Modulation of activity of NADH oxidase from Thermus thermophilus through change in flexibility in the enzyme active site induced by Hofmeister series anions.

The conformational dynamics of NADH oxidase from Thermus thermophilus was modulated by the Hofmeister series of anions (H2PO4-, SO42-, CH3COO-, Cl-, Br-, I-, ClO4-, SCN-) in the concentration range 0-3 M. Both chaotropic and kosmotropic anions, at high concentration, inhibit the enzyme by different mechanisms. Chaotropic anions increase the apparent Michaelis constant and decrease the activation barrier of the reaction. Kosmotropic anions have the opposite effect. Anions from the middle of the Hofmeister series do not significantly affect the enzyme activity even at high concentration. We detected no significant changes in ellipticity of the aromatic region in the presence of the anions studied. There is a decreased Stern-Volmer quenching constant for FAD fluorescence quenching in the presence of kosmotropic anions and an increased quenching constant in the presence of chaotropic anions. All of this indicates that active site flexibility is important in the function of the enzyme. The data demonstrate that both the high rigidity of the active site in the presence of kosmotropic anions, and its high flexibility in the presence of chaotropic anions have a decelerating effect on enzyme activity. The Hofmeister series of anions proved to be suitable agents for altering enzyme activity through changes in flexibility of the polypeptide chain, with potential importance in modulating extremozyme activity at room temperature.     

Effects of chaotropic anions on bicuculline inhibition of high affinity γ-aminobutyric acid binding in bovine retina and rat brain membranes

Chaotropic anions (ions that favour transfer of apolar groups to water) increased bicuculline inhibition of ³H-γ-aminobutyric acid binding to bovine retinal membranes as previously reported for rat forebrain membranes. The increased bicuculline inhibition was reversible when the chaotropic anion was removed thus ruling out the possibility that ‘endogenous regulators’ were being removed by the chaotropic anions. Another possible explanation for the enhanced bicuculline inhibition is an increase in the solubility of bicuculline, an organic compound that is sparingly soluble in water; however, when bicuculline-methiodide, a more water soluble form of bicuculline was used, no difference was noted in this enhancement.Although the chaotropic anions temporarily increase the bicuculline inhibition of γ-aminobutyric acid binding, they do not increase ³H-γ-aminobutyric acid receptor binding as previously suggested. Thiocyanate or perchlorate have no effect on ³H-γ-aminobutyric acid receptor binding to rat forebrain or cerebellar membranes. Although thiocyanate slightly inhibits γ-aminobutyric acid receptor binding to bovine retinal membranes, perchlorate has no effect. The previous observation that sodium perchlorate enhanced γ-aminobutyric acid binding in bovine retina was due to the enhancement of sodium-dependent binding to a nipecotic acid-sensitive binding site (perhaps an uptake site).The mechanism of action of chaotropic anions in vitro on γ-aminobutyric acid binding may be through an alteration in the interaction of the receptor with the antagonists, bicuculline or bicuculline-methiodide, but is not through the removal of a component that blocks the binding site or regulates the receptor's properties, as evidenced by the reversibility of the chaotropic anion effect and the lack of effect on agonist binding.     

Effect of kosmotropicity of ionic liquids on the enzyme stability in aqueous solutions

This paper examined the effect of several pyridinium and imidazolium-based ionic liquids (ILs) on the protease stability in aqueous solutions. In general, the enzyme was found quite active at low concentrations of hydrophilic ILs. In aqueous environment, the enzyme was stabilized by the kosmotropic anions (such as CF3COO- and CH3COO-) and chaotropic cations (such as [BuPy]+ and [EMIM]+), but was destabilized by chaotropic anions (such as tosylate and BF4-) and kosmotropic cations (such as [BMIM]+).     

Absence of substrate inhibition and freezing-inactivation of the mosquito acetylcholinesterase are caused by alterations of hydrophobic interactions

Membrane-bound acetylcholinesterase (AChE) from mosquito showed the characteristic substrate inhibition of this enzyme, but 105,000 x g supernatants of freshly extracted enzyme did not. Addition of chaotropic anions, a freeze-thaw cycle and autolysis of the amphiphilic acetylcholinesterase to its non-amphiphilic derivatives resulted in return of the substrate inhibition feature along with an apparent increment in the enzyme activity. These results suggested that the lipidic environment of the mosquito AChE is temporarily perturbed when extracted. The enzyme is probably trapped in non-sedimenting mixtures composed of endogenous amphiphilic molecules. The occurrence of this phenomenon was not affected by the presence of Triton X-100 and other detergents, either alone or in combination with sodium chloride. Freezing in the presence of strong chaotropic anions (perchlorate, iodide and thiocyanate) caused the irreversible inactivation of the mosquito AChE. Crude and incomplete purified fractions of the enzyme were more sensitive than a more purified preparation. With both the purified AChE and the non-purified AChE, amphiphilic AChE was more freeze labile. Freezing at -10 degrees C enhanced inactivation of non-purified fractions. At this temperature, even weak chaotropic anions (fluoride, chloride and nitrate), while in combination with non-ionic detergents that solubilized mosquito AChE efficiently, reduced the enzyme activity of these fractions. In this case, recovery of the enzyme activity by incubation at 25 degrees C was inversely correlated with the effectiveness of the chaotropic anion. Gel filtration failed to show any change in the hydrodynamic radius of the freezing-inactivated AChE. Therefore, this phenomenon is explained as different degrees of denaturation of the enzyme in direct association with the chaotropic strength. Thus, antichaotropic anions, such as sulfate, should improve the stability of the mosquito acetylcholinesterase during extraction, purification and storage.     

Effect of the chaotropic anions thiocyanate and perchlorate on the entry of ricin into Vero cells.

The effect of different anions on the sensitivity of Vero cells to ricin was investigated. The cells were equally sensitive to ricin when NaCl was replaced by NaBr, NaI, Na2SO4 or with iso-osmotic concentrations of mannitol. In contrast, NaSCN and NaClO4 strongly protected against ricin at pH 7.2, but not at pH 7.6. The possibility that the protective effect is due to the ability of chaotropic anions to decrease the pH close to the membrane is discussed.     

The effect of chaotropic anions on the activation and the activity of spinach chloroplast fructose-1,6-bisphosphatase

The effect of chaotropic anions was studied on processes that constitute the chloroplast fructose-1,6-bisphosphatase reaction, i.e. enzyme activation and catalysis. The specific activity of chloroplast fructose-1,6-bisphosphatase was enhanced by preincubation with dithiothreitol, fructose 1,6-bisphosphate, Ca2+, and a chaotropic anion. When chaotropes were ranked in the order of increasing concentrations required for maximal activation they followed a lyotropic (Hofmeister) series: SCN- less than Cl3C-COO- less than ClO4- less than I- less than Br- less than Cl- less than SO4(2-). On the contrary, salts inhibited the catalytic step. The stimulation of chloroplast fructose-1,6-bisphosphatase by chaotropic anions arose from a decrease of the activation kinetic constants of both fructose 1,6-bisphosphate and Ca2+; on the other hand, in catalysis neutral salts caused a decrease of kcat because the S0.5 for both fructose 1,6-bisphosphate and Mg2+ remained unaltered. The molecular weight of chloroplast fructose-1,6-bisphosphatase did not change after the activation by incubation with dithiothreitol, fructose 1,6-bisphosphate, Ca2+, and a chaotrope; consequently, the action of these modulators altered the conformation of the enzyme. Modification in the relative position of aromatic residues of chloroplast fructose-1,6-bisphosphatase was detected by UV differential spectroscopy. In addition, the concerted action of modulators made the enzyme more sensitive to (a) trypsin attack and (b) S-carboxymethylation by iodoacetamide. These results provide a new insight on the mechanism of light-mediated regulation of chloroplast fructose-1,6-bisphosphatase; concurrently to the action of a sugar bisphosphate, a bivalent cation, and a reductant, modifications of hydrophobic interactions in the structure of chloroplast fructose-1,6-bisphosphatase play a crucial role in the enhancement of the specific activity.     

Effect of ions and other compatible solutes on enzyme activity, and its implication for biocatalysis using ionic liquids

The effect of ions on enzyme activity and stability usually follows the Hofmeister series (or the kosmotropicity order): kosmotropic anions and chaotropic cations stabilize enzymes while chaotropic anions and kosmotropic cations destabilize them. The effect of ionic liquids (ILs) on the enzyme activity/stability/enantioselectivity is complicated especially when there is no or little water presence in the IL media. However, when aqueous solutions of hydrophilic ILs are employed as reaction media, the enzyme seems to follow the Hofmeister series since ILs dissociate into individual ions in water.     

Effects of chaotropic agents versus detergents on dihydroorotate dehydrogenase.

As chaotropic salts are generally believed to affect water structure in a manner which increases lipophilicity of water, they may seem to be capable of substituting for detergents in the solubilization of particulate enzyme. Although solubilization either by detergents or by chaotropic salts has been demonstrated with several membrane proteins, the effects these agents have on the properties and activity of an enzyme may be quite different. This is illustrated by the effects on mammalian mitochondrial dihydroorotate dehydrogenase. Stability of the solubilized enzymic activity is dependnet on the presence of a detergent and maximum enzymic activity is observed at the critical micelle concentration of the detergent. Addition of low concentrations of various anions of the chaotropic series further enhances activity while higher concentrations of these anions, although increasing solubility of the enzyme, irreversibly inhibit catalysis.     

Mechanism of lysis of Escherichia coli by ethanol and other chaotropic agents.

Ethanol has been shown to inhibit the assembly of cross-linked peptidoglycan and to induce cell lysis in Escherichia coli. These effects of ethanol appear to result from the weakening of hydrophobic interactions by ethanol rather than from the intercalation of ethanol into membranes. Other chaotropic agents also inhibited cross-linking and induced lysis. The potency of chaotropic anions with regard to this effect followed the expected chaotropic series. Antichaotropic agents, which strengthened hydrophobic interactions, antagonized ethanol-induced lysis. The weakening of hydrophobic interactions by ethanol is proposed as a general mechanism by which ethanol and other chaotropic agents could affect membrane-associated enzyme activities.     

The chaotropic anions thiocyanate and nitrate inhibit the electric current through the tonoplast ATPase of isolated vacuoles from suspension cells of Chenopodium rubrum

Using the whole-vacuolar mode of the patch clamp technique, we studied the effect of the chaotropic anions thiocyanate and nitrate on the electric currents generated by the proton pumping tonoplast ATPase and pyrophosphatase (PPiase), respectively, in vacuoles from suspension cells of Chenopodium rubrum L. Addition of KNO₃ (150–250 m M ) or KSCN (70–150 m M ), and ATP (5 m M , obligatory) irreversibly inhibited the subsequent electric current through the tonoplast ATPase driven by 1 m M ATP, whereas PPiase-activity by 50 μ M PPi remained unaffected. The kinetics of inhibition, indicative of ATPase disintegration by the chaotropic anions, follows a single exponential (τ= 3.44 min). However, apparent ATPase disintegration did not measurably increase the tonoplast conductance. We conclude that, by contrast to organellar F-ATPases, upon disintegration the transmembrane proteolipid of the V-ATPase does not act as a proton conductor which, in the presence of chaotropic anions, like chloride or nitrate, would severely perturb solute compartmentation in the plant cell.     

A protein factor extracted from murine brains confers physiological Ca2+ sensitivity to exocytosis in sea urchin eggs.

Exocytosis in sea urchin eggs can be reconstituted in vitro using the cell ghosts (the isolated cortices). When the isolated cortices were handled in the medium primarily composed of non-chaotropic ions, exocytosis can be induced by a micromolar level of Ca2+. However, when the cortices are exposed to chaotropic anions such as Cl-, it is induced only at higher Ca2+ concentrations of 10(-5) to 10(-4) M, due to the chaotropic anionic effect, by which a specific protein(s) is dissociated from the cortex. The dissociated protein can be added back to the cortex to restore the original Ca2+ sensitivity [(1984) Dev. Biol. 101, 125-135]. A protein which has the similar effect on the isolated cortex was also found in the extract of murine brain. This protein was neither calmodulin, a G-protein or a kinase. The data suggest the general regulatory mechanism of the Ca2+ sensitivity of exocytosis by a protein factor widely distributed among cells.     

The use of chaotropic salts for separation of ribonucleic acids and proteins from yeast nucleoproteins

The effect of chaotropic salts on the dissociation of ribonucleic acid from yeast nucleoprotein complex was studied. The effectiveness of various salts on the dissociation of ribonucleic acid followed the chaotropic series; i.e., Cl(3)CCOONa = NaClO(4) > NaBr > NaCl. Treatment of the nucleoprotein complex with 0.5M Cl(3)CCOONa or NaClO(4) resulted in RNA removal of about 80%, whereas NaCl and NaBr removed only about 10 and 25%, respectively. Based on the results presented, a simple and novel method for industrial-scale preparation of single-cell proteins with low levels of nucleic acid is proposed.     

Effect of phosphate and other inorganic anions on the activity of chicken liver cytosolic aspartate aminotransferase

Chicken liver aspartate aminotransferase was inhibited by several inorganic anions. The inhibitory effect of the anions was related to their chaotropic character. Apparent Km (2-oxoglutarate) and Km (L-aspartate) values depended on the molarity of the buffer. The profile of the curves obtained did not depend on the nature of the enzyme sample assayed. Phosphate slightly inhibited the holoaspartate aminotransferase and was a strong inhibitor of apoaspartate aminotransferase with respect to pyridoxal phosphate.     

Ion-induced alterations of the local hydration environment elucidate Hofmeister effect in a simple classical model of Trp-cage miniprotein

Protein stability is known to be influenced by the presence of Hofmeister active ions in the solution. In addition to direct ion-protein interactions, this influence manifests through the local alterations of the interfacial water structure induced by the anions and cations present in this region. In our earlier works it was pointed out that the effects of Hofmeister active salts on the stability of Trp-cage miniprotein can be modeled qualitatively using non-polarizable force fields. These simulations reproduced the structure-stabilization and structure-destabilization effects of selected kosmotropic and chaotropic salts, respectively. In the present study we use the same model system to elucidate atomic processes behind the chaotropic destabilization and kosmotropic stabilization of the miniprotein. We focus on changes of the local hydration environment of the miniprotein upon addition of NaClO₄ and NaF salts to the solution. The process is separated into two parts. In the first, ‘promotion’ phase, the protein structure is fixed, and the local hydration properties induced by the simultaneous presence of protein and ions are investigated, with a special focus on the interaction of Hofmeister active anions with the charged and polar sites. In the second, ‘rearrangement’ phase we follow changes of the hydration of ions and the protein, accompanying the conformational relaxation of the protein. We identify significant factors of an enthalpic and entropic nature behind the ion-induced free energy changes of the protein-water system, and also propose a possible atomic mechanism consistent with the Collins’s rule, for the chaotropic destabilization and kosmotropic stabilization of protein conformation.     

Investigation of anion binding to neutral lipid membranes using 2H NMR.

The binding of aqueous anions (ClO4-, SCN-, I-, and NO3-) to lipid bilayer membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated using deuterium (2H) and phosphorus-31 (31P) nuclear magnetic resonance (NMR) spectroscopy. The ability of these anions to influence the 2H NMR quadrupole splittings of POPC, specifically labeled at the alpha or beta position of the choline head group, increased in the order NO3- much less than I- less than SCN- less than ClO4-. In the presence of these chaotropic anions, the quadrupole splitting increased for alpha-deuterated POPC and decreased for beta-deuterated POPC, indicating a progressive accumulation of negative charge at the membrane surface. Calibration of the 2H NMR quadrupole splittings with the amount of membrane-bound anion permitted binding isotherms to be generated for perchlorate, thiocyanate, and iodide, up to concentrations of 100 mM. The binding isotherms were analyzed by considering electrostatic contributions, according to the Gouy-Chapman theory, as well as chemical equilibrium contributions. For neutral POPC membranes, we obtained ion association constants of 32, 80, and 115 M-1 for iodide, thiocyanate, and perchlorate, respectively. These values increase in the order expected for a Hofmeister series of anions. We conclude that the factor determining whether a particular anion will bind to lipid bilayers is the ease with which that anion loses its hydration shell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of protic ionic liquids on the structure and stability of succinylated Con A

We report the synthesis of a series of ionic liquids (ILs) from various ions having different kosmotropicity including dihydrogen phosphate (H(2)PO(4)(-)), hydrogen sulfate (HSO(4)(-)) and acetate (CH(3)COO(-)) as anions and chaotropic cation such as trialkylammonium cation. To characterize the biomolecular interactions of ILs with protein, we have explored the stability of succinylated Con A (S Con A) in the presence of these aqueous ILs, which are varied combinations of kosmotropic anion with chaotropic cation such as triethylammonium dihydrogen phosphate [(CH(3)CH(2))(3)NH][H(2)PO(4)] (TEAP), trimethylammonium acetate [(CH(3))(3)NH][CH(3)COO] (TMAA), trimethylammonium dihydrogen phosphate [(CH(3))(3)NH][H(2)PO(4)] (TMAP) and trimethylammonium hydrogen sulfate [(CH(3))(3)NH][HSO(4)] (TMAS). Circular dichroism (CD) and fluorescence experiments have been used to characterize the stability of S Con A by ILs. Our data distinctly demonstrate that the long alkyl chain IL TEAP is a strong stabilizer for S Con A. Further, our experimental results reveal that TEAP is an effective refolding enhancer for S Con A from a thermally denatured protein structure.     

Influence of counterions on the interaction of pyridinium salts with model membranes

The interaction of pyridinium salts (PS) with red blood cells and planar lipid membranes was studied. The aim of the work was to find whether certain cationic surfactant counterion influence its possible biological activity. The counterions studied were Cl-, Br-, I-, ClO4-, BF4- and NO3-. The model membranes used were erythrocyte and planar lipid membranes (BLM). At high concentration the salts caused 100% erythrocyte hemolysis (C100) or broke BLMs (CC). Both parameters describe mechanical properties of model membranes. It was found that the efficiency of the surfactant to destabilize model membranes depended to some degree on its counterion. In both, erythrocyte and BLM experiments, the highest efficiency was observed for Br-, the lowest for NO3-. The influence of all other anions on surfactant efficiency changed between these two extremities; that of chloride and perchlorate ions was similar. Some differences were found in the case of BF4- ion. Its influence on hemolytic possibilities of PS was significant while BLM destruction required relatively high concentration of this anion. Apparently, the influence of various anions on the destructive action of PS on the model membrane used may be attributed to different mobilities and radii of hydrated ions and hence, to different possibilities of particular anions to modify the surface potential of model membranes. This can lead to a differentiated interaction of PS with modified bilayers. Moreover, the effect of anions on the water structure must be taken into account. It is important whether the anions can be classified as water ordering kosmotropes that hold the first hydration shell tightly or water disordering chaotropes that hold water molecules in that shell loosely.     

Ion selectivity of aqueous leaks induced in the erythrocyte membrane by crosslinking of membrane proteins

The aqueous leak induced in the human erythrocyte membrane by crosslinking of spectrin via disulfide bridges formed in the presence of diamide (Deuticke, B., Poser, B., Lütkemeier, P. and Haest, C.W.M. (1983) Biochim. Biophys. Acta 731, 196-210) was further characterized with respect to its ion selectivity by means of (a) measurements of cell volume changes or hemolysis, (b) determination of membrane potentials and (c) analysis of potential-driven ion fluxes. The leak turned out to be slightly cation-selective (PK:PCl approximately equal to 4:1). It discriminates mono- from divalent ions (PNa:PMg greater than 100:1, PCl:PSO4 greater than 10:1) and to a much lesser extent monovalent ions among each other. The selectivities for monovalent ions follow the sequence of free solution mobilities, increasing in the order Li+ less than or equal to Na+ less than K+ less than or equal to Rb+ less than Cs+ and F- less than Cl- less than Br- less than I-. Polyatomic anions also fit into that order. Quantitatively, the ratios of permeabilities of the leak are larger than those of the ion mobilities in free solution. The ion permeability of the leak is concentration-independent up to at least 150 mM. The ion milieu, however, has marked effects on leak permeability, most pronounced for chaotropic ions (guanidinium, nitrate, thiocyanate), which increase leak fluxes of charged and uncharged solutes. The results support the view that, besides geometric constraints, weak coulombic or dipolar interactions between penetrating ions and structural elements of the leak determine permselectivity.     

Flexibility and enzyme activity of NADH oxidase from Thermus thermophilus in the presence of monovalent cations of Hofmeister series

Recently, we have shown that anions of Hofmeister series affect the enzyme activity through modulation of flexibility of its active site. The enzyme activity vs. anion position in Hofmeister series showed an unusual bell-shaped dependence. In the present work, six monovalent cations (Na(+), Gdm(+), NH(4)(+), Li(+), K(+) and Cs(+)) of Hofmeister series with chloride as a counterion have been studied in relation to activity and stability of flavoprotein NADH oxidase from Thermus thermophilus (NOX). With the exception of strongly chaotropic guanidinium cation, cations are significantly less effective in promoting the Hofmeister effect than anions mainly due to repulsive interactions of positive charges around the active site. Thermal denaturations of NOX reveal unfavorable electrostatic interaction at the protein surface that may be shielded to different extent by salts. Michaelis-Menten constants for NADH, accessibility of the active site as reflected by Stern-Volmer constants and activity of NOX at high cation concentrations (1-2 M) show bell-shaped dependences on cation position in Hofmeister series. Our analysis indicates that in the presence of kosmotropic cations the enzyme is more stable and possibly more rigid than in the presence of chaotropic cations. Molecular dynamic (MD) simulations of NOX showed that active site switches between open and closed conformations [J. Hritz, G. Zoldak, E. Sedlak, Cofactor assisted gating mechanism in the active site of NADH oxidase from Thermus thermophilus, Proteins 64 (2006) 465-476]. Enzyme activity, as well as substrate binding, can be regulated by the salt mediated perturbation of the balance between open and closed forms. We propose that compensating effect of accessibility and flexibility of the enzyme active site leads to bell-shaped dependence of the investigated parameters.     

Conformational study of globulin from rice (Oryza sativa) seeds by Fourier-transform infrared spectroscopy

The conformation of rice globulin (10%, w/v, in deuterated phosphate buffer, pD 7.4) under the influence of pH, chaotropic salts, several protein structure perturbants and heat treatments was studied by Fourier-transform infrared (FTIR) spectroscopy. Rice globulin exhibited seven major bands in the region of 1700-1600 cm-1 and the spectrum suggests high alpha-helical content with large quantities of beta-sheet and beta-turn structures. Highly acidic and alkaline pH conditions induced changes in band intensity attributed to intermolecular beta-sheet structure (1681 and 1619 cm-1). Addition of chaotropic salts led to progressive changes in band intensity, following the lyotropic series of anions, whereas several protein structure perturbants caused shifts in band positions. Heating at increasing temperature led to progressive decreases in alpha-helical content and increases in random coil structures, suggesting protein denaturation. This was accompanied by intensity increases in the intermolecular beta-sheet transitions.