Calculation of the isoelectric points of native proteins with spreading of pKa values
Authors:
- 1 January 1995
- journal article
- research article
- Published by Wiley in Electrophoresis
- Vol. 16 (1) , 1377-1380
Abstract
The isoelectric points (pI) of native proteins are important in several separation techniques. For estimating pI values the net charge of several proteins was calculated versus pH by use of the Henderson‐Hasselbalch equation. Amino acid composition, pKa values for amino acid side chains and for the N‐ and C‐terminal groups, and the presence of other charged groups were taken into account. A set of pKa values was chosen for amino acid residues with ionizable side chains. Each particular type of ionizable group was assumed to have pKa values distributed around the chosen value, thereby simulating the situation in proteins and polypeptides. The calculated pI values showed reasonably good agreement with experimental ones for most of 16 native proteins over a wide pH range (3.4–11) when charge contributions of heme groups, sialic acid residues, etc., were taken into account. The calculated pI for the human red cell glucose transporter (Glut 1) with one sialic acid residue was decreased from 8.8 to 8.5 by introducing pKa value spreading and became consistent with the experimental pI value of 8.4 ± 0.05 at 15°C determined in the presence of 6 M urea. The pI of the native Glut1 was lower, 8.0 ± 0.1, at 22°C. In general, the pI values for native proteins are affected by the three‐dimensional structure of the proteins, which causes greater differences between calculated and experimental pI values than in the case of polypeptides for which pI values are determined in the presence of urea.Keywords
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