Separation of rat pancreatic secretory proteins by cation-exchange fast protein liquid chromatography.
Rat pancreatic secretory proteins were separated by an automated liquid chromatography system utilizing a Mono S cation-exchange column. Optimal resolution was obtained with a multistep salt and pH gradient (0.01-2 M LiCl, pH 5.3-63). A total of fourteen well-separated peaks, as well as several minor peaks, were detected by UV absorption. The main pancreatic enzymes were resolved (two amylases, two chymotrypsinogens, two trypsinogens, proelastase, lipase, prophospholipase A2, procarboxypeptidase A, procarboxypeptidase B, and ribonuclease). In addition, proteins without enzymic activity, such as lithostathine and pancreatitis-associated protein, were identified. Activation of proenzymes did not occur during the separation. At a flow-rate of 0.5 ml/min, ca. 250 micrograms to 5 mg of protein could be applied with equal resolution. The reproducibility of retention volumes and peak areas was high (less than 1% or 5% variation, respectively). When radiolabeled proteins were separated, a comparable pattern of peaks was obtained. The technique described is, therefore, not only useful for analytical and preparative separation of pancreatic proteins but can additionally serve for quantitative determination of the pancreatic isoenzyme pattern.
Fuchs
M J
MJ
Medizinische Klinik, Klinikum Mannheim, Universität Heidelberg, Germany.
Keim
V
V
D000818:Animals; D002852:Chromatography, Ion Exchange; D004032:Diet; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification; D008970:Molecular Weight; D010179:Pancreas / Q000737:chemistry* / Q000201:enzymology; D010189:Pancreatic Juice / Q000737:chemistry; D010195:Pancreatitis / Q000378:metabolism; D000073718:Pancreatitis-Associated Proteins; D011506:Proteins / Q000302:isolation & purification*; D051381:Rats; D013056:Spectrophotometry, Ultraviolet
D007527:Isoenzymes; D000073718:Pancreatitis-Associated Proteins; D011506:Proteins; C000612141:REG3A protein, human