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

Abstract

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, P-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 a-lactalbumin and P-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 P-lactoglobulin compared with lysozyme and a-lactalbumin. A similar interaction was not observed between a-lactalbumin and 0-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 a-lactalbumin or lysozyme and P-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 P-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 P-lactoglobulin. For a-lactalbuminlysozyme mixtures, however, the modelling suggested that non-specific electrostatic binding may occur.

Effects of protein–protein interaction in ultrafiltration based fractionation processes

“…Another possible explanation for the decrease in transmembrane pressure due to BSA injection could be the formation of BSA-lysozyme co-precipitates. The co-precipitation of lysozyme along with other proteins such as alpha-lactalbumin and beta-lactoglobulin has been discussed by Howell et al (1995). The osmotic pressure exerted by a solute such as a protein is a function of the number of solute molecules present in solution.…”

“…However, it is more than likely that more BSA and lysozyme molecules could be involved in such complexes, which are referred to as co-precipitates. Co-precipitation is indicated by an increase in turbidity of the protein solution (Howell et al, 1995). When BSA and lysozyme were simultaneously present within the stirred cell at pH 9.0, some turbidity of the feed was observed.…”

Heteroprotein complex coacervation: A generic process

“…The first description of the complexation between LYS and -LA dates back to 1995 [70]. Several years later, in-depth studies were published describing LYS/-LA coacervation and its dependence on pH, protein flexibility, temperature, ionic strength and protein concentration.…”

“…Howell et al [70] observed the co-precipitation of LYS and -LA at pH 6.8 in unbuffered aqueous medium. More recently, the complexation of these two proteins was investigated in unbuffered aqueous solution adjusted at pH values ranging from 3 to 11 [71].…”

Analysis of lysozyme in cheese by immunocapture mass spectrometry

“…During cheese manufacturing, lysozyme is transferred to the curd, probably by association of the enzyme with the caseins [23]. Lysozyme added to kettle milk binds to the caseins [24], but also to the whey proteins ␣-lactalbumin and ␤-lactoglobulin [25]. The binding is particularly caused by electrostatic interactions between the positively charged lysozyme and the milk proteins, which are negatively charged at the pH-value of about 7 in the kettle milk.…”