High-performance liquid chromatography: purification and chromatographic behaviour of molecular variants of pepsinogen A from human urine.
By combining conventional DEAE chromatography with high-performance liquid chromatography on Sephacryl S-200 HR and Mono-Q columns, we have been able to isolate and fractionate human pepsinogen A (PGA) isozymogens from large amounts of urine. This method of fractionation is simple and allows one to obtain pepsinogen in a native non-denatured conformation. The isozymogens are homogeneous by electrophoretic and chromatographic criteria; this was confirmed by N-terminal amino acid sequencing. Purified PGA-3 and PGA-5 can be converted into an additional, more anionic, isoform on incubation at 37 degrees C. This isoform exists not only in vitro but also in vivo. The net negative charge of the PGA isozymogens is in the order PGA-5 less than deamidated PGA-5 less than PGA-3 less than deamidated PGA-3. Surprisingly, the elution order on the Mono-Q column was PGA-5/PGA-3/deamidated PGA-5/deamidated PGA-3. We have performed molecular modelling on PGA to investigate this phenomenon in terms of surface charge (not net charge) of the proteins. The model provides evidence that (1) only a fraction of the protein surface interacts with the support and (2) regions of localized charge at the protein surface may allow portions of the external surface to dominate chromatographic behaviour, resulting in a steering of the proteins with respect to the oppositely charged matrix. Pepsinogens may serve as model proteins for elucidating some of the variables that determine the chromatographic behaviour of proteins on ion-exchange columns.
Bank
R A
RA
Institute of Human Genetics, Faculty of Medicine, Vrije Universiteit, Amsterdam, Netherlands.
Eriksson
A W
AW
Pals
G
G
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000652:urine*; D010435:Pepsinogens / Q000737:chemistry / Q000652:urine*
D007527:Isoenzymes; D010435:Pepsinogens