Anions and the anomalous gel filtration behavior of notexin and scutoxin

Based on their mol. wts, notexin and scutoxin elute later than expected from gel filtration columns in multiple peaks [Francis et al. (1991) Toxicon 29, 85-96]. Notexins present in these peaks have identical amino acid sequences and unmodified amino acid side-chains. Scutoxin is an isoform of notexin which contains arginine at position 16 and glutamate at position 82. Like notexin, it also elutes in different fractions on a gel filtration column, yet the fractions show identical amino acid sequence. This perplexing chromatographic behavior appears to be caused by the association of these proteins with different anions, since dissolving notexin in buffers containing different anions produces up to a 30% change in elution volume. Certain anions promote an apparent reduction in the interaction of notexin with gel filtration matrix, hence earlier elution. These anions include citrate, 3-phosphoglycerate and 2-phosphoglycerate, which also inhibit the PLA2 activity of notexin. However, even under conditions which minimize protein-matrix interaction the toxins elute later than expected based on their mol. wt.