Charge determination of proteins with polyelectrolyte titration.
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.
D000818:Animals; D002918:Chymotrypsin / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D004563:Electrochemistry; D004573:Electrolytes; D004722:Endoribonucleases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010434:Pepsin A / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic; D014357:Trypsin / Q000378:metabolism
D003574:Cytochrome c Group; D004573:Electrolytes; D011506:Proteins; D004722:Endoribonucleases; D012259:Ribonuclease, Pancreatic; D002918:Chymotrypsin; D014357:Trypsin; D010434:Pepsin A