Effect of intracellular pH on rotational speed of bacterial flagellar motors

Weak acids such as acetate and benzoate, which partially collapse the transmembrane proton gradient, not only mediate pH taxis but also impair the motility of Escherichia coli and Salmonella at an external pH of 5.5. In this study, we examined in more detail the effect of weak acids on motility at various external pH values. A change of external pH over the range 5.0 to 7.8 hardly affected the swimming speed of E. coli cells in the absence of 34 mM potassium acetate. In contrast, the cells decreased their swimming speed significantly as external pH was shifted from pH 7.0 to 5.0 in the presence of 34 mM acetate. The total proton motive force of E. coli cells was not changed greatly by the presence of acetate. We measured the rotational rate of tethered E. coli cells as a function of external pH. Rotational speed decreased rapidly as the external pH was decreased, and at pH 5.0, the motor stopped completely. When the external pH was returned to 7.0, the motor restarted rotating at almost its original level, indicating that high intracellular proton (H+) concentration does not irreversibly abolish flagellar motor function. Both the swimming speeds and rotation rates of tethered cells of Salmonella also decreased considerably when the external pH was shifted from pH 7.0 to 5.5 in the presence of 20 mM benzoate. We propose that the increase in the intracellular proton concentration interferes with the release of protons from the torque-generating units, resulting in slowing or stopping of the motors.     

The Bacterial Flagellar Type III Export Gate Complex Is a Dual Fuel Engine That Can Use Both H+ and Na+ for Flagellar Protein Export

The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H⁺–protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF) in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na⁺ increased it dramatically. Phenamil, a blocker of Na⁺ translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na⁺ concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na⁺ channel. In wild-type cells, however, neither Na⁺ nor phenamil affected protein export, indicating that the Na⁺ channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na⁺ concentration.     

Oxidation-reduction properties of disulfide-containing proteins of the Rhodobacter capsulatus cytochrome c biogenesis system

Oxidation-reduction titrations for the active-site disulfide/dithiol couples of the helX- and ccl2-encoded proteins involved in cytochrome c biogenesis in the purple non-sulfur bacterium Rhodobacter capsulatus have been carried out. The R. capsulatus HelX and Ccl2 proteins are predicted to function as part of a dithiol/disulfide cascade that reduces a disulfide on the apocytochromes c so that two cysteine thiols are available to form thioether linkages between the heme prosthetic group and the protein. Oxidation-reduction midpoint potential (E(m)) values, at pH 7.0, of -300 +/- 10 and -210 +/- 10 mV were measured for the HelX and Ccl2 (a soluble, truncated form of Ccl2) R. capsulatus proteins, respectively. Titrations of the disulfide/dithiol couple of a peptide designed to serve as a model for R. capsulatus apocytochrome c(2) have also been carried out, and an E(m) value of -170 +/- 10 mV was measured for the model peptide at pH 7.0. E(m) versus pH plots for HelX, Ccl2, and the apocytochrome c(2) model peptide were all linear over the pH range from 5.0 to 8.0, with the -59 mV/pH unit slope expected for a reaction in which two protons are taken up for each disulfide that is reduced. These results provide thermodynamic support for the proposal that HelX reduces Ccl2 and that reduced Ccl2, in turn, serves as the reductant for the production of the two thiols of the CysXxxYyyCysHis heme-binding motif of the apocytochromes.     

Observations on intracellular pH during cleavage of eggs of Xenopus laevis

Direct measurements of intracellular pH was made with recessed-tip pH microelectrodes in fertilized eggs of the frog, Xenopus laevis, from approximately 1 h after fertilization to mid-blastula. The intracellular pH just before first cleavage was 7.65 +/- 0.04 (SD; n = 9). By stage 5 to the middle of stage 6, average intracellular pH was 7.70 +/- 0.06 (SD; n = 16). A statistically significant alkalization of 0.18 +/- 0.03 pH unit (SD; n = 5) was observed beginning in early blastula. A cycle of less than or equal to 0.05 pH unit was occasionally observed during the pre-blastula period, but its significance is unknown. By exposing the early cleavage embryo to saline buffered with sodium propionate, pH 4.7-5.0, it was possible to lower intracellular pH with some degree of control. Apparently, normal cleavage continued to occur when intracellular pH had been forced as much as 0.3 unit below normal. We conclude that this implies no specific involvement of intracellular pH in mitosis and cytokinesis. If intracellular pH was lowered further, cell division ceased at about pH 7.2, and furrow regression began at about pH 7.0. Once furrow regression occurred, subsequent development was usually arrested or abnormal when the embryo was transferred back to normal saline.     

Decolorization of the textile dyes using purified banana pulp polyphenol oxidase

Polyphenol oxidase (PPO) purified using DEAE-cellulose and Biogel P-100 column chromatography from banana pulp showed 12.72-fold activity and 2.49% yield. The optimum temperature and pH were found to be 30 degrees C and 7.0, respectively for its activity. Catechol was found to be a suitable substrate for banana pulp PPO that showed V(max), 0.041 mM min(-1) and K(m), 1.6 mM. The enzyme activity was inhibited by sodium metabisulfite, citric acid, cysteine, and beta-mercaptoethanol at 10 mM concentration. The purified enzyme could decolorize (90%) Direct Red 5B (160 microg mL(-1)) dye within 48 h and Direct Blue GLL (400 microg mL(-1)) dye up to 85% within 90 h. The GC-MS analysis indicated the presence of 4-hydroxy-benzenesulfonic acid and Naphthalene-1,2,3,6-tetraol in the degradation products of Direct Red 5B, and 5-(4-Diazenyl-naphthalene-1-ylazo)-8-hydroxy-naphthalene-2-sulfonic acid and 2-(4-Diazenyl-naphthalene-1-ylazo)-benzenesulfonic acid in the degradation products of Direct Blue GLL.     

Interaction of lidocaine with the cardiac sodium channel: effects of low extracellular pH are consistent with an external blocking site

Brief extracellular application of millimolar concentrations of lidocaine affected sodium currents recorded in isolated rat ventricular myocytes in two ways: 1) a reduction of the maximum current consistent with a channel blocking action, and 2) a negative shift in the voltage dependence of inactivation consistent with an interaction with the inactivated state of the channel. Both effects occurred very rapidly (< 1 s). Decreasing extracellular pH to 6.4 increased the potency for channel block (EC50 1.8 +/- 0.2 mM at pH 7.4 and 0.8 +/- 0.1 mM at pH 6.5) and decreased the potency to shift inactivation (V(1/2) shift -42 mV by 1 mM lidocaine at pH 7.4 and -12.6 mV at pH 6.5). Channel block was slightly less at +90 mV compared to -40 mV at either pH (not statistically significant). The increase in potency for block at decreased extracellular pH, while intracellular pH is buffered, and the lack of voltage dependence of block, suggest that the charged form of lidocaine can block the channel by interacting with a site near the extracellular mouth, although alternative explanations are discussed.     

Protons block BK channels by competitive inhibition with K+ and contribute to the limits of unitary currents at high voltages

Proton block of unitary currents through BK channels was investigated with single-channel recording. Increasing intracellular proton concentration decreased unitary current amplitudes with an apparent pKa of 5.1 without discrete blocking events, indicating fast proton block. Unitary currents recorded at pH(i) 8.0 and 9.0 had the same amplitudes, indicating that 10(-8) M H(+) had little blocking effect. Increasing H(+) by recording at pH(i) 7.0, 6.0, and 5.0 then reduced the unitary currents by 13%, 25%, and 53%, respectively, at +200 mV. Increasing K(+)(i) relieved the proton block in a manner consistent with competitive inhibition of K(+)(i) action by H(+)(i). Proton block was voltage dependent, increasing with depolarization, indicating that block was coupled to the electric field of the membrane. Proton block was not described by the Woodhull equation for noncompetitive voltage-dependent block, but was described by an equation for cooperative competitive inhibition that included voltage-dependent block from the Woodhull equation. Proton block was still present after replacing the eight negative charges in the ring of charge at the entrance to the intracellular vestibule by uncharged amino acids. Thus, the ring of charge is not the site of proton block or of competitive inhibition of K(+)(i) action by H(+)(i). With 150 mM symmetrical KCl, unitary current amplitudes increased with depolarization, reaching 66 pA at +350 mV (pH(i) 7.0). The increase in amplitude with voltage became sublinear for voltages >100 mV. The sublinearity was unaffected by removing from the intracellular solutions Ca(2+) and Ba(2+) ions, the Ca(2+) buffers EGTA and HEDTA, the pH buffer TES, or by replacing Cl(-) with MeSO(3)(-). Proton block accounted for approximately 40% of the sublinearity at +200 mV and pH 7.0, indicating that factors in addition to proton block contribute to the sublinearity of the unitary currents through BK channels.     

Oxidation-reduction midpoint potentials of the molybdenum center in spinach NADH:nitrate reductase

Oxidation-reduction midpoint potentials for the molybdenum center in assimilatory NADH:nitrate reductase isolated from spinach (Spinacia oleracea) have been determined at pH 7.0 in the presence of dye mediators using EPR spectroscopy to monitor formation of Mo(V). Values for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples were determined to be -8 and -42 mV, respectively.     

Sodium channel kinetic changes that produce Brugada syndrome or progressive cardiac conduction system disease

Some mutations of the sodium channel gene Na(V1.5) are multifunctional, causing combinations of LQTS, Brugada syndrome and progressive cardiac conduction system disease (PCCD). The combination of Brugada syndrome and PCCD is uncommon, although they both result from a reduction in the sodium current. We hypothesize that slow conduction is sufficient to cause S-T segment elevation and undertook a combined experimental and theoretical study to determine whether conduction slowing alone can produce the Brugada phenotype. Deletion of lysine 1479 in one of two positively charged clusters in the III/IV inter-domain linker causes both syndromes. We have examined the functional effects of this mutation using heterologous expression of the wild-type and mutant sodium channel in HEK-293-EBNA cells. We show that DeltaK1479 shifts the potential of half-activation, V(1/2m), to more positive potentials (V(1/2m) = -36.8 +/- 0.8 and -24.5 +/- 1.3 mV for the wild-type and DeltaK1479 mutant respectively, n = 11, 10). The depolarizing shift increases the extent of depolarization required for activation. The potential of half-inactivation, V(1/2h), is also shifted to more positive potentials (V(1/2h) = -85 +/- 1.1 and -79.4 +/- 1.2 mV for wild-type and DeltaK1479 mutant respectively), increasing the fraction of channels available for activation. These shifts are quantitatively the same as a mutation that produces PCCD only, G514C. We incorporated experimentally derived parameters into a model of the cardiac action potential and its propagation in a one dimensional cable (simulating endo-, mid-myocardial and epicardial regions). The simulations show that action potential and ECG changes consistent with Brugada syndrome may result from conduction slowing alone; marked repolarization heterogeneity is not required. The findings also suggest how Brugada syndrome and PCCD which both result from loss of sodium channel function are sometimes present alone and at other times in combination.     

Changes in the proton-motive force in Escherichia coli in response to external oxidoreduction potential.

The pH homeostasis and proton-motive force (Deltap) of Escherichia coli are dependent on the surrounding oxidoreduction potential (ORP). Only the internal pH value and, thus, the membrane pH gradient (DeltapH) component of the Deltap is modified, while the membrane potential (DeltaPsi) does not change in a significant way. Under reducing conditions (Eh < 50 mV at pH 7.0), E. coli decreases its Deltap especially in acidic media (21% decrease at pH 7.0 and 48% at pH 5.0 for a 850-mV ORP decrease). Measurements of ATPase activity and membrane proton conductance (CH+m) depending on ORP and pH have shown that the internal pH decrease is due to an increase in membrane proton permeability without any modification of ATPase activity. We propose that low ORP values de-energize E. coli by modifying the thiol : disulfide balance of proteins, which leads to an increase in the membrane permeability to protons.     

Vasoconstrictor hormones depolarize renal glomerular mesangial cells by activating chloride channels

Mesangial cells are smooth muscle-like cells of the renal glomerulus which contract and produce prostaglandins in response to vasopressin and angiotensin. These responses serve to regulate the glomerular capillary filtering surface area. We have used the membrane potential-sensitive fluorescent dye bis-oxonol and the intracellular fluorescent calcium-sensitive probe Indo-1 to study the changes in membrane potential (Em) and intracellular free calcium concentration ([Ca2+]i) in cultured rat mesangial cells in response to vasoconstrictor hormones. Basal [Ca2+]i was 227 +/- 4 nM, and stimulation by maximal concentrations of either vasopressin or angiotensin resulted in a transient 4-6-fold rise. Resting membrane potential was 45.8 +/- 0.9 mV and vasoconstrictor hormones caused a depolarization of 14-18 mV. The following extracellular ion substitutions indicated that chloride efflux was the predominant ion flux responsible for depolarization: 1) depolarization persisted when sodium in the medium was substituted with N-methylglucamine; 2) substitution of medium sodium chloride with sodium gluconate, which enhances the gradient for chloride efflux, augmented vasoconstrictor-stimulated depolarization; 3) suspension of cells in potassium chloride medium resulted in depolarization, following which, stimulation by either vasopressin or angiotensin resulted in hyperpolarization; and 4) this hyperpolarization did not occur when potassium gluconate medium was used to depolarize the cells. The calcium ionophore ionomycin also resulted in membrane depolarization. However, prevention of the rise in [Ca2+]i by prior exposure to ionomycin in calcium-free medium or by loading mesangial cells with the intracellular calcium buffer BAPTA did not abrogate the depolarization response to vasoconstrictor hormones. This indicates that a rise in intracellular calcium is not necessary for depolarization. In contrast, prior depolarization of the cells using varying concentrations of KCl in the external medium, which dissipated the electrochemical gradient for chloride efflux, resulted in a corresponding prolongation of the transient calcium response to vasopressin and angiotensin. These findings indicate that angiotensin and vasopressin depolarize mesangial cells by activating chloride channels and that this activation can occur by both calcium-dependent and -independent mechanisms. In addition, activation of chloride channels with resulting depolarization may serve to modulate the calcium signal.     

Amiloride at pH 7.0 inhibits the Na(+)-driven flagellar motors of Vibrio alginolyticus but allows cell growth.

Amiloride, a specific inhibitor for the Na(+)-driven flagellar motors of alkalophilic Bacillus, is known to inhibit secondarily the growth of alkalophiles. The motility of a marine Vibrio, V. alginolyticus, was almost completely inhibited by 2 mM amiloride either at pH 7.0 or 8.5. We found that this concentration of amiloride inhibited the cell growth completely at pH 8.5 but only slightly at pH 7.0. Kinetic analysis of the inhibition of motility by amiloride at pH 7.0 showed that the inhibition was competitive with Na+ in the medium. Thus, amiloride at pH 7.0 is really a specific and useful tool for the analysis of the Na(+)-driven flagellar motors of Vibrio.     

Redox characteristics of the tungsten DMSO reductase of Rhodobacter capsulatus

The dimethylsulfoxide reductase (DMSOR) from Rhodobacter capsulatus is known to retain its three-dimensional structure and enzymatic activity upon substitution of molybdenum, the metal that occurs naturally at the active site, by tungsten. The redox properties of tungsten-substituted DMSOR (W-DMSOR) have been investigated by a dye-mediated reductive titration with the concentration of the W(V) state monitored by EPR spectroscopy. At pH 7.0, E(m)(W(VI)/W(V)) is -194 mV and E(m)(W(V)/W(IV)) is -134 mV. Each E(m) value of W-DMSOR is significantly lower (220 and 334 mV, respectively) than that of the corresponding couple of Mo-DMSOR. These redox potentials are consistent with the ability of Mo-DMSOR to catalyze both the reduction of DMSO to DMS and the back reaction, whereas W-DMSOR is very effective in catalyzing the forward reaction, but shows no ability to catalyze the oxidation of DMS to DMSO.     

Hydrogen ion gradients across the mitochondrial, endosomal and plasma membranes in bloodstream forms of trypanosoma brucei solving the three-compartment problem.

Conditions for the use of both [14C]methylamine and 5, 5-dimethyl[14C]oxa-azolidine-2,4-dione (DMO) to measure the H+ concentration of intracellular compartments of monomorphic long thin bloodstream forms of Trypanosoma brucei were established. Neither probe was actively transported or bound to internal components of the cell and both probes equilibrated passively with a t1/2 close to 8 min. DMO was excluded from cells, while methylamine was accumulated but not metabolized. Solution of the three-compartment problem revealed that, when cells were respiring aerobically on glucose at an external pH of 7.5, the cytoplasmic pH was in the range 6.99-7.03, the pH of the mitochondrial matrix was 7.71-7.73, and the algebraic average pH of the various endosomal compartments was 5.19-5.50. Similar values were found when cells were respiring aerobically on glycerol. However, bloodstream forms of T. brucei could not maintain a constant internal H+ concentration outside the external pH range 7.0-7.5, and no evidence for the presence of an H+/Na+ exchanger was found. Full motility and levels of pyruvate production were maintained as the external pH was raised as high as 9.5, suggesting that these cells tolerate significant internal alkalinisation. However, both motility and pyruvate production were severely inhibited under acidic conditions, and the cells deteriorated rapidly below an external pH of 6.5. Physiologically, the plasma membrane of T. brucei had low permeability to H+ and the internal pH was unaffected by changes in Deltapsip, which is dominated by the potassium diffusion potential. However, in the presence of FCCP, the internal pH fell rapidly about 0.5 pH unit and came into equilibrium with Deltapsip. Oligomycin abolished the mitochondrial pH gradient (DeltapHm) selectively, whereas chloroquine abolished only the endosomal pH gradient (DeltapHe). The pH gradient across the plasma membrane (DeltapHp) alone could be abolished by careful osmotic swelling of cells. The plasma membrane had an inwardly directed proton-motive force (DeltaPp) of -52 mV and an inwardly directed sodium-motive force (DeltaNp) of -149 mV, whereas the mitochondrial inner membrane had only an inwardly directed DeltaPm of -195 mV. The pH gradient across the endosomal membranes was not accompanied by an electrical gradient. Consequently, endosomal membranes had an algebraically average outwardly directed DeltaPl within the range + 89 to +110 mV, depending on the measurement method.     

Ionic regulation of the plasma membrane potential of rainbow trout (Salmo gairdneri) spermatozoa: role in the initiation of sperm motility

The ionic dependence of the trout sperm plasma membrane potential was analysed by measuring the accumulation of the lipophilic ions 3H-tetraphenylphosphonium (TPP) and 14C-thiocyanate (SCN) following dilution in artificial media isotonic to the seminal fluid. Our data showed that the trout sperm plasma membrane has a mixed conductance: the plasma membrane potential is sensitive upon the transmembrane gradients of K+, Na+, and H+. This potential is negative (less than -40 mV) in a 125 mM choline chloride media (ChM) at pH 8.5. Replacement of choline by sodium has a small depolarizing effect. The membrane potential is about -15 mV in a 125 mM potassium chloride and falls near zero mV only if valinomycin is added. In ChM changing the external pH (pHe) greatly affects the membrane potential: its value rises from less than -40 mV at pHe 9.0 to -17 mV at pHe 5.0. This pH effect is observed also in presence of sodium or potassium. A decrease in the transmembrane proton gradient produced by increasing internal pH without changing pHe induces also a depolarisation of the plasma membrane. In the different media in which trout sperm remain immotile after dilution (media with [K+] greater than 20-40 mM or a pH less than 7.5) the plasma membrane is more depolarized than in media allowing motility, suggesting a relationship between the state of membrane polarization and the intracellular effectors of the axonemal movement.     

Rhodamine B as a mitochondrial probe for measurement and monitoring of mitochondrial membrane potential in drug-sensitive and -resistant cells

In order to get more insight into the energetic state of multidrug-resistance (MDR) cell compared with its corresponding sensitive cell, a noninvasive fluorescence method for determining and monitoring the mitochondrial membrane potential (DeltaPsi(m)), using rhodamine B and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was established. Rhodamine B distributes across biological membranes in response to the electrical transmembrane potential. P-glycoprotein- and MRP1-protein-mediated efflux do not create a concentration gradient, leading the cell-rhodamine B system to reach a steady state, where the ratio of cytosolic to extracellular rhodamine B was equal to 1. The mitochondrial matrix rhodamine B concentration was precisely determined as a decrease of rhodamine B fluorescence in the presence of formazan, a rhodamine B fluorescence quencher, which locally accumulates in the matrix of mitochondria. The kinetics of decrease in rhodamine B fluorescence (V(i)) can be used to estimate DeltaPsi(m) using the Nernst equation: DeltaPsi(m)=-61.54 log V(i)-258.46. The DeltaPsi(m) values determined were -160+/-4 mV for K562 cell, -146+/-6 mV for K562/adr cell, -161+/-10 mV for GLC4 cell and -168+/-2 mV for GLC4/adr cell. An increase or a decrease in DeltaPsi(m) consequently followed an increase or a decrease in the cellular ATP contents. An increase ATP content in the two MDR cell lines can protect cells from cytotoxicity induced by pirarubicin.     

Redox control in heme proteins: electrostatic substitution in the active site of leghemoglobin

The effects of electrostatic substitutions on the spectroscopic, ligand binding, and redox properties of the heme in leghemoglobin have been examined by replacement of the proximal leucine 88 residue with an aspartic acid residue (Leu88Asp). Electronic and resonance Raman spectra of the ferric derivative of Leu88Asp indicate a mixture of 6-coordinate, high-spin and 6-coordinate, low-spin hemes, analogous to that observed in the recombinant wild-type protein (rLb). At alkaline pH, formation of hydroxide-bound heme is indicated for Leu88Asp; the pK(a) for this transition (8.7 +/- 0.2, micro = 0.10 M, 25.0 degrees C) is 0.4 pH units higher than for rLb. Equilibrium dissociation constants (sodium phosphate, pH 7.0, micro = 0.10 M, 25.0 +/- 0.1 degrees C) for binding of anionic ligands (N(-)(3), nicotinate) to Leu88Asp are higher (K(d,nicotinate) = 6.8 +/- 0.2 microM; K(d,azide) = 33 +/- 0.6 microM) than the corresponding values for rLb (K(d,nicotinate) = 1.4 +/- 0.3 microM (pH 5.5, micro = 0.10 M, 25.0 +/- 0.1 degrees C); K(d,azide) = 4.8 +/- 0.2 microM). Resonance Raman spectra (sodium phosphate, pH 7.0, micro = 0.10 M) for the ferrous derivatives of Leu88Asp and rLb exhibit a strong nu(Fe-His) stretching frequency at 223 cm(-1) in both cases, indicating that the hydrogen bonding structure on the proximal side is not substantially altered in the variant. The reduction potential of Leu88Asp is -14 +/- 2 mV vs standard hydrogen electrode (SHE) (25.0 degrees C, micro = 0.10 M, pH 7.0), a decrease of 35 mV over the corresponding value for the wild-type protein under the same conditions (21 +/- 3 mV vs SHE). An assessment of these data in terms of electrostatic and hydrogen bonding considerations is presented.     

Interaction of cytidine 3'-monophosphate and uridine 3'-monophosphate with ribonuclease a at the denaturation temperature

Differential scanning calorimetry (DSC) measurements were performed on the thermal denaturation of ribonuclease a and ribonuclease a complexed with an inhibitor, cytidine or uridine 3'-monophosphate, in sodium acetate buffered solutions. Thermal denaturation of the complex results in dissociation of the complex into denatured ribonuclease a and free inhibitor. Binding constants of the inhibitor to ribonuclease a were determined from the increase in the denaturation temperature of ribonuclease a in the complexed form and from the denaturation enthalpy of the complex. Binding enthalpies of the inhibitor to ribonuclease a were determined from the increase in the denaturation enthalpy of ribonuclease a complexed with the inhibitor. For the cytidine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 87 +/- 8 M-1 (pH 7.0) to 1410 +/- 54 M-1 (pH 5.0), while the binding enthalpies increase from 17 +/- 13 kJ mol-1 (pH 4.7) to 79 +/- 15 kJ mol-1 (pH 5.5). For the uridine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 104 +/- 1 M-1 (pH 7.0) to 402 +/- 7 M-1 (pH 5.5), while the binding enthalpies increase from 16 +/- 5 kJ mol-1 (pH 6.0) to 37 +/- 4 kJ mol-1 (pH 7.0). The binding constants and enthalpies of the cytidine inhibitor in 0.05 M sodium acetate buffered solutions increase respectively from 328 +/- 37 M-1 (pH 6.5) to 2200 +/- 364 M-1 (pH 5.5) and from 22 kJ mol-1 (pH 5.5) to 45 +/- 7 kJ mol-1 (pH 6.5). the denaturation transition cooperativities of the uncomplexed and complexed ribonuclease a were close to unity, indicating that the transition is two state with a stoichiometry of 1.(ABSTRACT TRUNCATED AT 250 WORDS)