Polyelectrolyte Behaviour of Phosvitin

Abstract

The results of spectroscopic and microcalorimetric measurements on dilute aqueous solutions of phosvitin are described. Our infrared and circular dichroism spectra show that the phosphoprotein chains may assume either random-coiled or partially helical or /?-structures depending upon the pH. The transition to the low p H 8-form occurs with a distinct cooperative character.The strongly interdependent ionization of fixed charges densely spaced along phosvitin chains, typical of highly charged polyelectrolytes, is well illustrated by the calorimetric enthalpy of protonation data.The interaction of phosvitin with acridine orange in dilute aqueous solution (pH -5) has been studied by means of spectral and equilibrium dialysis experiments. For relatively low polymer to dye concentration ratios, bound acridine orange exhibits a y-type spectrum thus suggesting aggregation of dye molecules onto the protein. Gradual decrease in the extent of bound acridine orange aggregation is produced by increasing the concentration of the protein ; the monomerically bound form is hardly obtained, however, even at relatively high phosvitin concentrations. Binding curves reveal a cooperativity character for the dye-protein interaction.It is suggested that about 60°/, only of phosphomonoester side groups of phosvitin are available for acridine orange binding, these being characterized by a high "stacking" coefficient.The results are tentatively discussed on the basis of present information on phosvitin chemical composition and conformation.The phosphoprotein phosvitin is an atypical biopolymer in that, owing mainly to the high negative charge density along the chain, it is present in aqueous solution at around pH 7 in an essentially unordered conformation [l -31. Gradual protonation of the secondary acidic functions of the phosphomonoester side-groups of phosvitin (0-phosphoserine accounts for more than half of the amino acid residues, while carboxyl groups account for another 12O/, of the total residues [4]) would permit, by reducing interchain electrostatic interactions, the formation of some ordered regions along the polypeptide backbone [2,3]. Finally, by lowering the pH of a dilute aqueous solution of phosvitin down to about 1.8, where also the dissociation of the primary acidic function of the phosphomonoester groups is suppressed, the protein undergoes a conformational transition to a &form We wish to report here the results of a research carried out i n our laboratory on different aspects of the behaviour of phosvitin in dilute aqueous solution. The first section of the paper includes the resultv of a series of microcalorimetric measurements of the enthalpy of protonation of phosvitin in a wide pH range, and infrared and circular dichroism (CD) studies in the entire accessible range of the degree of protonation of the protein. These results provide new information on the polyelectrolyte behaviour of phosvitin and on the ability of its chain to assume Werent conformations depending upon the pH of the solutions. Evidence is presente...

The effect of complex-formation with polyanions on the redox properties of cytochrome c

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