Trypsinogen chromatography

Figure B3.1.1 A 15% SDS-polyacrylamide gel stained with Coomassie brilliant blue. Protein samples were assayed for the purification of a proteinase, cathepsin L, from fish muscle according to the method of Seymour et al. (1994). Lane 1, purified cathepsin L after butyl-Sepharose chromatography. Lane 2, cathepsin L complex with a cystatin-like proteinase inhibitor after butyl-Sepharose chromatography. Lane 3, sarcoplasmic fish muscle extract after heat treatment and ammonium sulfate precipitation. Lane 4, sarcoplasmic fish muscle extract. Lanes M, low-molecular-weight standards aprotinin (Mr 6,500), a-lactalbumin (Mr 14,200), trypsin inhibitor (Mr 20,000), trypsinogen (Mr 24,000), carbonic anhydrase (Mr 29,000), gylceraldehyde-3-phosphate dehydrogenase (Mr 36,000), ovalbumin (Mr 45,000), and albumin (Mr 66,000) in order shown from bottom of gel. Lane 1 contains 4 pg protein lanes 2 to 4 each contain 7 pg protein.

Fig. 1. Chromatography of bovine pancreatic juice on DEAE-cellulose (anionic proteins) and Amberlite IRC-50 (cationic proteins) (1). RNAase, ribonuclease ChTg-a, chymotrypsinogen A Tg, trypsinogen ProCp-B and Cp-B, procarboxypeptidase B and carboxypeptidase B DNAase, deoxyribonuclease ProCp-A, procarboxypeptidase A.

Keller and Cohen (36) also subjected to chromatography acidic extracts of cattle pancreatic microsomes and ribosomes. In microsomes they found the expected amounts of all enzymes which are known to be stable in acid, viz., chymotrypsinogen A, trypsinogen, deoxyribonuclease, and ribonuclease. The amounts of chymotrypsinogen B were abnormally low and ribonuclease B was perhaps not present. The results concerning ribosomes were made somewhat uncertain by the ability of these particles to incorporate proteins from the medium. Nevertheless, a series of characteristic enzymes could be isolated from what appears to be the very site of their biosynthesis. 

Fig. 11. Second chromatography of porcine chymotrypsinogen A and trypsinogen (85). Both elutions are performed with buffers of constant composition (equilibrium chromatography). On the left chymotrypsinogen A (specific activity, 2.9). CM-cellu-lose column equilibrated and eluted with 0.03 M citrate, pH 5.0. On the right trypsinogen (specific activity, 0.35-0.37). CM-cellulose column equilibrated and eluted with pH 6.0 buffer 0.015 M in citrate and 10 < M in DFP. Ordinates and abscissas are the same as in Figs. 9 and 10. Solid line, activity. Dotted line, proteins.

Figures 10 and 11 show how porcine trypsinogen can be prepared in high yield by chromatography on CM-cellulose. The amino acid composition of this protein is given in Table III, with two sets of values for the bovine precursor. Analytical results have also been obtained with a commercial sample of bovine trypsin (123).

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