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Mitochondria are the major source of cellular energy metabolism. In the cardiac cells, mitochondria produce byway of the oxidative phosphorylation more than 90% of the energy supply in the form of ATP, which is utilized in many ATP- dependent processes, like cycling of the contractile proteins or maintaining ion gradients. Reactive oxygen species (ROS) are by-products of cellular metabolism and their levels are controlled by intracellular antioxidant systems. Imbalance between ROS and the antioxidant defense leads to oxidative stress and oxidative changes to cellular biomolecules. Molecular hydrogen ([H.sub.2]) has been proved as beneficial in the prevention and therapy of various diseases including cardiovascular disorders. It selectively scavenges hydroxyl radical and peroxynitrite, reduces oxidative stress, and has anti-inflammatory and anti-apoptotic effects. The effect of [H.sub.2] on the myocardial mitochondrial function and coenzyme Q levels is not well known. In this paper, we demonstrated that consumption of [H.sub.2]-rich water (HRW) resulted in stimulated rat cardiac mitochondrial electron respiratory chain function and increased levels of ATP production by Complex I and Complex II substrates. Similarly, coenzyme [Q.sub.9] levels in the rat plasma, myocardial tissue, and mitochondria were increased and malondialdehyde level in plasma was reduced after HRW administration. Based on obtained data, we hypothesize a new metabolic pathway of the [H.sub.2] effect in mitochondria on the Q-cycle and in mitochondrial respiratory chain function. The Q- cycle contains three coenzyme Q forms: coenzyme Q in oxidized form (ubiquinone), radical form (semiquinone), or reduced form (ubiquinol). [H.sub.2] may be a donor of both electron and proton in the Q-cycle and thus we can suppose stimulation of coenzyme Q production. When ubiquinone is reduced to ubiquinol, lipid peroxidation is reduced. Increased [CoQ.sub.9] concentration can stimulate electron transport from Complex I and Complex II to Complex III and increase ATP production via mitochondrial oxidative phosphorylation. Our results indicate that [H.sub.2] may function to prevent/treat disease states with disrupted myocardial mitochondrial function. Key words: coenzyme [Q.sub.9], energy, mitochondrial respiratory chain, molecular hydrogen, myocardium. Les mitochondries sont la principale source d'energie pour le metabolisme de la cellule. Dans les cellules cardiaques, les mitochondries produisent, grace a la phosphorylation oxydative, plus de 90 % de l'apport energetique sous forme d'ATP utilisee dans de nombreux processus dependant de l'ATP, comme le cycle des proteines contractiles et le maintien des gradients ioniques. Les derives reactifs de l'oxygene proviennent du metabolisme cellulaire et leurs taux sont maintenus par les systemes antioxydants intracellulaires. Un desequilibre entre les derives reactifs de l'oxygene et la defense antioxydante mene a du stress oxydatif et a des modifications oxydatives des biomolecules cellulaires. On a montre que l'hydrogene moleculaire ([H.sub.2]) apporte des bienfaits dans la prevention et le traitement de diverses maladies, y compris les maladies cardiovasculaires. Il piege selectivement le radical hydroxyl et le peroxynitrite, attenue le stress oxydatif, est dote de proprietes anti-inflammatoires et anti- apoptotiques. On ne connait pas bien l'effet de l'[H.sub.2] sur la fonction mitochondriale myocardique et les taux de coenzyme Q. Dans cet article, nous avons montre que la consommation d'eau a teneur elevee en [H.sub.2] entraine la stimulation du fonctionnement de la chaine respiratoire electronique dans les mitochondries cardiaques chez le rat, avec une augmentation des degres de production d'ATP par les substrats Complexe I et Complexe II. De maniere similaire, apres l'administration d'eau a teneur elevee en [H.sub.2] chez le rat, les taux de coenzyme Q9 dans le plasma, ainsi que dans le tissu et les mitochondries myocardiques etaient en hausse, avec un abaissement des taux de malondialdehyde dans le plasma. A partir des donnees obtenues, nous presumons la presence d'une nouvelle voie metabolique de l'effet de l'[H.sub.2] dans les mitochondries sur le cycle Q, ainsi que sur le fonctionnement de la chaine respiratoire mitochondriale. Le cycle Qcomprend trois formes de coenzyme Q: le coenzyme Qsous forme oxydee (ubiquinone), sous forme radicale (semiquinone) ou sous forme reduite (ubiquinol). L'[H.sub.2] pourrait etre un donneur d'electrons comme de protons dans le cycle Q, et nous pouvons donc presumer qu'il stimule la production de coenzyme Q. Quand l'ubiquinone est reduite en ubiquinol, la peroxydation lipidique diminue. L'augmentation de la concentration de CoQ9 peut stimuler le transport des electrons du Complexe I et du Complexe II vers le Complexe III, et faire augmenter la production d'ATP par l'intermediaire de la phosphorylation oxydative mitochondriale. Nos resultats montrent que par son fonctionnement, l'[H.sub.2] pourrait permettre de prevenir ou de traiter des pathologies comprenant des perturbations du fonctionnement des mitochondries myocardiques. [Traduit par la Redaction] Mots-cles: coenzyme Q9, energie, chaine respiratoire mitochondriale, hydrogene moleculaire, myocarde., Introduction Mitochondria play an important role in the physiology of the heart function and in the pathogenesis and development of various types of cardiovascular diseases (CVD) that belong to the [...]

Generation of free radicals through incomplete reduction of oxygen during ischemia-reperfusion (I/R) is well described. On the other hand, molecular hydrogen ([H.sub.2]) reduces oxidative stress due to its ability to react with strong oxidants and easily penetrate cells by diffusion, without disturbing metabolic redox reactions. This study was designed to explore cardioprotective potential of hypoxic postconditioning (HpostC) against I/R (30 min global I-120 min R) in isolated rat hearts using oxygen-free Krebs-Henseleit buffer (KHB). Furthermore, the possibility to potentiate the effect of HpostC by [H.sub.2] using oxygen-free KHB saturated with [H.sub.2] ([H.sub.2] + HpostC) was tested. HPostC was induced by 4 cycles of 1-minute perfusion with oxygen-free KHB intercepted by 1-minute perfusion with normal KHB, at the onset of reperfusion. [H.sub.2] + HPostC was applied in a similar manner using [H.sub.2]-enriched oxygen-free KHB. Cardioprotective effects were evaluated on the basis of infarct size (IS, in % of area at risk, AR) reduction, post-I/R recovery of heart function, and occurrence of reperfusion arrhythmias. HPostC significantly reduced IS/AR compared with non-conditioned controls. [H.sub.2] present in KHB during HPostC further decreased IS/AR compared with the effect of HPostC, attenuated severe arrhythmias, and significantly restored heart function (vs. controls). Cardioprotection by HpostC can be augmented by molecular hydrogen infusion. Key words: ischemia-reperfusion injury, ischemic postconditioning, hypoxic postconditioning, molecular hydrogen, antioxidants. La production de radicaux libres par la reduction incomplete de l'oxygene pendant l'ischemie-reperfusion (I/R) est un phenomene qui est bien decrit. Par ailleurs, l'hydrogene moleculaire ([H.sub.2]) permet de reduire le stress oxydatif en raison de sa capacite de reagir avec des oxydants puissants et de penetrer facilement dans les cellules par diffusion, sans perturber les reactions d'oxydoreduction metabolique. Cette etude a ete concue en vue d'explorer le potentiel cardioprotecteur du postconditionnement hypoxique (postCH) contre l'I/R (I globale de 30 min -Rde 120 min) dans des creurs isoles de rat a l'aide de tampon Krebs-Henseleit (TKH) sans oxygene. En outre, nous avons etudie la possibilite de potentialiser l'effet du postCH par l'[H.sub.2] a l'aide de TKH sans oxygene sature d'[H.sub.2] (postCH + [H.sub.2]). Nous avons obtenu le postCH en procedant a 4 cycles de perfusion d'une minute avec du TKH sans oxygene interceptes par des perfusions d'une minute avec du TKH normal au debut de la reperfusion. Nous avons applique la postCH + [H.sub.2] d'une maniere semblable a l'aide de TKH sans oxygene enrichi d'[H.sub.2]. Nous avons evalue les effets cardioprotecteurs sur la base de la reduction de la taille de l'infarctus (TI, en % de la zone a risque, ZR), de la recuperation de la fonction cardiaque apres l'I/R et de l'apparition d'arythmies de reperfusion. La postCH a permis de reduire le rapport TI/ZR de facon plus marquee que chez les temoins non conditionnes. L'[H.sub.2] present dans le TKH pendant la postCH a permis de diminuer encore plus le rapport TI/ZR qu'avec la postCH, d'attenuer les arythmies graves et de retablir la fonction cardiaque de facon notable (vs. les temoins). En somme, il est possible d'augmenter la cardioprotection par le postconditionnement hypoxique en perfusant de l'hydrogene moleculaire. [Traduit par la Redaction] Mots-cles : lesion d'ischemie-reperfusion, postconditionnement ischemique, postconditionnement hypoxique, hydrogene moleculaire, antioxydants., Introduction The strategies that may improve prognosis of patients after infarction or cardiosurgical interventions are intensively studied. Ischemic preconditioning (IP) induced by short-term ischemic periods has been shown to enhance [...]

We describe the steady-state function of the ubiquitous mammalian Na/H exchanger (NHE) 1 isoform in voltage-clamped Chinese hamster ovary cells, as well as other cells, using oscillating pH-sensitive microelectrodes to quantify proton fluxes via extracellular pH gradients. Giant excised patches could not be used as gigaseal formation disrupts NHE activity within the patch. We first analyzed forward transport at an extracellular pH of 8.2 with no cytoplasmic Na (i.e., nearly zero-trans). The extracellular Na concentration dependence is sigmoidal at a cytoplasmic pH of 6.8 with a Hill coefficient of 1.8. In contrast, at a cytoplasmic pH of 6.0, the Hill coefficient is

The photosynthetic bacterium Rhodobacter capsulatus contains two [NiFe] hydrogenases: an energy-generating hydrogenase, HupSL, and a regulatory hydrogenase, HupUV. The synthesis of HupSL is specifically activated by [H.sub.2] through a signal transduction cascade comprising three proteins: the [H.sub.2]-sensing HupUV protein, the histidine kinase HupT, and the transcriptional regulator HupR. Whereas a phosphotransfer between HupT and HupR was previously demonstrated, interaction between HupUV and HupT was only hypothesized based on in vivo analyses of mutant phenotypes. To visualize the in vitro interaction between HupUV and HupT proteins, a six-His ([His.sub.6])-HupU fusion protein and the HupV protein were coproduced by using a homologous expression system. The two proteins copurified as a [His.sub.6]-HupUHupV complex present in dimeric and tetrameric forms, both of which had [H.sub.2] uptake activity. We demonstrated that HupT and HupUV interact and form stable complexes that could be separated on a native gel. Interaction was also monitored with surface plasmon resonance technology and was shown to be insensitive to salt concentration and pH changes, suggesting that the interactions involve hydrophobic residues. As expected, [H.sub.2] affects the interaction between HupUV and HupT, leading to a weakening of the interaction, which is independent of the phosphate status of HupT. Several forms of HupT were tested for their ability to interact with HupUV and to complement hupT mutants. Strong interaction with HupUV was obtained with the isolated PAS domain of HupT and with inactive HupT mutated in the phosphorylable histidine residue, but only the wild-type HupT protein was able to restore normal [H.sub.2] regulation.

Atom depth, originally defined as the distance between a protein atom and the nearest water molecule surrounding a protein, is a simple but valuable geometrical descriptor of the protein interior. It can be easily computed from the 3D structure of a protein, thus complementing the information provided by the calculation of the solvent accessible surface area and buried surface area. Depth has been found to be correlated with several molecular, residue and atomic properties, such as average protein domain size, protein stability, free energy of formation of protein complexes, amino acid type hydrophobicity, residue conservation and hydrogen/deuterium amide proton exchange rates.

A sulfur reductase (SR) and a hydrogenase were purified from solubilized membrane fractions of anaerobically grown cells of the sulfur-dependent archaeon Acidianus ambivalens and the corresponding genes were sequenced. The SR reduced elemental sulfur with hydrogen as electron donor [45 U [(mg protein).sup.-1]] in the presence of hydrogenase and either 2,3-dimethylnaphthoquinone (DMN) or cytochrome c in the enzyme assay. The SR could not be separated from the hydrogenase during purification without loss of activity, whereas the hydrogenase could be separated from the SR. The specific activity of the hydrogenase was 170 U [(mg protein).sup.-1] with methyl viologen and 833 U [(mg protein).sup.-1] with DMN as electron acceptors. Both holoenzymes showed molecular masses of 250 kDa. In SDS gels of active fractions, protein bands with apparent masses of 110 (SreA), 66 (HynL), 41 (HynS) and 29 kDa were present. Enriched hydrogenase fractions contained 14 [micro]mol Fe and 2 [micro]mol Ni [(g protein).sup.-1]; in addition, 2-5 [micro]mol Mo [(g protein).sup.-1] was found in the membrane fraction. Two overlapping genomic cosmid clones were sequenced, encoding a five-gene SR cluster (sre) including the 110 kDa subunit gene (sreA), and a 12-gene hydrogenase cluster (hyn) including the large and small subunit genes and genes encoding proteins required for the maturation of NiFe hydrogenases. A phylogenetic analysis of the SR amino acid sequence revealed that the protein belonged to the DMSO reductase family of molybdoenzymes and that the family showed a novel clustering. A model of sulfur respiration in Acidianus developed from the biochemical results and the data of the amino acid sequence comparisons is discussed.

Research has been conducted on PS120 cells expressing Na (super)+/H (super)+ exchanger isoforms and their mutants. The study of the hyperosmolarity-induced changes in cell volume and cytoplasmic pH in these cells is described.

The sulfur nutrition of five benthic animals living in or on reductive, tidal-flat sediments has been studied by using the sulfur isotopic signatures of their soft tissues. The mean [delta][sup.34]S values (+6.1 [per thousand] to +13.8 [per thousand], relative to the Canyon Diablo troilite reference) of these animals' tissues are lower than those of many common marine animals, which have values close to those of seawater sulfate-sulfur (+21 [per thousand]). This indicates that these animals use a [sup.34]S-depleted sulfur source, which may be derived from bacterial sulfide in the sediments (less than -20[per thousand]). The animals that inhabit such sediments are adapted to an anoxic environment, where toxic hydrogen sulfide prevails. Due to the sulfide-rich habitat, benthic animals are expected to assimilate hydrogen sulfide directly during detoxification, or to prefer a diet which has a low [delta][sup.34]S signature. Variations in [delta][sup.34]S values of the sampled molluscs were fairly small, whereas migratory shrimp and fish tended to have larger variations, implying that migratory animals feed on diverse prey, while the molluscs feed on a common food, or their assimilative capacity for sulfide-sulfur does not vary among individuals. A gastropod inhabiting the surface of the reductive sediment showed a distinctively low [delta][sup.34]S value (+5.8 [per thousand] to + 6.4 [per thousand]), indicating that the animal fed on detritus from reeds and halophytes with low [delta][sup.34]S values (-19.1 [per thousand] to -2.1[per thousand]). This study suggests that some benthic animals incorporate sulfide-sulfur for their nutrition and that sulfur isotope composition can be a useful indicator in the analysis of the food habitat of animals.

Research has been conducted on alpha crystallins. The interactions of adenosine 5'-triphosphate with these crystallins have been investigated via the use of hydrogen-deuterium exchange to measure adenosine 5'-triphosphate effect on amide hydrogen accessibility, and the details are reported.

Research has been conducted on catalytic hydrogen evolution and oxidation by Allochromatium vinosum [NiFe]-hydrogenase. The mechanism of these evolution and oxidation has been investigated via the use of protein film voltammetry, and the catalytic cycle energetics have been determined and discussed.

Research has been conducted on cytoplasmic and periplasmic Na (super)+/H (super)+ antiporter loops. The proximity between pair of Cys replacements in these loops has been investigated and the results indicate that the N-terminus and loop VIII-IX are in proximity within Na (super)+/H (super)+ antiporter protein.

Research has been conducted on Rhodobacter sphaeroides prrA mutant. Experiments demonstrate that this mutant can be involved in dark chemoautotrophic growth in H (sub)2- CO (sub)2 -O (sub)2 atmosphere.

Research has been conducted on urea and guanidine hydrochloride. Results indicate that these compounds stabilize ferrocytochrome c at subdenaturing concentrations of denaturants and reveal the nature of protein-denaturant interactions.

Research has been conducted on Ralstonia eutropha tetrameric cytoplasmic [NiFe] hydrogenase. The site-directed mutagenesis has been extended to the conserved regions of active site-containing subnunit HoxH, and the resulting [NiFe] hydrogenase mutants have been characterized with respect to their structural and catalytic properties.

Research has been conducted on Staphylococcus aureus intact cells harvested during stationary or exponential growth conditions. The study of these cells carried out via the use of cross-polarization magic-angle spinning and rotational-echo double resonance NMR experiments in the presence of vancomycin allows to determine peptidoglycan cross-link concentrations and suggests that vancomycin interrupts the synthesis of peptidoglycans via interference with transglycosylation.

Research has been conducted on enoyl-CoA hydratase. The ability of this hydratase to catalyze Cr-CoA hydration to 3(S)-hydroxybutyryl with an exceptional degree of stereospecificity has been confirmed and the results indicate that the correct product enantiomer formation requires oxyanion hole and substrate's optimal positioning.

Research has been conducted on the transhydrogenase domain I of Rhodospirillum rubrum transhydrogenase. The high-resolution structures of this domain have been determined with NADH bound and in the absence of NAD(H) and the single-crystal spectrophotometry has been used to determine NADH presence.

Research has been conducted on the coupled protein motion role in enzymic catalysis. The relationship between protein motions and the hydride rate transfer has been investigated via the study of additional residues involved in the coupled network and the details are reported.

Research has been conducted on the cyclic organihydroborate zirconium monocyclopentadienyl complexes. The preparation of these complexes in diethyl ether has been carried out and the details are reported.

Research has been conducted on the duplexes containing A-tracts. The role of NH (sub)4 (super)+ in the structure, chemical shifts and imino proton exchange kinetics of these duplexes has been investigated and the results are discussed.

Research has been conducted on the down regulated in adenoma gene product. The hypothesis that this gene product can bind to the PDZ domains of NHE3 building a structural link between Na (super)+ /H (super)+ and Cl (super)- /HCO (sub)3 (super)- exchangers has been tested and the results are reported.

Research has been conducted on native-state hydrogen exchange-directed protein engineering. The use of this protein engineering in populating partially unfolded forms for their structural studies is described.

Research has been conducted on the protein folding and unfolding. The analysis of the amount of information transferred from this folding and unfolding to another member of the same family has been carried out via the characterization of the dimeric enzyme serine hydroxymethyltransferase' equilibrium unfolding process and the details are presented.

Research has been conducted on the Sorghum bicolor hydroxynitrile lyase. The crystal structure of this lyase has been investigated in complex with the inhibitor benzoic acid and the effect of this structure on the catalysis evolution in the hydroxynitrile lyase super family has been analyzed and discussed.

Using the pH-sensitive fluorescent dye BCECF, we compared the effects of hyperosmotic urea on basolateral [Na.sup.+]/[H.sup.+] exchange (NHE) with those of hyperosmotic mannitol in isolated nonperfused proximal tubule S2 segments from mice lacking both the mdr1a and mdr1b genes (KO) and wild-type (WT) mice. All the experiments were performed in C[O.sub.2]/HC[O.sub.3]-free HEPES solutions. Osmolality of the peritubular solution was raised from 300 to 500 mosmol/kg[H.sub.2]O by adding mannitol or urea. NHE activity was assessed by the [Na.sup.+]-dependent acid extrusion rate ([J.sub.H]) after an acid load with N[H.sub.4]CI prepulse. In WT mice, hyperosmotic mannitol had no effect on [J.sub.H] at over the entire range of intracellular pH (p[H.sub.i] studied (6.20-6.90), whereas in KO mice it increased [J.sub.H] at a p[H.sub.i] range of 6.20-6.45. In contrast, in both WT and KO mice, hyperosmotic urea increased [J.sub.H] at a phi range of 6.20-6.90. In KO mice, [J.sub.H] in a hyperosmotic urea solution were similar to those in a hyperosmotic mannitol solution at a phi range of 6.20-6.40 but were greater than in a hyperosmotic mannitol solution at a phi range of 6.45-6.90. In WT mice, hyperosmotic urea caused an increase in [V.sub.max] without changing Km for peritubular [Na.sup.+]. Staurosporine (the PKC inhibitor) inhibited hyperosmotic mannitol-induced NHE activation in KO mice, whereas it had no effect on hyperosmotic urea-induced NHE activation in WT or KO mice. Genistein (the tyrosine kinase inhibitor) inhibited hyperosmotic urea-induced NHE activation in WT and KO mice, whereas it caused no effect on hyperosmotic mannitol-induced NHE activation in KO mice. We conclude that hyperosmotic urea activates basolateral NHE via tyrosine kinase in tubules from both WT and KO mice, whereas hyperosmotic mannitol activates it via PKC only in tubules from KO mice. mdr1a; mdr1b; isolated nonperfused tubule; intracellular pH measurement; PKC; tyrosine kinase

Myocardial ischemia-reperfusion activates the [Na.sup.+]/[H.sup.+] exchanger, which induces arrhythmias, cell damage, and eventually cell death. Inhibition of the exchanger reduces cell damage and lowers the incidence of arrhythmias after ischemia-reperfusion. The [omega]-3 polyunsaturated fatty acids (PUFAs) are also known to be cardioprotective and antiarrhythmic during ischemia-reperfusion challenge. Some of the action of PUFAs may occur via inhibition of the [Na.sup.+]/[H.sup.+] exchanger. The purpose of our study was to determine the capacity for selected PUFAs to alter cardiac sarcolemmal (SL) [Na.sup.+]/[H.sup.+] exchange. Cardiac membranes highly enriched in SL vesicles were exposed to 10-100 [micro]M eicosapentanoic acid (EPA) or docosahexanoic acid (DHA). [H.sup.+]-dependent [sup.22][Na.sup.+] uptake was inhibited by 30-50% after treatment with [greater than or equal to] 50 [micro]M EPA or [greater than or equal to] 25 [micro]M DHA. This was a specific effect of these PUFAs, because 50 [micro]M linoleic acid or linolenic acid had no significant effect on [Na.sup.+]/[H.sup.+] exchange. The SL vesicles did not exhibit an increase in passive [Na.sup.+] efflux after PUFA treatment. In conclusion, EPA and DHA can potently inhibit cardiac SL [Na.sup.+]/[H.sup.+] exchange at physiologically relevant concentrations. This may explain, in part, their known cardioprotective effects and antiarrhythmic actions during ischemia-reperfusion. ischemia-reperfusion; myocardial cell death; antiarrhythmic; eicosapentanoic acid; docosahexanoic acid

After massive small bowel resection (MSBR), the remnant small intestine adapts to restore Na absorptive function. The possibility that this occurs through increases in cellular Na absorptive capacity was examined by assessing the regional effects of 50% proximal MSBR on the function and expression of the apical membrane Na/H exchangers (NHEs) NHE2 and NHE3. Morphometric analysis confirmed adaptive changes consistent with villus hypertrophy, particularly distal to the anastomosis. Villus epithelium prepared by light mucosal scrapings from 2-wk-postresected and -posttransected control rats exhibited comparable brush-border hydrolase activities, total cell protein per DNA, and villin expression but increased basolateral Na-K-ATPase activity. Parallel increases of two- to threefold in protein and mRNA abundance of NHE2 and NHE3 were observed only in ileal regions distal to the anastomosis of resected rats. Basolateral NHE1 expression was unchanged. After 80% resection, increases in NHE2 and NHE3 became evident in proximal colon. We conclude that increased enterocyte expression and function of apical membrane NHEs in regions distal to the anastomosis play a role in the adaptive process after MSBR. The increased luminal Na load to distal bowel regions after proximal resection may stimulate increases in apical membrane NHE gene transcription and protein expression. sodium transport; sodium/hydrogen exchange; intestinal surgery; intestinal adaptation; diarrhea; malabsorption; epithelial cell; intestinal physiology

Raman spectroscopy is applied to an investigation of the interfacial chemistry of silica-immobilized 8-hydroxyquinoline (8HQ) for binding of metal ions over a wide range of solution conditions. Since the derivatized silica has a high specific binding capacity, the mass of silica equilibrated with solution needs to be small for studies of reactions with trace-level ([micro]M) metal ions; otherwise, the solution volume required to reach equilibrium becomes excessive. To address this problem, a small-volume flow cell is designed for this work using a fiber-optic Raman probe inserted directly into the packed end of a micro-column, allowing excitation and collection of Raman scattering from less than 10 mg of derivatized silica. This cell is attached to a flow system that allows control of solution conditions while the response of the 8HQ-silica material is acquired by continuous monitoring of Raman scattering from the sample. Raman spectra of the deprotonated, neutral, protonated, and copper-complexed forms of the ligand can be distinguished, allowing proton-transfer and metal ion binding reactions of the ligand to be investigated. To account for the effects of changing surface potential on these reactions, [xi]-potential measurements are made on the 8HQ-silica particles under the same solution conditions that are employed in the Raman scattering measurements. The observed pH dependence of metal ion binding was corrected for the effect of surface potential using the Boltzmann equation, and the resulting equilibrium constant for binding of [Cu.sup.2+] was independent of metal ion concentration over a 100-fold range from 30 [micro]M to 5 mM.