Charge determination of proteins with polyelectrolyte titration.

D Horn,C C Heuck

doi:10.1016/s0021-9258(18)33037-0

Abstract

A recently developed photometric version of polyelectrolyte titration was applied for the determination of the number of charged residues on globular proteins. Based on the observation that oppositely charged polyelectrolytes form, in general, stoichiometric polyelectrolyte complexes, the protein solutions were incubated in excess with an oppositely charged polyelectrolyte, and the residual amount was back-titrated using o-toluidine blue for end point detection. It was found that within the range pH 2 to pH 9 the interaction of the polyelectrolytes, potassium polyvinylsulfate, polydiallylammonium chloride, and N-methylglycolchitosan iodide, with various proteins of known amino acid composition (ribonuclease A, trypsin, chymotrypsin A, pepsin, cytochrome c) occurs stoichiometrically through 1:1 ion pair interaction, irrespective of the spatial distribution of the interacting ionic sites. The close correspondence between the experimental data for the net charge and the calculated balance of ionized residues for the proteins at a given pH indicates that in the native structure of these proteins oppositely charged ionic functions are largely neutralized by the formation of intramolecular salt linkages. It is concluded that polyelectrolyte titration offers an easy access to the determination of the surface charge of proteins and other biopolymers. The data further support the notion of the importance of electrostatic cooperative interactions in biological systems.

Highlights

A recently developed photometric version of polye- Interaction of synthetic polyelectrolytes or biological micellar lectrolyte titration was applied for the determination colloids with polyelectrolyte properties andcells may contribof the number of chargresdidues on globular proteins. ute toproliferation [13]and/or regulation of metabolic events
The close cor- surface charge density of biopolymers may result in a change respondence between the experimental data for tnheet of the turnover of the macromolecules[22].Hydrogen ion charge and the calculated balance of ionized residues titration and conductometry are well established techniques for the proteins at a given pH indicatesthatinthe for the quantitative assessment of the surface charge of bionative structure of these proteins oppositely charged polymers [23,24,25]
The data further hamper the application of these techniques in cases where support the notion of the importance of electrostatic only limited amounts of material are available

Summary

Charge Determination of Proteins with Polyelectrolyte Titration*

From the Hauptlahoratorium der BASF AG, Ludwigshafen/RheinG, FR and the Klinisches Znstitut fur Herzinfarktfo;schung, Heidelberg, GFR. The close cor- surface charge density of biopolymers may result in a change respondence between the experimental data for tnheet of the turnover of the macromolecules[22].Hydrogen ion charge and the calculated balance of ionized residues titration and conductometry are well established techniques for the proteins at a given pH indicatesthatinthe for the quantitative assessment of the surface charge of bionative structure of these proteins oppositely charged polymers [23,24,25] Both methods require relatively ionic functions are largely neutralized by the formathiiognh concentrations of the substratesfor reliable results.Furof intramolecularsalt linkages. The charge equivalence of the polycation was calculated from the amount of KPVS needed to reach the point of steepest slope, indicating complete 1:l ion pair interaction in the polyelectrolyte complex formation. 100 200 300 100 500 600 700 860 pl MGC

Theoretical charge density uersus pH curves were calculated with

RESULTS

IKPVSIPDDAI nlKPVSlMGCI

Cytochrome c

DISCUSSION