Please use this identifier to cite or link to this item:

The study of the active center of chymopapain

File Description SizeFormat 
uhm_phd_6613721_uh.pdfVersion for UH users3 MBAdobe PDFView/Open
uhm_phd_6613721_r.pdfVersion for non-UH users. Copying/Printing is not permitted3.05 MBAdobe PDFView/Open

Item Summary

Title: The study of the active center of chymopapain
Authors: Tsunoda, Joyce Nishimura
Keywords: Chymopapain
Issue Date: 1966
Publisher: [Honolulu]
Abstract: The proteolytic enzyme chymopapain was first isolated from fresh papaya latex by Jansen and Balls (J. Biol. Chem., 137, 459, 1951). Subsequently, a modified procedure for purification of chymopapain from dried papaya latex was developed by Ebata (J. Biol. Chem., 237, 1086, 1962). Chymopapain was shown to be a sulfhydryl enzyme on the basis of its susceptibility to sulfhydryl reagents and its requirement for "thiol activators" such as cysteine and cyanide for maximum activity. Titration of cyanide activated chymopapain with p-chloro mercuribenzoate (CMS) revealed the presence of 1.4-1.6 moles of SH per mole of enzyme (m.w., 30,000). The controlled addition of the colored sulfhydryl reagent, N-4-dimethylamino- 3,5-dinitropheriyl)-maleimide (Witter and Tuppy, Biochim. Biophys. Acta, 45, 429, 1960), and enzymic hydrolysis of the S-DDPS-chymopapain thus formed led to the isolation of a deca-peptide containing the labeled half cysteine residue. The amino acid sequence of this peptide was found to be: Lys-Arg-Val-Pro-Asp-Ser-Gly-Glu-Cys-Tyr (Cys = the labeled half of cysteine residue). This sequence differed from those of the peptides containing the reactive SH groups of papain (Light et al., Proc. Nat'l. Acad. Sci, U.S.A., 52, 1276, 1964) and ficin (Wong and Liener, Biochem. Res. Comm., 11, 470, 1964). v Possible reasons for this difference in the sulfhydryl peptides from papain and ficin and that from chymopapain are discussed. Preliminary studies (Ebata et al., Biochem. Biophys. Res. Comm., 9, 173, 1962) indicated that chymopapain was inhibited by diisopropylfluorophosphate (DFP) under certain conditions. The present investigation showed that there was a corresponding loss of the CMB-titratable SH group and enzymic activity with increasing concentration of DFP added. This corroborated the work of Gould and Liener (Biochemistry, 1, 349, 1965) who reported that an impurity in the DFP preparation reacted with the essential SH group of ficin thus inactivating the enzyme. However, cyanide-activated chymopapain was phosphorylated by DFP (organic phosphorus/protein, 0,89, mole/mole). Unactivated chymopapain was not phosphorylated under the same conditions. The site of phosphorylation was the hydroxyl group of a serine residue. The partial structure of the phosphorylated peptide from tryptic digest of DIP-chymopapain was Ser-Gly(Cys,Asp,Thr,Ser,Glu,Ala)-Lys Methylene-blue catalyzed photooxidation of CMB-chymopapain resulted in the loss of all three histidine residues of chymopapain with only 50% loss of enzymic activity. A tentative mechanism for the reaction between chymopapain and substrate is proposed on the basis of the available data.
Description: Typescript.
Thesis (Ph. D.)--University of Hawaii, 1966.
Bibliography: leaves 135-139.
vi, 139 l illus
Rights: All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Appears in Collections:Ph.D. - Biomedical Sciences (Biochemistry)

Items in ScholarSpace are protected by copyright, with all rights reserved, unless otherwise indicated.