Influence of NaCl and sorbitol on the stability of conformations of cytochrome c

Influence of ionic (NaCl) and non-ionic (sorbitol) additives on structural transitions of cytochrome c was investigated by circular dichroism, optical and EPR spectroscopy. Transformations of cytochrome c, induced by the acidification of solution and temperature perturbation, were monitored in the heme pocket together with changes in the secondary structure. NaCl and sorbitol exhibited antagonistic effect on the acid-induced transition of the protein. Sorbitol enhanced the stability of native conformation while NaCl destabilized this state. The midpoints of acid-induced transitions in the axial coordination of heme as well as in the secondary structure occurred nearly at the same pH values. However, temperature-induced transitions in the unfolding of the secondary structure were almost coincidental with the cleavage of Met80–Fe bond only in the sorbitol solutions. In the salt solution the Met80–Fe bond was markedly more labile than the secondary structure.     

Metal ions- and pH-induced conformational changes of acutolysin A from Agkistrodon acutus venom probed by fluorescent spectroscopy

Acutolysin A isolated from the venom of Agkistrodon acutus is a protein of 22 kDa with marked haemorrhagic and proteolytic activities. The metal ions- and pH-induced conformational changes of acutolysin A have been studied by following fluorescence and activity measurements. Here, we provide evidence for the fact that native holo-acutolysin A adopts two subtly different conformations, native state a (Na) stable in the weak acidic pH range from 6.0 to 7.0 with low activity and native state b (Nb) stable in the weak alkaline pH range from 7.5 to 9.0 with high activity. Holo-acutolysin A has an optimum pH of 8.5 for caseinolytic activity, and the protein adopts the most stable conformation with the maximum fluorescence at pH 8.5. The Ca2+ and Zn2+ ions have significant effects on both the pH-induced denaturing transition curve and the pH-dependent activity curve. Addition of 1 mM Ca2+ to holo-acutolysin A shifts both the acid-induced denaturing transition curve and the end zone of acid-induced inactivation curve towards lower pH value, and shifts both the alkali-induced denaturing transition curve and the end zone of alkali-induced inactivation curve towards higher pH value. Addition of 1 mM Zn2+ also shifts both the alkali-induced denaturing transition curve and the end zone of alkali-induced inactivation curve towards higher pH value and shifts the acid-induced denaturing transition curve to lower pH value, but has little effect on the acid-induced inactivation. Removal of Ca2+ and Zn2+ from the protein enhances its sensitivity to pH and significantly reduces its overall stability during acid-induced denaturation. It is also evident from the present work that the free Zn2+ -induced inactivation in the pH range from 8.0 to 9.0 should be attributed to the effect of Zn(OH)2 precipitation on the protein.     

Detection and characterization of sorbitol dehydrogenase from apple callus tissue

Sorbitol dehydrogenase (l-iditol:NAD(+) oxidoreductase, EC 1.1.1.14) has been detected and characterized from apple (Malus domestica cv. Granny Smith) mesocarp tissue cultures. The enzyme oxidized sorbitol, xylitol, l-arabitol, ribitol, and l-threitol in the presence of NAD. NADP could not replace NAD. Mannitol was slightly oxidized (8% of sorbitol). Other polyols that did not serve as substrate were galactitol, myo-inositol, d-arabitol, erythritol, and glycerol. The dehydrogenase oxidized NADH in the presence of d-fructose or l-sorbose. No detectable activity was observed with d-tagatose. NADPH could partially substitute for NADH.Maximum rate of NAD reduction in the presence of sorbitol occurred in tris(hydroxymethyl)aminomethane-HCl buffer (pH 9), or in 2-amino-2-methyl-1,3-propanediol buffer (pH 9.5). Maximum rates of NADH oxidation in the presence of fructose were observed between pH 5.7 and 7.0 with phosphate buffer. Reaction rates increased with increasing temperature up to 60 C. The K(m) for sorbitol and xylitol oxidation were 86 millimolar and 37 millimolar, respectively. The K(m) for fructose reduction was 1.5 molar.Sorbitol oxidation was completely inhibited by heavy metal ions, iodoacetate, p-chloromercuribenzoate, and cysteine. ZnSO(4) (0.25 millimolar) reversed the cysteine inhibition. It is suggested that apple sorbitol dehydrogenase contains sulfhydryl groups and requires a metal ion for full activity.     

Metal-ion- and pH-induced conformational changes of acutolysin D from Agkistrodon acutus venom probed by fluorescent spectroscopy

Acutolysin D isolated from the venom of Agkistrodon acutus is a protein of 44 kDa with marked hemorrhagic and proteolytic activities. The metal-ion- and pH-induced conformational changes of acutolysin D have been studied by following fluorescence and activity measurements. Here we provide evidence for the fact that native holo-acutolysin D adopts two different conformations, native state a, stable in the weak acidic pH range from 5.5 to 7.0 with low activity, and native state b, stable in the weak alkaline pH range from 8.0 to 9.0 with high activity. Holo-acutolysin D has an optimum pH of 9.0 for caseinolytic activity and a maximum fluorescence at pH 9.0. The protein adopts the most stable conformation at pH 9.0. The addition of 1 mM Zn(2+) shifts both the alkali-induced unfolding transition curve and the alkali-induced inactivation curve toward higher pH value but has little effect on the acid-induced unfolding transition curve. No obvious effects on the pH-induced unfolding transition curve and the pH-dependent activity curve have been observed after the addition of 1 mM Ca(2+) to holo-acutolysin D. The results indicate that Zn(2+) is essential for its CA, while Ca(2+) is not essential for its CA. Removal of Ca(2+) and Zn(2+) from the protein enhances its sensitivity to pH and significantly reduces its overall stability during acid-induced denaturation. The kinetic results of the demetalization of holo-acutolysin D show that the demetalization rate constant K(1) for a slower reaction linearly decreases with the pH increase from 5.0 to 9.0, while K(2) for the faster reaction linearly increases with the pH change from 5.0 to 7.0. It is also evident from the present work that the free Zn(2+)-induced inactivation in the pH range from 8.0 to 9.0 should be attributed to the effect of Zn(OH)(2) precipitation on the protein.     

Protonation-dependent inactivation of Na,K-ATPase by hydrostatic pressure developed at high-speed centrifugation

Irreversible inactivation of membranous Na,K-ATPase by high-speed centrifugation in dilute aqueous solutions depends markedly on the protonation state of the protein. Pig kidney Na,K-ATPase is irreversibly inactivated at pH 5 but is fully protected at pH 7 and above. Shark rectal gland Na,K-ATPase is irreversibly inactivated at neutral or acidic pH and partially protected at an alkaline pH. The overall Na,K-ATPase activity and the K-dependent pNPPase activity were denatured in parallel. Cryoprotectants such as glycerol or sucrose at concentrations of 25-30% fully protect both enzymes against inactivation. The specific ligands NaCl and KCl protect the Na,K-ATPase activity partially and the pNPPase activity fully at concentrations of 0.2-0.3 M. Electron microscope analysis of the centrifuged Na,K-ATPase membranes revealed that the ultrastructure of the native membranes is preserved upon inactivation. It was also observed that the sarcoplasmic reticulum Ca-ATPase and hog gastric H, K-ATPase are susceptible to inactivation by high-speed centrifugation in a pH-dependent fashion. H,K-ATPase is protected at alkaline pH, whereas Ca-ATPase is protected only in the neutral pH range.     

Purification and characterization of a NAD+-dependent sorbitol dehydrogenase from Japanese pear fruit

NAD+-dependent sorbitol dehydrogenase NAD-SDH, EC 1.1.1.14) from Japanese pear fruit was purified to apparent homogeneity (single band by SDS-PAGE with silver staining), and had a specific activity of 916.7 nKatal/mg protein. The molecular of the native enzyme was calculated to be 160 kDa by gel filtration, whereas SDS-PAGE gave a subunit size of 40 kDa, indicating that the native enzyme is a homotetramer. The protein immunologically reacted with an antibody raised in rabbit against the fusion protein expressed in E. coli harboring an apple NAD-SDH cDNA. The Km, values for sorbitol and fructose were 96.4+/-8.60 and 4239+/-33.5 mM, respectively, and optimum pH for sorbitol oxidation was 9.0 and 7.0 for fructose reduction. Pear NAD-SDH had a very narrow substrate specificity, that is, sorbitol, L-iditol, xylitol and L-threitol were oxidized but not any of the other alcohols tested. These data suggest the structural importance of an S configuration at C-2 and an R configuration at C-4 in the substrate(s). Its enzymatic activity was strongly inhibited both by heavy metal ions such as mercury, and by thiol compounds, such as L-cysteine. However, the addition of zinc ion reversed the enzyme inactivation caused by addition of L-cysteine.     

Biochemical characteristics of erythrocyte sorbitol dehydrogenase from selected mammals

The erythrocyte sorbitol dehydrogenase (EC 1.1.1.14) activity, regarding its action on sorbitol oxidation to fructose, was studied in 19 species of mammals, showing a striking variability, with high activity in rodents. Enzyme activity was studied against other polyols, namely xylitol, inositol, manitol and dulcitol. Most animals showed activity against all the polyols studied, but hamster and red deer only presented activity on sorbitol and xylitol. Michaelis-Menten constant determinations for sorbitol were performed, and it was observed that animals which presented high activity had a high Km. pH curves were obtained from 8 animals, with an optimum pH ranging from pH 8.0 to pH 10.0; four of the animals presented an optimum pH at 8.5.     

An in vitro study of the pH-lowering potential of salivary lactobacilli associated with dental caries

AIMS: Lactobacilli are known to produce acids from sucrose or glucose. This acid production can cause a drop in pH which is sufficiently significant to demineralize the hard tissues of the teeth. Some authors have demonstrated the benefits of substituting sorbitol or xylitol for sucrose. The aim of this work was to study the acid production of salivary lactobacilli with one test sugar (glucose) and two polyols (sorbitol and xylitol). METHODS AND RESULTS: The pH-lowering potential of three strains of oral lactobacilli was recorded with glucose or one of the polyols at three different concentrations. The results showed that polyols were broken down by certain strains of lactobacilli. When this degradation took place, the pH dropped to values sufficiently low to demineralize the hard tissues of the teeth. CONCLUSIONS: Further studies must be carried out on the metabolism of polyols before encouraging their widespread substitution for sucrose.     

More stable structure of wheat germ lipase at low pH than its native state

Wheat germ lipase is a cereal lipase which is a monomeric protein. In the present study we sought to structurally characterize this protein along with equilibrium unfolding in solution. Conformational changes occurring in the protein with varying pH, were monitored by circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy, binding of hydrophobic dye, 1-anilino 8-naphthalenesulfonic acid (ANS) and dynamic light scattering (DLS). Our study showed that acid denaturation of lipase lead to characterization of multiple monomeric intermediates. Native protein at pH 7.0 showed far-UV spectrum indicating mixed structure with both alpha and beta-type of characteristics. Activity of lipase was found to fall on either sides of pH 7.0–8.0. Acid-unfolded state was characterized at pH 4.0 with residual secondary structure, disrupted tertiary spectrum and red-shifted fluorescence spectrum with decreased intensity. Further decrease in pH lead to formation of secondary structure and acid-induced molten globule state was found to be stabilized at pH 1.4, with exposed tryptophan residues and hydrophobic patches. Notably, interesting finding of this study was characterization of acid-induced state at pH 0.8 with higher secondary structure content than native lipase, regain in tertiary spectrum and induction of compact conformation. Although enzymatically inactive, acid-induced state at pH 0.8 was found to be structurally more stable than native lipase, as shown by chemical and thermal denaturation profiles.     

Temperature dependence of the preferential interactions of ribonuclease A in aqueous co-solvent systems: thermodynamic analysis.

The temperature dependence of preferential solvent interactions with ribonuclease A in aqueous solutions of 30% sorbitol, 0.6 M MgCl2, and 0.6 M MgSO4 at low pH (1.5 and 2.0) and high pH (5.5) has been investigated. This protein was stabilized by all three co-solvents, more so at low pH than high pH (expect 0.6 M MgCl2 at pH 5.5). The preferential hydration of protein in all three co-solvents was high at temperatures below 30 degrees C and decreased with a further increase in temperature (for 0.6 M MgCl2 at pH 5.5, this was not significant), indicating a greater thermodynamic instability at low temperature than at high temperature. The preferential hydration of denatured protein (low pH, high temperature) was always greater than that of native protein (high pH, high temperature). In 30% sorbitol, the interaction passed to preferential binding at 45% for native ribonuclease A and at 55 degrees C for the denatured protein. Availability of the temperature dependence of the variation with sorbitol concentration of the chemical potential of the protein, (delta mu(2)/delta m3)T,p,m2, permitted calculation of the corresponding enthalpy and entropy parameters. Combination with available data on sorbitol concentration dependence of this interaction parameter gave (approximate) values of the transfer enthalpy, delta H2,tr, and transfer entropy delta S2,tr. Transfer of ribonuclease A from water into 30% sorbitol is characterized by positive values of the transfer free energy, transfer enthalpy, transfer entropy, and transfer heat capacity. On denaturation, the transfer enthalpy becomes more positive. This increment, however, is small relative to both the enthalpy of unfolding in water and to the transfer enthalpy of the native protein from water a 30% sorbitol solution.     

Bile salt hydrolase, the member of Ntn-hydrolase family: differential modes of structural and functional transitions during denaturation

Conformational transitions and functional stability of the bile salt hydrolase (BSH; cholylglycine EC: 3.5.1.24) from Bifidobacterium longum (BlBSH) cloned and expressed in E. coli were studied under thermal, chemical and pH-mediated denaturation conditions using fluorescence and CD spectroscopy. Thermal and Gdn-HCl-mediated denaturation of BlBSH is a multistep process of inactivation and unfolding. The inactivation and unfolding of the enzyme was found to be irreversible. Enzyme activity seems sensitive to even minor conformational changes at the active site. Thermal denaturation as such did not result in any insoluble protein aggregates. However, on treating with 0.25 - 1 M Gdn-HCl the enzyme showed increasing aggregation at temperatures of 40 - 55 degrees C indicating more complex structural changes taking place in the presence of chemical denaturants. The enzyme secondary structure was still intact at acidic pH (pH 1 - 3). The perturbation in the tertiary structure at the acidic pH was detected through freshly formed solvent exposed hydrophobic patches on the enzyme. These changes could be due to the formation of an acid-induced molten globule-like state.     

Sorbitol as the Primary Carbon Source for the Growth of Embryogenic Callus of Maize

The effects of various carbon sources on initiation and maintenance of embryogenic callus of maize (Zea mays L.) and on the regeneration of plants from embryogenic callus were studied. Growth of embryogenic callus tissue on media containing sucrose was typified by the subsequent growth of both embryogenic (regenerable) and nonembryogenic (nonregenerable) callus. Growth of embryogenic callus on sorbitol was unique among the carbon sources tested in that sorbitol supported the subsequent growth of only embryogenic callus. Further experiments demonstrated that embryogenic callus grown on sorbitol had a greater regenerative capacity (more plants produced per gram fresh weight of callus) than callus grown on sucrose. Sorbitol dehydrogenase was detected in embryogenic callus of maize at a specific activity roughly equivalent to that found in zygotic embryos of developing seeds. Nonembryogenic callus did not contain significant levels of sorbitol dehydrogenase activity.     

Photodynamic Action on Native and Denatured Transforming Deoxyribonucleic Acid from Haemophilus influenzae

The photodynamic inactivation of native or denatured transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae is described. The inactivation at the same pH was higher for denatured than native DNA. At acidic pH, the inactivation both for native and denatured DNA was faster than at alkaline pH. The guanine content of photoinactivated native DNA at neutral pH was less than untreated DNA. The inactivation of biological activity was more extensive than the alteration of guanine. The absorption spectrum of photoinactivated native or denatured DNA was only slightly different than the control DNA at the different experimental conditions.     

Further improvement of the ACD-AG protective solution for blood. III. Improvement of the oxygen transport function of erythrocytes in sorbitol xylitol pyruvate solutions with elevated Ph]

If the pH value in the ACD or in the ACD-AG storage solution is enhanced, the glucose in the autoclaving with undergo a caramelizing process. For this reason glucose was replaced by sorbite in the storage solutions with a pH value of 6.0 and additions of xylitol and pyruvate. The initial pH value in the blood amounted to 7.3. The content of 2.3 DPG of the erythrocytes remained fully preserved in the blood with sorbitol and additions of xylitol and pyruvate during the first 2 weeks of storage and decreased to 30% only in the third week. There were only slight amounts of 2.3 DPG in the ACD-AG blood at that time of storage. Up to the third week of storage the ATP content of erythrocytes as well as the haemoglobin level in the plasma revealed no essential differences between stored blood with sorbitol and xylitol as a substrate or glucose + xylitol respectively. The quick decrease of the ATP level to zero and the simultaneous strong increase of haemolysis in the sorbitol blood within the fourth week of storage is discussed in connection with a lowering of the NAD/NADH2 quotient. For the purpose of keeping the 2.3 DPG level of erythrocytes a storage solution with sorbite and xylitol (ASCX-AG-Pyr 10mM) seems to be well suited for a storing time of 2---3 weeks at first.     

The effect of chemical crosslinking of invertase with dimethyl suberimidate on its pH stability

The effect of chemical crosslinking of invertase with a homo-bifunctional bisimidoester on its pH stability was studied. Dimethyl suberimidate (DMS) was used as the crosslinker and pH inactivation was investigated in the pH range of 2.5–10. The inactivation mechanisms of both native and DMS crosslinked invertases were observed to follow first-order kinetics during prolonged incubation. Although DMS crosslinking of invertase increased the pH stability of the enzyme over the complete pH range, it was especially effective over the acidic range. The half-lives of invertase increased almost twofold, on average, by crosslinking at the neutral to the acidic range. The effect of crosslinking was especially pronounced at pH 5 since both the half-life of the native invertase and the stabilization factor were very high. Higher activation free energies of inactivation were obtained for DMS crosslinked invertases over the whole pH range which also indicates the stabilization of invertase by crosslinking.     

Fermentation pattern of Zymomonas mobilis at high sucrose concentrations

Summary Zymomonas mobilis was grown in batch concentrations between 200 and 400 g/l sucrose. The fermentation pattern revealed that the efficiency of sucrose hydrolysis dropped only from 94 to 78.6% whereas the efficiency with which the hydrolyzed products were converted to ethanol decreased from 94 to 43%. The ethanol yields were relatively constant for final concentrations which lay between 80 and 132 g/l. Fermentation times increased to 72 hours at the higher sucrose concentrations. Sorbitol and fructose were identified as the major by-products. Preliminary evidence suggests that the ratio between the two by-products depends on the pH of the culture medium. Results suggest the possibility of processes producing ethanol plus fructose, ethanol plus fructose and sorbitol, or ethanol plus sorbitol in a single-stage batch fermentation.     

Hydrophobic core variant ubiquitin forms a molten globule conformation at acidic pH

The conformational properties of hydrophobic core variant ubiquitin (Val26 to Ala mutation) in an acidic solution were studied. The intrinsic tryptophan fluorescence emission spectrum, far-UV and near-UV circular dichroic spectra, the fluorescence emission spectrum of 8-anilinonaphthalene-1-sulfonic acid in the presence of V26A ubiquitin, and urea-induced unfolding measurements indicate this variant ubiquitin to be in the partially folded molten globule conformation in solution at pH 2. The folding kinetics from molten globule to the native state was nearly identical to those from the unfolded state to the native state. This observation suggests that the equilibrium molten globule state of hydrophobic core variant ubiquitin is an on-pathway folding intermediate.     

Testing polyols' compatibility with Gibbs energy of stabilization of proteins under conditions in which they behave as compatible osmolytes

It is generally believed that compatible osmolytes stabilize proteins by shifting the denaturation equilibrium, native state <--> denatured state toward the left. We show here that if osmolytes are compatible with the functional activity of the protein at a given pH and temperature, they should not significantly perturb this denaturation equilibrium under the same experimental conditions. This conclusion was reached from the measurements of the activity parameters (K(m) and k(cat)) and guanidinium chloride-induced denaturations of lysozyme and ribonuclease-A in the presence of five polyols (sorbitol, glycerol, mannitol, xylitol and adonitol) at pH 7.0 and 25 degrees C.