Rolf J. Mehlhorn

The free radical theory of aging: A critical review

Abstract Although the free radical theory of aging was proposed several decades ago the involvement of radicals in the aging process remains obscure. Considerable progress has been made in detecting oxygen free radicals in biological environments and such radicals are now known to be generated during a variety of metabolic processes. The failure to achieve substantial lifespan extensions with antioxidants casts doubt on the validity of the theory as originally formulated. Further doubts about the theory arise from studies with cultured cell aging models, which fail to exhibit an expected sensitivity to the oxygen concentration in their growth environments. Only one nutritional manipulation, caloric restriction, is known to exert substantial life-extending effects; however its relationship to free radicals has not been resolved. Thus, present knowledge does not argue for a predominant role of free radicals in aging. However, compelling evidence exists for the involvement of free radicals in life-shortening diseases, including autoimmunity, cancer, atherosclerosis, and Parkinsons disease. Further studies of the effects of normally-occurring free radicals are warranted; quantitative data on damage associated with these species may reveal that previous analyses failed to identify critical cellular targets.

[89] Measurements of volumes and electrochemical gradients with spin probes in membrane vesicles

Publisher Summary This chapter discusses the measurements of volumes and electrochemical gradients with spin probes in membrane vesicles. Volumes and electrochemical gradients in cells and vesicles can be measured by several available methods. It is important to employ at least two independent techniques for measuring these parameters to avoid misinterpretation of data because of artifacts. Spin probe techniques complement traditional methods which focus upon extravesicular and extracellular water. By observing probe concentrations inside sealed membrane systems, these methods provide an important check on other techniques in terms of criteria of complementarity—that is, whether inferred changes of electrochemical potentials on two sides of a membrane are opposite and equal after correcting for volume differences. Spin probes also give some unique information: volume measurements are independent of assumptions of cell shape; probe binding is quantitated and corrected for; and with several probes, kinetic measurements appear to be feasible.

Mutagenicity of nitroxide-free radicals.

Stable nitroxides were found to be mutagenic using Salmonella typhimurium tester strain TA 104, a strain chosen on the basis of its high sensitivity to oxidative damage. Nitroxide mutagenicity was dramatically increased in the presence of the superoxide radical generating system, xanthine oxidase/hypoxanthine, and it was suppressed in cells carrying the oxyR1 mutation, which causes induction of enzymes protecting against oxidative stress. As nitroxide-free radicals occur biologically, e.g., in the metabolism of aromatic amines, these radical-induced mutations could be a model for the carcinogenicity observed with these compounds.

[25] Electron spin resonance spin destruction methods for radical detection

Publisher Summary It is important to establish only whether or not free radicals are involved in some reaction. Nitroxides can be useful for this purpose if it can be shown that loss of an electron spin resonance (ESR) signal occurs and that the nonreoxidizable reaction product is not the secondary amine, R 2 NH. Nitroxides (R 2 NO:) react rapidly with many free radicals to form diamagnetic products and thus have potential utility for studies of free radical reactions. To ensure efficient trapping––that is, maximal accumulation of free radicals by nitroxides––it is advisable to avoid competing formation of peroxy radicals by conducting experiments anaerobically. A convenient procedure for performing such experiments in an erythrocyte sedimentation rate (ESR) instrument is to place the reaction mixture into gas permeable tubing under a stream of nitrogen gas. This procedure proved useful for light-generated radicals when the reaction mixture was alternately made aerobic and anaerobic to consume all of the hydroxylamine reaction products that can accumulate via electron- or hydrogen-transfer reactions. Nitroxides can also provide evidence for free-radical chain reactions involving peroxy intermediates if it can be shown that oxygen consumption in an aerobic process is decreased in the presence of nitroxides. The nitroxide concentration should exceed that of oxygen by a factor of about 10 to compete effectively for reactive radicals.

MOLECULAR ASPECTS OF LIGHT‐INDUCED UPTAKE AND RELEASE OF PROTONS BY PURPLE MEMBRANES

Abstract— The precise molecular description of the time dependent steps in the uptake, translocation and release of protons by bacteriorhodopsin following photon absorption requires information on the time resolved changes in protonation of the side chains of specific amino acid residues and the correlation of these changes with photocycle kinetics. Thus far, the use of chemical modification to probe the role of amino acid side chains in this process has proven of value in demonstrating a role for tyrosine residues in release and uptake of protons associated with early and later stages (before and after M412 formation) of the photocycle. In addition, it has demonstrated the essential role of ionic interactions between negatively charged carboxyl groups and positively charged guanidinium groups of arginine, and amino groups of lysine. The transmembrane regulatory effect of ΔμH+ on the M412 species of the photocycle provides additional evidence for the participation of reversible protonation of amino acid side chains at the surfaces of the purple membrane in the mechanism of proton translocation. Thus, our studies relate molecular events of proton translocation to the bioenergetics of the purple membrane.

[17] Preparation of tocopheroxyl radicals for detection by electron spin resonance

Publisher Summary This chapter discusses the preparation of tocopheroxyl radicals for detection by electron spin resonance (ESR). In principle, ESR analysis of the perturbation of free radical signals by paramagnetic ions or the perturbation of nuclear magnetic resonance (NMR) signals of fatty acyl chain residues are tools that can be used for studying the location of the tocopheroxyl radical in the membrane–lipid bilayer. ESR studies can also be used to analyze the kinetics of radical formation and decay, and this approach is yielding valuable information about processes that allow tocopherol to act catalytically to scavenge lipid free radicals. A general principle governing optimal tocopheroxyl production is that the oxidation system should efficiently produce the one-electron oxidation product of tocopherol but should not involve molecular species that can readily react with the resulting tocopheroxyl radical. Operationally, particularly for studies of the tocopheroxyl radical in liposomes and natural membranes, a suitable oxidations system is one that produces ESR signals of sufficient magnitude to be quantitated and that allows the tocopheroxyl radical to be observed for prolonged periods in the absence of reducing agents or molecular species that can react with it. In solvents, even inefficient oxidation methods or drastic oxidation procedures that rapidly destroy the tocopheroxyl radical as soon as it is formed may be adequate to produce detectable tocopheroxyl radicals, provided the solvents allow the high concentrations of tocopherol to be homogeneously dispersed in them. For this reason, the experiments of tocopherols in solvent permit much more latitude in the choice of oxidation systems than experiments with aqueous membranes, where far lower tocopherol concentrations are generally attainable.

[55] Measurement of transmembrane proton movements with nitroxide spin probes

Publisher Summary This chapter explores the way nitroxide spin probes employed to study the permeability of halobacterial envelope vesicle and erythrocyte membranes to spin-labeled weakly polar molecules, ions, amines, and weak acids. Membrane proton pumps, such as bacteriorhodopsin, generate electrochemical potentials that can be measured with a combination of probes responsive to transmembrane electrical and pH gradients. Weakly polar spin probes with fewer than two hydrogen-bonding residues permeate most membranes too rapidly to measure with available stop-flow electron spin resonance (ESR) techniques. One of the nitroxides, 4-oxo-2,2,6,6,-tetramethylpiperidine- N -oxyl (TEMPONE), is used to measure volume changes in many envelope and cell preparations and permeates at least one cell appreciably more rapidly than water does. Improved biophysical techniques are needed that will give higher resolution in space and time of the initial cascade of energy-conserving reactions or structural changes. The spin-probe approach for improving the kinetics and spatial resolution of the measurements of electrochemical potentials across biological membranes is discussed in the chapter. It also discusses the volume measurement technique, proton gradients, and electrical potentials. Phosphonium probes, which excessively slow diffusion, invariably distort measurements. The syntheses of other hydrophobic spin-labeled ions with the goal of improving the permeability constants, and thus reducing errors are required.

New amino-nitroxide spin labels.

Stable free mono- and diradicals containing reactive primary or secondary amino groups in the side-chain have been synthesized by transesterification of amino-substituted esters with paramagnetic alcohols or from spin-labeled acid derivatives and amines. In the second approach the new radical 18 (1-oxyl-3-(2-bromoethoxycarbonyl)-2,2,5,5-tetramethylpyrroline) is proposed as an efficient alkylating species. The nitroxides described are pH-sensitive spin probes and spin labels potentially useful for a diversity of ESR applications in chemistry and biology. New spin-labeled tyramine 16 (N-(1-oxyl-3-carbonyl-2,2,5,5-tetramethyl-pyrroline)tyramine) was successfully employed in a novel assay of protein oxidative damage.

[73] Assay for free radical reductase activity in biological tissue by electron spin resonance spectroscopy

Publisher Summary This chapter describes an assay for free radical reductase activity in biological tissue by electron spin resonance (ESR) spectroscopy. The assay is based on one-electron reduction of nitroxide radicals. This assay has been applied to epidermis and skin homogenares, because the skin is a readily accessible tissue and a target organ of oxidative stress that is subjected to a variety of pathologic conditions that have been associated with free radical processes. The epidermal surface of 4 mm skin biopsies is treated with 2 μl of a 10 mM solution of nitroxide, incubated for 5 minutes, and washed for 15 seconds with isotonic saline. Subsequently, the sample is immediately transferred to the electron paramagnetic resonance (EPR) cavity and the nitroxide spectrum is recorded. Nitroxide uptake into the epidermal membranes varies considerably and is roughly a function of the octanol–water partition coefficient. The epidermis is pretreated with skin-permeable thiol group inhibitors, such as N-ethylmaleimide (NEM), treated with zinc sulfate or heated briefly. A heat-, zinc-, and NEM-sensitive free radical reducing component is identified during the assay in the epidermis with the membrane-binding cationic nitroxide CAT-4 (2,2,6,6-tetramethyl-1-piperidinoxy-4-N,N-dimethyl-N-butylammonium bromide). Results confirm a free radical reductase at the surface of the epidermis.

[54] Measurement of bioenergetics phenomena in cyanobacteria with magnetic resonance techniques

Publisher Summary This chapter deals with the application of magnetic resonance methods to the problem of cyanobacterial bioenergetics, particularly as it pertains to mechanisms of salt tolerance. Electron spin (ESR) and nuclear magnetic (NMR) resonance methods can yield information about intracellular concentrations of organic and inorganic solutes without cell disruption. ESR provides substantially greater sensitivity than NMR, but relies on probes, which may adversely affect structure or function. NMR detects molecules and ions that are present naturally, or employs isotopes of natural molecules that do not perturb biological structure significantly. Thus, each method has its advantages. For organic solutes being observed with carbon-13 NMR, sensitivity is greatly enhanced with enrichment of this isotope. Enrichment provides the added advantage that pulse-chase strategies can be used to monitor carbon turnover processes. The ESR method has recently been expanded to include measurements with multiple probes that are sensitive to pH; this has raised the possibility of determining the pH values of several subcellular compartments and, in principle, the volumes of these compartments in unbroken cells.