Standard midpoint potentials have been determined for p-benzoquinone, methoxy-p-benzoquinone and 2,3-, 2,5-, and 2,6-dimethoxy-p-benzoquinones in aqueous solution. ESR studies have been made of the ascorbate and semiquinone radicals produced when these quinones interact with sodium ascorbate. Direct correlations are found between the electrochemical potentials, generated semiquinone lifetimes, and cytotoxic action in Ehrlich ascites-bearing mice.This work follows from earlier considerations of the oxidationreduction and cytotoxic properties of some methoxyquinones (1) and ofcharge-transfer reactions and electronic delocalization effects in biological systems (2-4). The compounds of interest in these particular studies have been the methoxy-substituted p-quinones, two ofwhich (methoxy-and 2,6-dimethoxy-p-benzoquinone) occur naturally in wheat germ (5).Bachur et aL (6) have suggested that the cytotoxic action of quinone anticancer drugs is mediated through their free-radical metabolites. An important first step in such activity is the production of relatively stable semiquinone free radicals. Radicals result from one-electron rather than two-electron redox reactions and the radical concentration and rate ofproduction should depend on the difference between the electrochemical potentials of the electron donor and acceptor molecules involved. In this work, electrochemical and ESR measurements have been made to determine the correlation between the cytotoxic activity of several quinone/ascorbate mixtures and their ability to produce stable semiquinone radicals. MATERIALS AND METHODSPure samples of p-benzoquinone, methoxy-p-benzoquinone, and the three dimethoxy-(2,3-, 2,5-, 2,6-) p-benzoquinones, as well as the corresponding hydroquinones, were supplied by G. Fodor (West Virginia University, Morgantown).The redox potentials of the various quinone-hydroquinone couples were determined at 25°C by using electrodes of bright platinum wire (24 gauge). These were subjected to a nonoxidizing cleaning procedure (7) by placing them for 10 min in boiling 10% sodium bisulfite and then washing in distilled water. The reference half-cell consisted of a saturated solution ofquinhydrone (Eastman Kodak) in nitrogen-saturated aqueous 0.01 M HC1/0.09 M KC1, and a 0.5 M KC1/5% agar salt bridge was used as the connection to the test half-cell. The combined electrode and salt-bridge junction errors were measured to be less than 0.2 mV and, when calibrated against a platinized platinum/hydrogen electrode, the standard potential (pH 0) of the quinhydrone reference electrode was determined as 700 ± 1 mV, in good agreement with the values 699.58 mV and 699.92 mV obtained by earlier workers (8, 9). The electrode potentials were measured by using a Keithley 616 digital electrometer (input impedance > 0.1 PfQ) that had been calibrated to within ±0.1 mV by using a Data Precision (model 8100) voltage calibrator. Fresh quinhydrone reference electrodes were prepared for each new experiment, which typically took less than 3 hr to perform. ...
Many problems are left open in this article. Its publication may be excused by the suffering cancer causes, which urges the researcher to publish as soon as he thinks he may have found a new trail, which also may be taken by others. What emerges clearly is that SH groups, with their specific reactivities, offer a hopeful target in the search for cancerostatic substances, among which the natural repressor of cell division may hold out the most promise. The glyoxal derivatives also have antiviral properties (7, 16) and may be in the center of a hitherto unknown system of equilibria which deserves a thorough study. The low molecular weight of the glyoxal derivative reported justifies the hope of an early clarification of its structure, as well as its synthesis (17).
Spectroscopic measurements are reported for the effects of pH, time, solvent, and chemical modification of arginine and lysine side chains on the reaction of proteins with methylglyoxal. The reaction responsible for the appearance of a brown coloration and increased submolecular electronic activity in the proteins involves the e-amino groups of the lysine residues. It is concluded that the primary step in the reaction involves the formation of a Schiff base linkage between the lysine side chain and methylglyoxal. These findings reaffirm the concept that, by the formation of Schiff bases, aldehydes can act as electron acceptors in charge transfer interactions with proteins.Considerations (1, 2) of the possible biological role of aldehydes and ketones in acting as electron acceptors for proteins have led us to study the interactions of proteins with methylglyoxal. These studies have shown that when proteins such as casein, collagen, bovine serum albumin, and lysozyme react with methylglyoxal they assume a stable brown color and exhibit a greatly enhanced electronic conductivity and electron spin resonance activity compared with the normal (white) unreacted proteins (3-5).Scintillation counts by W. M. Arnold and J. Behi using 14C-labeled methylglyoxal (personal communication) and weight increase measurements have shown that after reaction with methylglyoxal for 5 days the resulting brown proteins contain on average 8-12% methylglyoxal on a dry weight basis. Amino acid analyses for the adducts of bovine serum albumin and of casein with methylglyoxal, performed by G. E. Siefring, Jr., at Northwestern University (personal communication) indicate that the arginine (;z100% reacted) and lysine residues (80-90% reacted) are the ones that react most strongly with methylglyoxal. We have blocked the arginine side chains of albumin with cyclohexanedione, using the procedure described by Smith (6), and this has produced no observable difference in the way the protein reacts with methylglyoxal to produce the brown color and electronic activity. However, when the E-amino groups of the lysine side chains of albumin and casein have been reductively dimethylated by the method of Lin et al. (7), upon reaction with methylglyoxal the proteins remain white and do not exhibit enhanced electronic activity. These results indicate that the reaction of particular importance in these studies involves the protein lysine residues, and the following spectroscopic studies were made as a step toward understanding the nature of this reaction. METHODSMeasurements were obtained by using a dual beam Beckman model 35 spectrophotometer and quartz sample cells (optical pathlength, 1 cm). For the spectra described here, 0.15 mM bovine serum albumin was allowed to interact with approxi- mately 10 mM methylglyoxal in water at 297 + 1 K; and the reference system consisted of an aqueous solution of methylglyoxal at the same concentration and pH as in the albumin/ methylglyoxal sample. The bovine serum albumin (fraction V) was obtained from Sigma. The...
The rate of quenching by Ehrlich ascites cells of anionic 2,6-dimethoxy-p-semiquinone and ascorbyl free radicals is investigated as a function of cell concentration, the blocking of cell-surface sulfhydryl groups by N-ethylmaleimide, and the reduction of cell-surface charge by neuraminidase. The rate of quenching is found to be proportional to cell viability and to the number of free cell-surface sulfhydryl groups. The enzymatic action of neuraminidase results in an increase of the free radical quenching rate, indicating that this rate can be used as a probe of cell-surface charge. Measurements as a function of the ionic strength of the suspending electrolyte gave a value of -1.22 ,uC-cm-2 for the charge density at the ascites cell surface. This is equivalent to a surface membrane potential of -14 mV for a 150 mM NaCl electrolyte and is a value in good agreement with published electrophoresis data.Previous studies (1) have shown that a direct correlation exists between the cytotoxic action of quinone/ascorbate mixtures against Ehrlich ascites tumor cells and the ability of such mixtures to produce stable populations of semiquinone and ascorbyl free radicals. These findings were considered to support the proposal by Bachur et al. (2) that the cytotoxic action of quinone anticancer drugs such as adriamycin is mediated through the production of semiquinone free radical metabolites. The main direction of our work follows from earlier considerations of the oxidation-reduction and cytotoxic properties of naturally occurring methoxyquinones (3,4).We report here investigations of the nature and kinetics of the interaction of semiquinone and ascorbyl radicals with Ehrlich ascites cells in physiological media. Evidence has been obtained to show that the rate at which the free radicals are quenched by the ascites cells is a function of cell viability and is controlled by the cell-surface electrical potential. The charge transfer interactions responsible for the free radical quenching appear to be dominated by the presence of cellmembrane sulfhydryl groups. Attention has been given to the 2,6-dimethoxyquinone (2,6-DMQ)/ascorbate mixture as this combination was observed (1) to produce the largest dynamic population of free radicals and the most effective treatment of ascites tumors in mice. MATERIALS AND METHODSFor all the studies reported here, 20 mM sodium ascorbate (Sigma)/superoxide dismutase (2800 units-ml-') (Sigma S-8254) was mixed with a suspension of Ehrlich ascites cells.These cells were obtained from a cell line maintained in our laboratory by transplantation into female CD1 mice (Charles River Breeding Laboratories). A previous determination had been made of cell concentration, and trypan blue and neutral red staining (5) had been used to determine cell viability.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2088Then, 2 mM 2,6-dimethoxy-p-quin...
Two substances, one promoting growth (promine) of ascites tumors in mice and the other inhibiting it (retine) have both been found in several tissues, namely, thymus, aorta, muscle, and tendon. In spite of similar solubilities in the solvents used for their extraction, the substances could be roughly separated. The value of the ratio between these substances in the same tissue may be significant.
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