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. 1973 Apr;131(4):809–822. doi: 10.1042/bj1310809

Human cathepsin B1. Purification and some properties of the enzyme

Alan J Barrett 1
PMCID: PMC1177541  PMID: 4124667

Abstract

1. Cathepsin B1 was purified from human liver by a method involving autolysis, fractional precipitation with acetone, adsorption on, and stepwise elution from, CM-cellulose and an organomercurial adsorbent, gel chromatography and finally equilibrium chromatography on CM-cellulose. 2. The early stages of the procedure, including the use of the organomercurial adsorbent, were suitable for the simultaneous isolation of cathepsin D. The two cathepsins were sharply separated on the organomercurial column, and particular attention was given to the method for the preparation and use of this adsorbent. 3. A method is described for the staining of analytical isoelectric-focusing gels for cathepsin B1 activity, as well as protein. By this method it was shown that cathepsin B1 was represented by at least six isoenzymes during the greater part of the purification procedure. After the gel-chromatography step this group of isoenzymes was obtained essentially free of other proteins, in good yield. The isoenzymes were resolved from this mixture by chromatography on CM-cellulose. The purified enzyme was stable for several weeks at slightly acid pH values in the absence of thiol compounds; it was unstable above pH7. 4. The pI values of the isoenzymes of cathepsin B1 extended from pH4.5 to 5.5, that of the major isoenzyme tending to increase from 5.0 to 5.2 during the purification procedure. Gel chromatography indicated a molecular weight of 27500 for all of the isoenzymes, whereas polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate gave a value of 24000. 5. An antiserum raised in sheep against the purified enzyme reacted specifically with the alkali-denatured molecule. Purified cathepsin B1 contained no material precipitable by an anti-(human cathepsin D) serum. 6. The enzyme hydrolysed several N-substituted derivatives of l-arginine 2-naphthylamide, as well as haemoglobin, azo-haemoglobin, azo-globin and azo-casein. Greatest activity was obtained near pH6.0. 7. The sensitivity of human cathepsin B1 to chemical inhibitors was generally similar to that of other thiol proteinases. The enzyme was inactivated by the chloromethyl ketones derived from tosylphenylalanine, tosyl-lysine, acetyltetra-alanine and acetyldialanylprolylalanine. 8. The hydrolysis of α-N-benzoyl-dl-arginine 2-naphthylamide by extracts of human liver at pH6 was attributable entirely to cathepsin B1.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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