Characterisation of heat-induced protein aggregation in whey protein isolate and the influence of aggregation on the availability of amino groups as measured by the ortho-phthaldialdehyde (OPA) and trinitrobenzenesulfonic acid (TNBS) methods

Whey protein isolate (WPI) solutions, with different levels of aggregated protein, were prepared by heating (5% protein, pH 7, 90 °C for 30 min) WPI solutions with either 20 mM added NaCl (WPI + NaCl), 5 mM N-ethylmaleimide (WPI + NEM) or 20 mM added NaCl and 5 mM NEM (WPI + NaCl + NEM). Gel electrophoresis demonstrated that the heated WPI and WPI + NaCl solutions had higher levels of aggregated protein, due to more covalent interactions between proteins, than the heated WPI + NEM and WPI + NaCl + NEM solutions. There were marked differences in the levels of amino groups between all heated WPI solutions when measured by the OPA and TNBS methods, with lower levels being measured by the TNBS method than by the OPA method. These results demonstrate that the measurement of available amino groups by the OPA method is less impacted than by the TNBS method after heat-induced structural changes, arising from disulfide or sulfhydryl-disulfide bond-mediated aggregation of whey protein molecules.