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 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.

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 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.

Inhibition studies in situ of yeast catalases.

The catalase activity of the intact yeast cells towards external substrate is generally lower than the 'cryptic' activity which is revealed after cell lysis. The physiological basis for the reduced catalytic activity of the intact cell ('patent' activity) has been investigated by establishing the inhibition profiles of catalases in situ using selected probes; to this end we utilized either non-penetrating acids and/or catalase poisons able to cross the plasmic membrane. Owing to the peculiar features of the reaction mechanism, competitive inhibitors, which are known to interact with the prosthetic group of catalases, show an efficiency that is unlinked to the hydrogen peroxide concentration under the usual assay conditions ([H2O2] much less than Km). This mode of interaction, which also characterizes the action of the penetrating probes HCOOH and HCN, is particularly well adapted to the study of the behaviour of the cytoplasmic catalases in situ. By this experimental approach, it has been shown that the catalase of the inner cellular region contributes, together with an isoenzyme present at the cell surface, to the patent activity. The mathematical processing of the data, which takes into account a rate-limiting diffusion of external substrate into the intact yeast cell, has allowed us to predict accurately the resulting apparent efficiency of inhibitors as a function of the physiological variations of the intracellular enzyme concentration.

The interaction of organic phosphates with human and chicken hemoglobin.

In this study of the binding properties of inositol hexaphosphate and 2,3-bisphosphoglycerate to chicken and human deoxyhemoglobin and carboxyhemoglobin were compared. It appeared that in all cases the binding to chicken hemoglobin is much stronger than to human hemoglobin. This is very probably due to the fact that 4 out of the 12 residues, responsible for the binding of phosphates in chicken hemoglobin, are arginines. These are absent in human hemoglobin, where the binding site is made up to only 8 residues. For chicken hemoglobin one strong binding site could be observed in both unliganded and liganded hemoglobin. From these observations we conclude that the same binding site is involved in both the oxy- and deoxy structure showing different affinity to phosphates in the two conformational states. For human hemoglobin we reached the same conclusion.

Quantitative determination of polyethylene glycol based upon its salting out and partitioning of a dye into the resulting aqueous two-phase system.

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.

Under proper control, oxidation of proteins with known chemical structure provides an accurate and absolute method for the determination of their molar concentration.

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.

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.