Kinetic and spectroscopic evidence of cation-induced conformation changes in yeast K+ -activated aldehyde dehydrogenase.

The activity, stability and spectroscopic properties of yeast K+ -activated aldehyde dehydrogenase were measured at various times after removal from, and after returning to a solution containing K+. Enzyme activity is rapidly lost on removal of most of the K+ and rapidly regained if K+ is replaced immediately. These activity changes are slower than likely rates of K+ dissociation and association. These rapid changes in concentration result in altered enzyme stability with enzyme in K+ the more stable. U.v. difference spectra are produced whenever enzyme in an activating environment (K+ or Tl+) is compared with enzyme in a non-activating environment (Tris+ or Li+). These spectral changes occur within 10s. The saturation characteristics with K+ are hyperbolic for all three phenomena of activation, stabilization and spectral change, with estimated apparent dissociation constants (Ks) for K+ of 7.5 mM, 5.5 mM and 6 mM respectively. Continued incubation of enzyme in the absence of K+ results in the accumulation of an enzyme form that re-activates only slowly on replacing K+. Stability characteristics in various concentrations of K+ over equivalent time scales are consistent with the existence of additional conformations. Spectroscopic evidence also indicates such additional slow conformation changes. Results have been interpreted in terms of two separate conformation transitions induced or stabilized by K+.     

The role of tightly bound ADP on chloroplast ATPase

Isolated chloroplast coupling factor 1 ATPase is known to retain about 1 mol of tightly bound ADP/mol of enzyme. Some experimental results have given evidence that the bound ADP is at catalytic sites, but this view has not been supported by observations of a slow replacement of the bound ADP when CaATP or MgATP is added. The experiments reported in this paper show why a slow replacement of ADP bound at a catalytic site can occur. When coupling factor 1, labeled with tightly bound [3H]ADP, is exposed to Mg2+ or Ca2+ prior to the addition of MgATP or CaATP, a pronounced lag in the onset of ATP hydrolysis is observed, and only slow replacement of the [3H]ADP occurs. Mg2+ or Ca2+ can induce inhibition very rapidly, as if an inhibited form of the enzyme results whenever the enzyme with tightly bound ADP encounters Mg2+ or Ca2+ prior to ATP. The inhibited form can be slowly reactivated by incubation with EDTA, although some irreversible loss in activity is encountered. In contrast, when MgATP or CaATP is added to enzyme depleted of Mg2+ and Ca2+ by incubation with EDTA, a rapid onset of ATP hydrolysis occurs and most of the tightly bound [3H]ADP is released within a few seconds, as expected for binding at a catalytic site. The Mg2+-induced inhibition of both the ATPase activity and the lack of replacement of tightly bound [3H] ADP can be largely prevented by incubation with Pi under conditions favoring Pi addition to the site containing the tightly bound ADP. Our and other results can be explained if enzyme catalysis is greatly hindered when MgADP or CaADP without accompanying Pi is tightly bound at one of the three catalytic sites on the enzyme in a high affinity conformation.     

Solvent effects on ouabain binding to the (Na+,K+)-ATPase of rat brain

The effects of the solvents deuterated water (2H2O) and dimethyl sulfoxide (Me2SO) on [3H]ouabain binding to (Na+,K+)-ATPase under different ligand conditions were examined. These solvents inhibited the type I ouabain binding to the enzyme (i.e., in the presence of Mg2+ + ATP + Na+). In contrast, both solvents stimulated type II (i.e., Mg2+ + Pi-, Mg2+-, or Mn2+-dependent) binding of the drug. The solvent effects were not due to pH changes in the reaction. However, pH did influence ouabain binding in a differential manner, depending on the ligands present. For example, changes in pH from 7.05 to 7.86 caused a drop in the rate of binding by about 15% in the presence of Mg2+ + Na+ + ATP, 75% in the Mg2+ + Pi system, and in the presence of Mn2+ an increase by 24% under similar conditions. Inhibitory or stimulatory effects of solvents were modified as various ligands, and their order of addition, were altered. Thus 2H2O inhibition of type I ouabain binding was dependent on Na+ concentration in the reaction and was reduced as Na+ was elevated. Contact of the enzyme with the Me2SO, prior to ligands for type I binding, resulted in a greater inhibition of ouabain binding than that when enzyme was exposed to Na+ + ATP first and then to Me2SO. Likewise, the stimulation of type II binding was greater when appropriate ligands acted on enzyme prior to addition of the solvent. Since Me2SO and 2H2O inhibit type I ouabain binding, it is proposed that this reaction is favored under conditions which promote loss of H2O, and E1 enzyme conformation; the stimulation of type II ouabain binding in the presence of the solvents suggests that this type of binding is favored under conditions which promote the presence of H2O at the active enzyme center and E2 enzyme conformation. This postulation of a role of H2O in modulating enzyme conformations and ouabain interaction with them is in concordance with previous observations.     

Kinetic investigations of the reactions of cytochrome c oxidase with hydrogen peroxide

The reaction of H2O2 with reduced cytochrome c oxidase was investigated with rapid-scan/stopped-flow techniques. The results show that the oxidation rate of cytochrome a3 was dependent upon the peroxide concentration (k = 2 X 10(4) M-1 X s-1). Cytochrome a and CuA were oxidised with a maximal rate of approx. 20 s-1, indicating that the rate of internal electron transfer was much slower with H2O2 as the electron acceptor than with O2 (k greater than or equal to 700 s-1). Although other explanations are possible, this result strongly suggests that in the catalytic cycle with oxygen as a substrate the internal electron-transfer rate is enhanced by the formation of a peroxo-intermediate at the cytochrome a3-CuB site. It is shown that H2O2 took up two electrons per molecule. The reaction of H2O2 with oxidised cytochrome c oxidase was also studied. It is shown that pulsed oxidase readily reacted with H2O2 (k approximately 700 M-1 X s-1). Peroxide binding is followed by an H2O2-independent conformational change (k = 0.9 s-1). Resting oxidase partially bound H2O2 with a rate similar to that of pulsed oxidase; after H2O2 binding the resting enzyme was converted into the pulsed conformation in a peroxide-independent step (k = 0.2 s-1). Within 5 min, 55% of the resting enzyme reacted in a slower process. We conclude from the results that oxygenated cytochrome c oxidase probably is an enzyme-peroxide complex.     

Effects of oxidative stress inducers, neurotoxins, and ganglioside GM1 on Na+, K+-ATPase in PC12 and brain synaptosomes

To elucidate mechanism of ganglioside neuroprotection, it is important to study their metabolic effects, specifically of action on Na+, K+ -ATPase. It has been shown that under effect of oxidative stress inductors and neurotoxins an oxidative inactivation of this enzyme takes place in PC12 cells and brain cortex synaptosomes, this inactivation being able to be prevented or decreased by ganglioside GM1. Thus, for instance, 24 h after action of 1 mM H2O2, activity of Na+, K+ -ATPase in PC12 cells decreased more than twice. However, in the case of preincubation of the cells with ganglioside GM1 prior to the H2O2 action this enzyme activity did not differ statistically significantly from control. Ganglioside GM1 also was able to increase significantly the enzyme activity decreased by action on the PC12 cells of amyloid beta-peptide (AP) causing lesion of neurons in Alzheimer's disease and at low H202 concentrations. Experiments on brain cortex synaptosomes have established that not only antioxidants--alpha-tocopherol and superoxide dismutase--but also ganglioside GM1 prevent the glutamateproduced Na+, K+ -ATPase oxidative inactivation. The obtained data agree with a suggestion that the ganglioside neuroprotective effect at action on nerve cells of such toxins as Abeta, glutamate or reactive oxygen species is due to their ability to inhibit the free-radical reactions.     

The inactivation of horseradish peroxidase isoenzyme A2 by hydrogen peroxide: an example of partial resistance due to the formation of a stable enzyme intermediate

The inactivation of horseradish peroxidase A2 (HRP-A2) with H2O2 as the sole substrate has been studied. In incubation experiments it was found that the fall in HRP-A2 activity was non-linearly dependent on H2O2 concentrations and that a maximum level of inactivation of approximately 80% (i.e. approximately 20% residual activity) was obtained with 2,000 or more equivalents of H2O2. Further inactivation was only induced at much higher H2O2 concentrations. Spectral changes during incubations of up to 5 days showed the presence of a compound III-like species whose abundance was correlated to the level of resistance observed. Inactivation was pH dependent, the enzyme being much more sensitive under acid conditions. A partition ratio (r1 approximately equals 1,140 at pH 6.5) between inactivation and catalysis was calculated from the data. The kinetics of inactivation followed single exponential time curves and were H2O2 concentration dependent. The apparent maximum rate constant of inactivation was lambdamax=3.56+/-0.07x10(-4)s(-1) and the H2O2 concentration required to give lambdamax/2 was K2=9.94+/-0.52 mM. The relationship lambdamax相似文献   

Catalytic properties of testicular hyaluronidase after gamma-irradiation

The effect of gamma-irradiation on ovine testicular hyaluronidase was studied in aqueous solution. Following irradiation, hyaluronidase is inhibited, and the kinetics of inhibition follow a pattern in which Km and Vmax decline as radiation dose is increased. It was indicated that the binding affinity of the residual activity of hyaluronidase with substrate is enhanced and depends upon radiation damage. Effects of various agents such as pH, salts, PCMB and glutathione on irradiated hyaluronidase have been compared with non-irradiated enzyme. The irradiated hyaluronidase was more sensitive to inhibition by CuSO4 than the non-irradiated enzyme. The residual activity after irradiation is less refractory to FeCl3 inhibition and less sensitive to NaCl stimulation compared to non-irradiated hyaluronidase. pH response curves of ovine testicular hyaluronidase show two maxima which become more evident after irradiation.     

Activity and kinetic characteristics of glutathione reductase in vitro in reverse micellar waterpool

The enzyme activity of glutathione reductase (NAD(P)H:oxidized-glutathione oxidoreductase, EC 1.6.4.2) incorporated in CTAB/H2O/CHCl3-isooctane (1:1, v/v) reverse micelles has been investigated. Enzyme follows the Michaelis-Menten kinetics within a specified concentration range. Effects of pH, waterpool (W0), and surfactant concentration on the activity of glutathione reductase have been studied in detail. Optimum pH for the maximum enzyme activity was found to be dependent on the size of the waterpool. Further, a substrate inhibition was observed when concentration of one of the substrates was present in large excess over the other substrate. Km values for the substrate, oxidized glutathione (GSSG) and NADPH in CTAB/H2O/CHCl3-isooctane (1:1, v/v) were determined at W0 values of 14.4, 20.0, 25.5 and 29.7, at pH 8.0. These values are close to those obtained in aqueous solution, whereas the kcat values vary with W0 values of 8.8 to 32.3. Studies on the storage stability in the reverse micelle at W0 29.7 and pH 8.0 showed that glutathione reductase retained about 80% of its activity even after a month. The enzyme showed a higher stability at high waterpool. Oxidized glutathione (GSSG) provides protection to glutathione reductase against denaturation on storage in reverse micellar solution. Apparently, the enzyme is able to acquire a suitable native conformation at waterpool 29.7 and pH 8.0 and thereby exhibits an activity and stability inside the micellar cavity that are almost equivalent to that in aqueous solution.     

醋酸酐对太平洋白对虾(Penaeus vannamei)β-N-乙酰- D-氨基葡萄糖苷酶的抑制动力学

β-N-乙酰-D-氨基葡萄糖苷酶与南美白对虾的食物消化吸收、蜕壳生长有着密切关系. 海水里存在的有机污染物将影响酶生理功能, 从而进一步影响虾的正常蜕壳,严重将导致对虾的死亡. 醋酸酐是常用的有机溶剂, 故本文应用动力学方法研究醋酸酐对南美白对虾β-N-乙酰-D-氨基葡萄糖苷酶催化pNP-NAG水解时酶活力的变化规律. 表明在醋酸酐浓度低于20.0 mmol/L, 酶的抑制作用是可逆的, 测得醋酸酐对酶抑制的IC50为9.0 mmol/L. 用双倒数作图法测定醋酸酐与游离酶(E)和酶-底物络合物(ES)的结合平衡常数, 结果显示醋酸酐是酶的非竞争性抑制剂. 用底物反应动力学方法观测在不同底物浓度下酶在0.0、3.0、6.0、9.0、12.0 mmol/L的醋酸酐溶液中的失活过程,分别测定了酶的微观失活速度常数k+0及复活速度常数k-0, 结果表明醋酸酐对酶的影响是快速结合再缓慢失活的过程. 比较微观失活速度常数k+0及复活速度常数k-0, 结果暗示在高浓度的醋酸酐溶液中, 酶将完全失活.     

Inhibitory kinetics of beta-N-acetyl-D-glucosaminidase from prawn (Litopenaeus vannamei) by zinc ion

Prawn (Litopenaeus vannamei) beta-N-acetyl-D-glucosaminidase (NAGase, EC 3.2.1.52) is involved in the digestion and molting processes. Zinc is one of the most important metals often found in the pollutant. In this article, the effects of Zn(2+) on prawn NAGase activity for the hydrolysis of pNP-NAG have been investigated. The results showed that Zn(2+) could reversibly and noncompetitively inhibit the enzyme activity at appropriate concentrations and its IC(50) value was estimated to be 6.00 +/- 0.25 mM. The inhibition model was set up, and the inhibition kinetics of the enzyme by Zn(2+) has been studied using the kinetic method of the substrate reaction. The inhibition constant was determined to be 11.96 mM and the microscopic rate constants were also determined for inactivation and reactivation. The rate constant of the inactivation (k(+0)) is much larger than that of the reactivation (k(-0)). Therefore, when the Zn(2+) concentration is sufficiently large, the enzyme is completely inactivated. On increasing the concentration of Zn(2+), the fluorescence emission peak and the UV absorbance peak are not position shifted, but the intensity decreased, indicating that the conformation of Zn(2+)-bound inactive NAGase is stable and different from that of native NAGase. We presumed that Zn(2+) made changes in the activity and conformation of prawn NAGase by binding with the histidine or cysteine residues of the enzyme.     

Kinetic study of the effects of calcium ions on cationic artichoke (Cynara scolymus L.) peroxidase: calcium binding, steady-state kinetics and reactions with hydrogen peroxide

The apparent catalytic constant (k(cat)) of artichoke (Cynara scolymus L.) peroxidase (AKPC) with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) increased 130-fold in the presence of calcium ions (Ca2+) but the affinity (K(m)) of the enzyme for ABTS was 500 times lower than for Ca2+-free AKPC. AKPC is known to exhibit an equilibrium between 6-aquo hexa-coordinate and penta-coordinate forms of the haem iron that is modulated by Ca2+ and affects compound I formation. Measurements of the Ca2+ dissociation constant (K(D)) were complicated by the water-association/dissociation equilibrium yielding a global value more than 1000 times too high. The value for the Ca2+ binding step alone has now been determined to be K(D) approximately 10 nM. AKPC-Ca2+ was more resistant to inactivation by hydrogen peroxide (H(2)O(2)) and exhibited increased catalase activity. An analysis of the complex H(2)O(2) concentration dependent kinetics of Ca2+-free AKPC is presented.     

太平洋白对虾(Penaeus vannamei)β-N-乙酰-D-氨基葡萄糖苷酶在二甲亚砜溶液中的失活动力学(英文)

应用动力学方法研究了太平洋白对虾(Penaeusvannamei)β-N-乙酰-D-氨基葡萄糖苷酶在二甲亚砜溶液中以pNP-β-D-GlcNAc为底物时酶活力的变化规律.表明酶在DMSO浓度低于4.20mol/L,酶的失活过程是可逆的,DMSO并不造成酶绝对量的减少,仅对酶的活力发生可逆的下降.测得DMSO对酶抑制的IC50为1.2mol/L.观测了在不同底物浓度下NAGase在0、0.35、0.70、1.05、1.40、1.75mol/L的DMSO溶液中的失活过程,分别测定了游离酶(E)和酶-底物络合物(ES)的微观失活速度常数k+0和k′+0比较结果(k+0值远远大于k′+0)表明,在DMSO溶液中游离酶比酶-底物络合物更易失活,即底物的存在对于酶被DMSO的失活具有明显的保护作用.随着DMSO浓度的增加,游离酶的逆向微观复活速度常数k-0却不断降低,说明在高浓度DMSO环境中,NAGase可逆恢复的能力逐渐微弱.     

The catalytic site of manganese peroxidase. Regiospecific addition of sodium azide and alkylhydrazines to the heme group.

Manganese peroxidase (MnP), which normally oxidizes Mn2+ to Mn3+, is rapidly and completely inactivated in an H2O2-dependent reaction by 2 equivalents of sodium azide. The inactivation is paralleled by formation of the azidyl radical and high yield conversion of the prosthetic heme into a meso-azido adduct. The meso-azido enzyme is oxidized by H2O2 to a Compound II-like species with the Soret band red-shifted 2 nm relative to that of native Compound II. The time-dependent decrease in this Compound II-like spectrum (t1/2 = 2.3 h) indicates that the delta-meso azido heme is more rapidly degraded by H2O2 than the prosthetic heme of control enzyme (t1/2 = 4.8 h). MnP is also inactivated by phenyl-, methyl-, and ethylhydrazine. The phenylhydrazine reaction is too rapid for kinetic analysis, but KI = 402 microM and kinact = 0.22/min for the slower inactivation by methylhydrazine. Reaction with phenylhydrazine at pH 4.5 does not yield iron-phenyl, N-phenyl, or meso-phenyl heme adducts. Ethylhydrazine inactivates the enzyme both at pH 4.5 and 7.0, but only detectably produces delta-meso-ethyl-heme at pH 7.0. Reconstitution of apo-MnP with hemin or delta-meso-ethylheme yields enzyme with, respectively, 50 and 5% of the native activity. The delta-meso-alkyl group thus suppresses most of the catalytic activity of the enzyme even though a Compound II-like species is still formed with H2O2. Finally, Co2+ inhibits the enzyme competitively with respect to Mn2+ but does not inhibit its inactivation by azide or the alkylhydrazines. The results argue that substrates interact with the heme edge in the vicinity of the delta-meso-carbon. They also suggest that Mn2+ and Co2+ bind to a common site close to the delta-meso-carbon without blocking the approach of small molecules to the heme edge. An active site model is proposed that accommodates these results.     

Inhibitory kinetics of mercuric ion on the activity of beta-N-acetyl-D-glucosaminidase from green crab (Scylla serrata)

Chemical modification of p-chloromercuribenzoate (PCMB) on beta-N-acetyl-d-glucosaminidase (NAGase, EC 3.2.1.52) from green crab (Scylla serrata) has been studied. The results show that sulfhydryl group is essential for the activity of the enzyme. Inhibitory kinetics of the enzyme by mercuric chloride (HgCl2) has been studied using the kinetic method of the substrate reaction during inhibitor of enzyme. The kinetic results show that the inhibition of the enzyme by mercuric ion (Hg2+) at lower than 1.0 microM is a reversible reaction with residual activity and the inhibition belongs to be competitive. The inhibition kinetics model of Hg2+ on the enzyme was set up and the microscopic rate constants were determined and the data obtained were well fitted with the model. It was also turned out that only one molecule of HgCl2 binds to the enzyme molecule to lead the enzyme lose its activity. The above results suggest that the cysteine residue is essential for activity and is situated at the active site of the enzyme.     

Transport of Ca2+ and Na+ across the chromaffin-granule membrane.

The soluble hydrogenase (hydrogen-NAD+ oxidoreductase, EC 1.12.1.2) of Alcaligenes eutrophus H16 was shown to be stabilized by oxidation with oxygen and ferricyanide as long as electron donors and reducing compounds were absent. The simultaneous presence of H2, NADH and O2 in the enzyme solution, however, caused an irreversible inactivation of hydrogenase that was dependent on the O2 concentration. The half-life periods of 4 degrees C under partial pressures of 0.1, 5, 20 and 50% O2 were 11, 5, 2.5 and 1.5 h respectively. Evidence has been obtained that hydrogenase produces superoxide free radical anions (O2-.), which were detected by their ability to oxidize hydroxylamine to nitrite. The correlation between O2 concentration, nitrite formation and inactivation rates and the stabilization of hydrogenase by addition of superoxide dismutase indicated that superoxide radicals are responsible for enzyme inactivation. During short-term activity measurements (NAD+ reduction, H2 evolution from NADH), hydrogenase activity was inhibited by O2 only very slightly. In the presence of 0.7 mM-O2 an inhibition of about 20% was observed.     

Unfolding and inactivation of Ampullarium crossean beta-glucosidase during denaturation by guanidine hydrochloride

Changes of activity and conformation of Ampullarium crossean beta-glucosidase in different concentrations of guanidine hydrochloride (GuHCl) have been studied by measuring the fluorescence spectra and its relative activity after denaturation. The fluorescence intensity of the enzyme decreased distinctly with increasing guanidine concentrations, the emission peaks appeared red shifted (from 338.4 to 350.8 nm), whereas a new fluorescence emission peak appeared near 310 nm. Changes in the conformation and catalytic activity of the enzyme were compared. A corresponding rapid decrease in catalytic activity of the enzyme was also observed. The extent of inactivation was greater than that of conformational changes, indicating that the active site of the enzyme is more flexible than the whole enzyme molecule. k(+0)>k(+0)' also showed that the enzyme was protected by substrate to a certain extent during guanidine denaturation.     

Alkyl Hydroperoxide Reductase Is the Primary Scavenger of Endogenous Hydrogen Peroxide in Escherichia coli

Hydrogen peroxide is generated during aerobic metabolism and is capable of damaging critical biomolecules. However, mutants of Escherichia coli that are devoid of catalase typically exhibit no adverse phenotypes during growth in aerobic media. We discovered that catalase mutants retain the ability to rapidly scavenge H(2)O(2) whether it is formed internally or provided exogenously. Analysis of candidate genes revealed that the residual activity is due to alkyl hydroperoxide reductase (Ahp). Mutants that lack both Ahp and catalase could not scavenge H(2)O(2). These mutants excreted substantial amounts of H(2)O(2), and they grew poorly in air. Ahp is kinetically a more efficient scavenger of trace H(2)O(2) than is catalase and therefore is likely to be the primary scavenger of endogenous H(2)O(2). Accordingly, mutants that lack Ahp accumulated sufficient hydrogen peroxide to induce the OxyR regulon, whereas the OxyR regulon remained off in catalase mutants. Catalase still has an important role in wild-type cells, because the activity of Ahp is saturated at a low (10(-5) M) concentration of H(2)O(2). In contrast, catalase has a high K(m), and it therefore becomes the predominant scavenger when H(2)O(2) concentrations are high. This arrangement is reasonable because the cell cannot provide enough NADH for Ahp to rapidly degrade large amounts of H(2)O(2). In sum, E. coli does indeed generate substantial H(2)O(2), but damage is averted by the scavenging activity of Ahp.     

α-淀粉酶在盐酸胍和碳酸胍变性中有关胍基作用的研究

利用紫外差谱、荧光光谱和园二色谱法对比地研究了α-淀粉酶盐酸胍和碳酸胍变性,分析了两种胍变性明显差异的原因。通过等同的胍基浓度下,α-淀粉酶两种胍变性的构象变化与活性关系的实验,表明同等摩尔浓度的两种胍盐变性能力上的明显差异并不主要是由于它们胍基含量上的不同。将盐酸胍从中性pH(6.5)调至碱性pH(10.4),其变性能力大增,紫外差谱与碳酸胍变性相似,出现了290nm的正肩和296nm的正峰,与此同时,酶的荧光强度大大降低,大部分酶活性丧失。由此推论,两种胍变性能力的明显差异的重要原因之一是在碱性介质中胍基的变性能力明显增强,并分析了其增强的原因。     

Functional effect of hydrogen peroxide on the sarcoplasmic reticulum membrane: uncoupling and irreversible inhibition of the Ca2+-ATPase protein

The chemical treatment of sarcoplasmic reticulum vesicles with H2O2 affects both Ca2+ transport and the hydrolytic activity supported by the Ca2+-ATPase protein. Ca2+ transport was much more sensitive to inhibition than ATPase activity and the decrease in Ca2+ transport was not the result of an increase in membrane permeability. The Ca2+/Pi uncoupling can be attributed to the own catalytic mechanism of the enzyme. Under conditions of high uncoupling, Ca2+ binding to the transport sites was barely affected and accumulation of phosphorylated species during the enzyme cycling gave almost maximal levels. These are features defining intramolecular uncoupling mediated by a phosphorylated form of the enzyme. Severe inhibition of the hydrolytic activity was observed when higher peroxide concentrations and leaky vesicles were used. These experimental conditions diminished maximal Ca2+ binding and the steady-state phosphoenzyme level. The low hydrolytic activity can be ascribed to a decrease in the rate of enzyme dephosphorylation.     

Bovine serum albumin in lithium chloride solutions.

The effects of li+ and H3O+ on the conformation of bovine serum albumin in azqueous solutions at room temperature are compared. At low pH (high concentration of H3O+) the change in conformation of the protein is demonstrated by an increase in effective volume, a decrease in helical content and a blue shift of tyrosyl residue. A similar change is observed for the protein in highly concentrated LiC1 solution (6.0-7.0M) at neutral pH. However, the H3O+ is 12,000 times more powerful than the Li+ in destabilizing the protein molecule. This is consistent with their thermodynamic and kinetic properties, since the H3O+ is often different from the Li+ in several orders of magnitude. While the changes in structural properties of the protein are almost identical in both the acidic solution and the highly concentrated LiC1 solution, further study using dioxane as a probe suggests different mechanisms under which the changes occur. The effect of H3O+ is related to electrostatic force, whereas the effect of Li+ is related to both the electrostatic hydrophobic forces. These two major forces are believed to be responsible for the conformation of protein molecules.