[Show abstract][Hide abstract] ABSTRACT: In the tropical species Carica papaya, the articulated and anastomosing laticifers form a dense network of vessels displayed in all aerial parts of the plant. Damaging the papaya tree inevitably severs its laticifers, eliciting an abrupt release of latex. Besides the well-known cysteine proteinases, papain, chymopapain, caricain and glycyl endopeptidase, papaya latex is also a rich source of other enzymes. Together, these enzymes could provide an important contribution to plant defence mechanisms by sanitising and sealing the wounded areas on the tree.
Cellular and Molecular Life Sciences CMLS 05/2001; 58(4):556-70. · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oxidation at 120 degrees C of inorganic and organic (including amino acids, di- and tripeptides) model compounds by K(2)Cr(2)O(7) in the presence of H(2)SO(4) (mass fraction: 0.572), Ag(2)SO(4) (catalyst), and HgSO(4) results in the quantitative conversion of their C-atoms into CO(2) within 24 h or less. Under these stressed, well-defined conditions, the S-atoms present in cysteine and cystine residues are oxidized into SO(3) while, interestingly, the oxidation states of all the other (including the N-) atoms normally present in a protein do remain quite unchanged. When the chemical structure of a given protein is available, the total number of electrons the protein is able to transfer to K(2)Cr(2)O(7) and thereof, the total number of moles of Cr(3+) ions which the protein is able to generate upon oxidation can be accurately calculated. In such cases, unknown protein molar concentrations can thus be determined through straightforward spectrophotometric measurements of Cr(3+) concentrations. The values of molar absorption coefficients for several well-characterized proteins have been redetermined on this basis and observed to be in excellent agreement with the most precise values reported in the literature, which fully assesses the validity of the method. When applied to highly purified proteins of known chemical structure (more generally of known atomic composition), this method is absolute and accurate (+/-1%). Furthermore, it is well adapted to series measurements since available commercial kits for chemical oxygen demand (COD) measurements can readily be adapted to work under the experimental conditions recommended here for the protein assay.
[Show abstract][Hide abstract] ABSTRACT: Papaya glutamine cyclotransferase (PQC), present in the laticiferous cells of the tropical species Carica papaya, was purified near to homogeneity. Starting from the soluble fraction of the collected plant latex, a combination of ion-exchange chromatography on SP-Sepharose Fast Flow, hydrophobic interaction chromatography on Fractogel TSK Butyl-650 and affinity chromatography on immobilized trypsin provided a purification factor of 279 with an overall yield of 80%. In the course of the purification procedure, the two solvent accessible thiol functions located on the hydrophobic surface of the enzyme were converted into their S-methylthioderivatives. Papaya QC, a glycoprotein with a molecular mass of 33 000 Da, contains a unique and highly basic polypeptide chain devoid of disulfide bridges as well as of covalently attached phosphate groups. Its absorption spectrum is dominated by the chromophores tyrosine which, nonetheless, do not contribute to the fluorescence emission of the plant enzyme. With a λmax of emission at 338 nm and a moderate susceptibility to be quenched by acrylamide, most of the tryptophyl residues of papaya QC appear to be sterically shielded by surrounding protein atoms. Fluorescence can thus be used to monitor unfolding 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 high resistance to proteolysis by the papaya cysteine proteinases, yet present in great excess (around 100 mol of proteinases per mol of PQC) in the plant latex. Altogether, these results awaken much curiosity and interest to further investigate how the structure of this plant enzyme is specified.
Biochimica et Biophysica Acta 10/1998; · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A class II chitinase is present in the latex of the tropical species Carica papaya. The enzyme may be readily purified by using a combination of hydrophobic interaction- and cation-exchange chromatography. This enzyme preparation is homogeneous with respect to the three physico-chemical criteria of charge, M(r) (28,000) and hydrophobicity. It is also completely free of any proteolytic and bacteriolytic activities. The enzyme was classified as a class II chitinase on the basis of its N-terminal amino acid sequence up to the 30th residue. In agreement with this classification, the enzyme preparation hydrolyses chitinase substrates only very slowly and several free thiol functions are present in the polypeptide chain. These free thiol functions are buried, and to be available for titration with 2,2'-dipyridyldisulphide, the enzyme must be denatured. Unfolding of papaya chitinase requires particularly drastic conditions, not less than 4 M guanidinium hydrochloride at 25 degrees and pH 6.8.
[Show abstract][Hide abstract] ABSTRACT: The major component of the whey fraction of bovine milk, beta-lactoglobulin (betaLG), has been transformed by grafting polyethylene glycol chains either on the thiol group (free and after reduction of the S-S bridges) of the cysteine residues, or on the amino group of the lysine residues and/or of the N-terminal amino acid. Acylation of the protein was achieved at a controlled pH of 7.0 using increasing ratios of activated PEG to betaLG. Transformation of the dimeric form into the monomer occurred at least for the fully pegylated adduct. The number of polymer chains fixed per mole of protein was determined by dosage of the free amino functions still present after reaction. The incidence of pegylation on the secondary structure of betaLG was evaluated using the Fourier Transform Infrared Spectroscopy (FTIR). Denaturation studies with guanidinium hydrochloride (Gu-HCl) by means of spectrofluorimetric measurements, showed an identical behavior of native as well as of pegylated betaLG.The antigenicity of the fully pegylated adduct was examined through antigenic competition towards native betaLG. The pegylated protein exhibited less than 1/100 of the native betaLG inhibition capacity, that could moreover never be complete. This is thus demonstrating the loss of accessibility for at least multiple conformational epitopes through pegylation procedure.Spectrofluorimetric measurements showed that betaLG-N-PEG(7) was still able to bind retinol while no effect on the intrinsic fluorescence could be detected by adding palmitic acid. Whether this last ligand binds or not to pegylated betaLG is discussed.
Biotechnology and Bioengineering 04/1997; 54(1):40-9. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Papaya proteinase III (PPIII) was purified, as the S-methylthio derivative from the latex of Carica papaya L., by ion-exchange chromatography. Separation of reactivable PPIII from the irreversibly oxidized molecular species of this enzyme was readily achieved after a selective conversion of the reactivated proteinase into the S-monomethoxypoly(ethylene glycol)thio derivative (S-mPEG thio PPIII). From this derivative, a PPIII preparation titrating 1 mol of thiol/mol of enzyme was regenerated. From the physicochemical properties of S-mPEG thio PPIII that were investigated, it is concluded that interactions between the mPEG and the PPIII chains occur only to a limited extent. In addition to its usefulness for purifying thiol-containing enzymes, the mPEG modification resulting from mixed disulfide bond formation may find other practical applications.
Applied Biochemistry and Biotechnology 04/1996; 56(3):243-63. · 1.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Papain is widely used in several laboratories as a reference enzyme and, as such, needs to be highly pure. A one-step purification of the enzyme is described here starting from spray-dried papaya latex. After protection of the essential SH group with a monomethoxypolyethylene glycol derivative synthesised in our laboratory, the mixture of papaya enzymes is submitted to fractionation on S-Sepharose Fast Flow. This procedure leads to the isolation of the S-pegylthiopapain conjugate devoid of any contamination nor by the enzyme's irreversibly oxidized form nor by one of the other proteinases present in the original mixture. Thereafter, S-pegylthiopapain may be quite easily converted into its fully active form.
Journal of Chromatography A 01/1996; 724:185-192. · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The four cysteine proteinases, papain, chymopapain, caricain, and endoproteinase Gly-C were isolated and purified as the catalytically
competent species from the commercially available latex of the tropical treeCarica papaya L. Their free thiol function (cysteine-25), which is essential for activity, was protected in the form of a mixed disulfide
containing a 5 kDa polyethylene glycol (PEG) chain. The second (nonessential) free thiol function (cysteine-117) of chymopapain
was blocked similarly. Caricain was also derivatized through acylation of its amino functions by PEG chains (average: 15 moles
of PEG per mole of enzyme). The Chromatographic behavior of these conjugates was examined on ion-exchange and hydrophobic
gels and compared to the Chromatographic behavior of the unpegylated proteinases. The results indicated that charge-shielding
effects by PEG chain(s), surrounding the different proteinases, plays(play) a key role in the course of separation of pegylated
and unpegylated species by ion-exchange chromatography. Similarly, PEG chain(s) is(are) able to mask hydrophobic regions on
the surface of the proteinases. However, the affinity showed by PEG itself for the hydrophobic ligands immobilized on the
matrix is the preponderant factor determining the behavior of the PEG-proteinases conjugates on Fractogel TSKButyl-650.
Applied Biochemistry and Biotechnology 01/1996; 60(2):167-183. · 1.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A method is described that allows quantitative determination of polyethylene glycol (PEG) concentrations by spectrophotometric measurement of fluorescein dye absorbance after its partitioning into an aqueous two-phase system containing mPEG (M(r) 5 kDa) in the upper phase and ammonium sulfate in the lower phase. The absorbance decrease of fluorescein in the lower phase is directly proportional to the mPEG concentration, with two proportionality constants equal to 4.42 x 10(5) and 2.84 x 10(5) M-1 cm-1 in the range of 0-0.4 and 0.4-1 microM, respectively. This experimental technique can be extended to PEGs of other molecular weights by means of calibration curves that give for each size of PEG the adequate proportionality constants. The results indicate that the quantitative determination is not affected by the presence of many substances such as proteins, reducing agents, and salts, at the usual concentrations.
[Show abstract][Hide abstract] ABSTRACT: The carboxyl function of pepstatin has been coupled, through an amide bond, to methoxypoly(ethylene glycol) (5 kDa), to which an amino function had been previously grafted. The mPEG-pepstatin conjugate inhibits hog pepsin (aspartic proteinase) in vitro as pepstatin itself, however, with a 400 times higher apparent Ki. The conjugate apparently does not inhibit proteinases belonging to other proteinase families such as serine (trypsin, carboxypeptidase Y), cysteine (Papaya proteinase III), or metallo (collagenase) proteinases.
Applied Biochemistry and Biotechnology 05/1994; 47(1):1-10. · 1.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Carbonylimidazol-l-yl-mPEG is obtained quantitatively by reacting methoxypoly(ethyleneglycol) (mPEG) with 1.1 Eq of N,N′-carbonyldiimidazole
in the presence of a stoechiometric amount of 4-dimethyl-aminopyridine used as hypernucleophilic acylation catalyst. Carbonylimidazol-l-yl-mPEG is quite stable in aqueous solutions with half-lives up to 70 h in pHs ranging from 6.0 to 7.0 at 25‡C. From reactivity
studies toward amines with various nucleophilic strengths, it is suggested that carbonylimidazol-l-yl-mPEG may be best used to modify α-amino terminal function of proteins selectively or to introduce amino function on PEG chains.
Applied Biochemistry and Biotechnology 07/1993; 42(2):127-135. · 1.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Monomethoxypoly(ethylene glycol)-N-succinimide carbonate (SC-PEG) was used to prepare PEG-lysozyme, PEG-papaya proteinase III, PEG-catalase, and PEG-lactoperoxidase
conjugates. SC-PEG produced extensively modified enzymes under mild conditions (pH 7.0; 25°C) within a couple of hours. PEG-enzyme
conjugates showed equal or even greater specific activity provided that low-molecularweight substrates were used to evaluate
the biological activities. However, papaya proteinase III and lysozyme lost their proteolytic and bacteriolytic activities,
respectively, on conjugation with PEG. This was most probably because of steric factors, since no drastic conformational changes
could be detected after conjugation of these enzymes with PEG chains. Unlike these enzymes, the secondary structures of the
two hemoproteins were somewhat affected by the covalent attachment of PEG chains as shown by FTIR experiments. These results
confirmed the potential usefulness of SC-PEG, for which a novel route of synthesis making use ofN,N′-disuccinimidyl carbonate was described.
Applied Biochemistry and Biotechnology 49(1):75-91. · 1.89 Impact Factor