PURIFICATION AND CHARACTERIZATION OF PAPAYA GLUTAMINE CYCLOTRANSFERASE, A PLANT ENZYME HIGHLY RESISTANT TO CHEMICAL, ACID AND THERMAL-DENATURATION

Papaya glutamine cyclotransferase (PQC), present in the laticiferous c ells of the tropical species Carica papaya, was purified near to homog eneity. Starting from the soluble fraction of the collected plant late x, a combination of ion-exchange chromatography on SP-Sepharose Fast F low, hydrophobic interaction chromatography on Fractogel TSK Butyl-650 and affinity chromatography on immobilized trypsin provided a purific ation factor of 279 with an overall yield of 80%. In the course of the purification procedure, the two solvent accessible thiol functions lo cated on the hydrophobic surface of the enzyme were converted into the ir S-methylthioderivatives. Papaya QC, a glycoprotein with a molecular mass of 33 000 Da, contains a unique and highly basic polypeptide cha in devoid of disulfide bridges as well as of covalently attached phosp hate groups. Its absorption spectrum is dominated by the chromophores tyrosine which, nonetheless, do not contribute to the fluorescence emi ssion of the plant enzyme. With a lambda(max) of emission at 338 nm an d a moderate susceptibility to be quenched by acrylamide, most of the tryptophyl residues of papaya QC appear to be sterically shielded by s urrounding protein atoms. Fluorescence can thus be used to monitor unf olding of this enzyme. Preliminary experiments show that papaya QC is exceptionally resistant to chemical (guanidinium hydrochloride), acid and thermal denaturation. At first sight also, this enzyme exhibits hi gh resistance to proteolysis by the papaya cysteine proteinases, yet p resent in great excess (around 100 mol of proteinases per mol of PQC) in the plant latex. Altogether, these results awaken much curiosity an d interest to further investigate how the structure of this plant enzy me is specified. (C) 1998 Elsevier Science B.V. All rights reserved.