysis of TP and acceptable for analysis of SSP. Discrepancies between the eOPA and standard methods for SSP were explained by sample preparation and not analytical factors. The eOPA method may be suitable for automation into a portable or miniaturised device.