Abstract
In rabbits, the depletion of cartilage matrix which occurs following intravenous administration of papain treated with iodacetamide is attributable to a portion of the enzyme in the disulfide form which has not undergone alkylation. It is this portion that is reactivated in cartilage in vivo and initiates the enzymatic breakdown of the protein-polysaccharide complex which forms a major component of the matrix. Evidence presented in support of these conclusions indicates that, contrary to an earlier hypothesis, papain acetamide is not reactivated in vivo. Following intravenous injection in amounts up to 4 mg./kg., active and inactive papain leaves the circulation at a rate proportional to the concentration, and it is likely that the initial rate of disappearance represents equilibration with the extracellular space. Following injection in the active or inactive form, a high proportion of papain in serum is bound to protein in the alpha globulin fraction. It is believed that in the case of fully active papain, the proportion which is not bound to alpha globulin becomes attached to other proteins of serum in extracellular fluid, such as albumin, by a process of enzyme substrate combination, and is thus prevented from diffusing into cartilage. In the case of inactive papain, a comparable excess remains free to enter cartilage, where it initiates depletion of matrix following reactivation within the tissue. These conclusions provide an explanation for the failure of fully active papain to cause depletion of cartilage matrix in vivo; the widespread changes seen after the injection of papain inactivated by iodoacetamide or by simple reversible oxidation are attributable to a small proportion of the injected material which enters cartilage in the disulfide form in a concentration of approximately 2 micrograms/gm. wet weight. The possibility that such a small amount of protease, when reactivated, can produce changes in cartilage matrix has been confirmed by studies on the effects of papain on isolated cartilage and chondromucoprotein in vitro. It has been shown that severe local injury results when active papain is injected into the skin of a rabbit in low concentrations. Since a much higher concentration of papain can be attained in the circulation without obvious adverse effects, it is evident that binding of the protease by alpha globulin and possibly other serum proteins may exemplify a mechanism whereby the tissues are protected from injury following entry into the circulation of other potentially harmful agents, such as proteolytic enzymes derived from cells or bacteria.
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Selected References
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- BRYANT J. H., LEDER I. G., STETTEN D., Jr The release of chondroitin sulfate from rabbit cartilage following the intravenous injection of crude papain. Arch Biochem Biophys. 1958 Jul;76(1):122–130. doi: 10.1016/0003-9861(58)90126-7. [DOI] [PubMed] [Google Scholar]
- DAVIS N. C., SMITH E. L. Assay of proteolytic enzymes. Methods Biochem Anal. 1955;2:215–257. doi: 10.1002/9780470110188.ch8. [DOI] [PubMed] [Google Scholar]
- KATCHMAN B. J., ZIPF R. E., HOMER G. M. Casein labelled with iodine-131 as a substrate for the measurement of trypsin and chymotrypsin. Nature. 1960 Jan 23;185:238–239. doi: 10.1038/185238a0. [DOI] [PubMed] [Google Scholar]
- KUNKEL H. G. Zone electrophoresis. Methods Biochem Anal. 1954;1:141–170. doi: 10.1002/9780470110171.ch6. [DOI] [PubMed] [Google Scholar]
- McCLUSKEY R. T., THOMAS L. The removal of cartilage matrix, in vivo, by papain; identification of crystalline papain protease as the cause of the phenomenon. J Exp Med. 1958 Sep 1;108(3):371–384. doi: 10.1084/jem.108.3.371. [DOI] [PMC free article] [PubMed] [Google Scholar]
- POTTER J. L., McCLUSKEY R. T., WEISSMANN G., THOMAS L. The effects of papain on cartilage in vivo: factors influencing the distribution of papain protease following intravenous injection. Ann N Y Acad Sci. 1960 Jun 30;86:929–942. doi: 10.1111/j.1749-6632.1960.tb42850.x. [DOI] [PubMed] [Google Scholar]
- SMITH E. L. Active site of papain and covalent high-energy bonds of proteins. J Biol Chem. 1958 Dec;233(6):1392–1397. [PubMed] [Google Scholar]
- SPICER S. S., BRYANT J. H. Cartilage changes in papain-treated rabbits. Am J Pathol. 1957 Nov-Dec;33(6):1237–1245. [PMC free article] [PubMed] [Google Scholar]
- THOMAS L. Reversible collapse of rabbit ears after intravenous papain, and prevention of recovery by cortisone. J Exp Med. 1956 Aug 1;104(2):245–252. doi: 10.1084/jem.104.2.245. [DOI] [PMC free article] [PubMed] [Google Scholar]
- TSALTAS T. T. Papain-induced changes in rabbit cartilage; alterations in the chemical structure of the cartilage matrix. J Exp Med. 1958 Oct 1;108(4):507–513. doi: 10.1084/jem.108.4.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
- WEISSMANN G., POTTER J. L., McCLUSKEY R. T., SCHUBERT M. Turbidity produced by hexamminecobaltic chloride in serum of rabbits injected intravenously with papain. Proc Soc Exp Biol Med. 1959 Oct-Dec;102:584–587. doi: 10.3181/00379727-102-25326. [DOI] [PubMed] [Google Scholar]