Studies on the electrostatic interactions of lysozyme with α‐lactalbumin and β‐lactoglobulin

Summary This paper reports the occurrence of protein‐protein co‐precipitation, through electrostatic interactions. As a preliminary overview the effect of mixing lysozyme with a range of proteins (BSA, β‐lactoglobulin, sodium caseinate and whey isolate) was tested by turbidity measurements. Turbidity increased when the proteins were mixed with lysozyme in water and not in phosphate buffer. The exception was the interaction of whey isolate and sodium caseinate which showed turbidity (but not precipitation) when mixed with lysozyme in phosphate buffer. Further detailed studies on the interaction between whey proteins α‐lactalbumin and β‐lactoglobulin and hen egg lysozyme by fast protein liquid chromatography (FPLC), have shown that, in aqueous solution of pH 6.8, a higher level of interaction occurred between lysozyme and β‐lactoglobulin compared with lysozyme and α‐lactalbumin. A similar interaction was not observed between α‐lactalbumin and β‐lactoglobulin. The predominant product of the interaction was an insoluble precipitate. In addition, a small amount of soluble complex was also recovered by ion‐exchange chromatography of the supernatant. The soluble complex was shown by SDS‐polyacrylamide gel electrophoresis to comprise the same components as the insoluble precipitate, i.e. lysozyme and α‐lactalbumin or lysozyme and β‐lactoglobulin. The interactions of the proteins and the amount of precipitation varied depending on the concentration of each protein in the mixture, the ionic strength and pH of the solution. Molecular modelling studies, using interactive docking of the crystal structures, indicated that for the β‐lactoglobulin‐lysozyme interaction the optimum visual fit could involve electrostatic interactions between Glutamate 35 and Aspartate 53 in the catalytic binding site on lysozyme and Lysines 138 and 141 at the dimerization site of β‐lactoglobulin. For α‐lactalbuminlysozyme mixtures, however, the modelling suggested that non‐specific electrostatic binding may occur.