Jeanne Brygier

Reversible modification of thiol-containing polypeptides with poly (ethylene glycol) through formation of mixed disulfide bonds. The case of papaya proteinase III.

Papaya proteinase III (PPIII) was purified, as the S-methylthio derivative from the latex ofCarica 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.

Potentiation of oxygen toxicity by menadione in Saccharomyces cerevisiae

The cytotoxicity of molecular oxygen can be sharply increased in the yeast Saccharomyces cerevisiae by the use of redox compounds capable of shunting electrons in vivo and of spontaneous reoxidation under aerobic conditions. Among these redox compounds, menadione (Vitamin K3) is particularly able to stimulate the cyanide-resistant respiration of the yeast cells. Under steady-state conditions, the efficiency of menadione is modulated by the physiological state of the yeast cells and also depends on the availability of reducing agents within the cell. Menadione shows lethal effects towards yeast cells in the presence of O2 only, as a result of the production of toxic metabolites like O2-. and H2O2 which are actually detected in the extracellular fluid. Inhibitors of the enzymes scavenging O2-. and H2O2 generally potentiate the lethal effects of this redox compound. On the other hand, superoxide dismutase and/or catalase supplemented into the incubation buffer have been found to protect the cells to various extents from the cytotoxic effects of menadione. Our data support the following conclusions: When the cellular enzymatic defences are functional, the moderate lethality induced by menadione is principally mediated by O2-. ions acting on the outer side of the cell (peripheral region). In the presence of cyanide, but not of azide, the loss of viability also results from additional damage occurring within the inner cell region. In this case, intracellular injury can be caused by H2O2 alone but our data also suggest that during redox cycling more reactive species--O2-. and probably OH.--are generally intracellularly and are involved in the cytotoxic process.

Preparation and characterization of a S-monomethoxypoly-(ethylene glycol) thioderivative of papain

Abstract A derivative of papain in which the free thiol group of the proteinase is attached to a poly(ethylene glycol) chain via a disulphide bond has been prepared. The poly(ethylene glycol) chain confers to the conjugate new physico-chemical properties, including a looser binding to ion exchange chromatographic supports such as CM-Sephadex C-50. The conjugate is thus readily separated from the irreversibly oxidized (unconjugated) forms of the enzyme. A papain preparation titrating 0.97 mol of thiol per mol of enzyme is obtained from the conjugate.

Covalent attachment of poly(ethyleneglycol) to peptides and proteins - Reevaluation of the synthesis, properties, and usefulness of carbonylimidazol-1-yl-methoxypoly(ethyleneglycol)

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.

Preliminary characterization of bovine beta-lactoglobulin after its conjugation to polyethylene glycol.

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.

Preparation and preliminary characterization of poly(ethylene glycol)-pepstatin conjugate.

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.

Acylation of amino functions of proteins with monomethoxypoly (ethylene glycol)-N-succinimide carbonate

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.

Sulfhydryl groups of chicken hemoglobins. Effect of the reaction with para-mercuribenzoate on subunits dissociation and oxygen affinity

Summary After quantitative reaction of the sulfhydryl groups of chicken hemoglobins with para mercuribenzoate, α and β chains cannot be separated by chromatography from either major or minor component. Ultracentrifugations show that dissociation into dimers occurs, but at protein concentrations much lower than those observed for mammalian hemoglobins. Oxygen equilibrium is affected: cooperative effects are weakened and oxygen affinity increases, the amplitude of the alteration being different for each component in presence or in absence of inositol hexaphosphate. Comparison with the behaviour of some mammalian hemoglobins is discussed.