Purification and characterization of cysteine protease from miswak Salvadora persica.

BACKGROUND: Generally, proteases in medicinal plants had different therapeutic effects such as anti-inflammatory effect; modulate the immune response and inhibitory effect toward tumor growth. In this study, protease was purified and characterized from miswak roots, as medicinal plant and natural toothbrush. RESULTS: Physical and chemical characterization of cysteine protease P1 were studied such as pH optimum (6.5), optimum temperature (50 °C), thermal stability (50 °C) and Km (3.3 mg azocasein/ml). The enzyme digested some proteins in the order of caseine > haemoglobin > egg albumin >gelatin > bovine serum albumin. Hg2+ had strong inhibitory effect on enzyme activity compared with other metal ions. Kinetic of inhibition for determination the type of protease was studied. Iodoactamide and p-Hydroximercuribenzaoic acid (p-HMB) caused strong inhibitory effect on enzyme activity indicating the enzyme is cysteine protease. CONCLUSIONS: The biochemical characterization of this enzyme will be display the suitable conditions for using of this enzyme in toothpaste in the future and the enzyme may be used in other applications.

Ovalbumin labeling with p-hydroxymercurybenzoate: The effect of different denaturing agents and the kinetics of reaction.

The aim of our study was to investigate how denaturing agents commonly used in protein analysis influence the labeling between a reactive molecule and proteins. For this reason, we investigated the labeling of ovalbumin (OVA) as a globular model protein with p-hydroxymercurybenzoate (pHMB) in its native state (phosphate buffer solution) and in different denaturing conditions (8 molL(-1) urea, 3 molL(-1) guanidinium thiocyanate, 6 molL(-1) guanidinium chloride, 0.2% sodium dodecyl sulfate, and 20% methanol). In addition to chemical denaturation, thermal denaturation was also tested. The protein was pre-column simultaneously denatured and derivatized, and the pHMB-labeled denatured OVA complexes were analyzed by size exclusion chromatography (SEC) coupled online with chemical vapor generation-atomic fluorescence spectrometry (CVG-AFS). The number of -SH groups titrated greatly depends on the protein structure in solution. Indeed, we found that, depending on the adopted denaturing conditions, OVA gave different aggregate species that influence the complexation process. The results were compared with those obtained by a common alternative procedure for the titration of -SH groups that employs monobromobimane (mBBr) as tagging molecule and molecular fluorescence spectroscopy as detection technique. We also investigated the labeling kinetics for denatured OVA and pHMB, finding that the 4 thiolic groups of OVA have a very different reactivity toward mercury labeling, in agreement with previous studies.

Differential effects of N-peptidyl-O-acyl hydroxylamines on dynorphin-induced antinociception in the mouse capsaicin test.

In the capsaicin test, intrathecal (i.t.) dynorphins are antinociceptive. Cysteine protease inhibitors such as p-hydroxymercuribenzoate (PHMB) given i.t. augment and prolong their activity. The effect of two novel cysteine protease inhibitors, N-peptidyl-O-acyl hydroxylamines, on the antinociception induced by i.t. administered dynorphin A or dynorphin B has been investigated. When administered i.t. 5 min before the injection of capsaicin (800 ng) into the plantar surface of the hindpaw, dynorphin A (62.5-1000 pmol) or dynorphin B (0.5-4 nmol) produced a dose-dependent and significant antinociceptive effect. The effect of dynorphin A (1 nmol) and dynorphin B (4 nmol) disappeared completely within 180 and 60 min, respectively. PHMB (2 nmol) and Boc-Tyr-Gly-NHO-Bz (BYG-Bz) (2 nmol) co-administered with dynorphin A or dynorphin B significantly prolonged antinociception induced by both. On the other hand, Z-Phe-Phe-NHO-Bz (ZFF-Bz) (1 and 2 nmol) only prolonged antinociception induced by dynorphin A. The results suggest that Z-Phe-Phe-NHO-Bz is an inhibitor of cysteine proteases preferring cleavage of dynorphin A, with less specificity towards dynorphin B in the mouse spinal cord.

Protective action of CLA against oxidative inactivation of paraoxonase 1, an antioxidant enzyme.

The effect of CLA on paraoxonase 1 (PON1), one of the antioxidant proteins associated with HDL, was investigated for its protective action against oxidative inactivation as well as its stabilization activity. When cis-9 (c9),trans-11 (t11)-CLA and t10,c12-CLA were examined for their protective activity against ascorbate/Cu(2+)-induced inactivation of PON1 in the presence of Ca2+, two CLA isomers exhibited a remarkable protection (Emax, 71-74%) in a concentration-dependent manner (50% effective concentration, 3-4 microM), characterized by a saturation pattern. Such a protective action was also reproduced with oleic acid, but not linoleic acid. Rather, linoleic acid antagonized the protective action of CLA isomers in a noncompetitive fashion. Additionally, the two CLA isomers also protected PON1 from oxidative inactivation by H2O2 or cumene hydroperoxide. The concentration-dependent protective action of CLA against various oxidative inactivation systems suggests that the protective action of CLA isomers may be mediated through their selective binding to a specific binding site in a PON1 molecule. Separately, the inactivation of PON1 by p-hydroxymercuribenzoate (PHMB), a modifier of the cysteine residue, was also prevented by CLA isomers, suggesting the possible existence of the cysteine residue in the binding site of CLA. The c9,t11-CLA isomer seems to be somewhat more effective than t10,c12-CLA in protecting against the inactivation of PON1 by either peroxides or PHMB, in contrast to the similar efficacy of these two CLA isomers in preventing ascorbate/Cu(2+)-induced inactivation of PON1. Separately, CLA isomers successfully stabilized PON1, but not linoleic acid. These data suggest that the two CLA isomers may play a beneficial role in protecting PON1 from oxidative inactivation as well as in its stabilization.

Interactions between ascorbyl free radical and coenzyme Q at the plasma membrane.

A role for coenzyme Q in the stabilization of extracellular ascorbate by intact cells has been recently recognized. The aim of this work was to study the interactions between reduced ubiquinone in the plasma membrane and the ascorbyl free radical, as an approach to understand ubiquinone-mediated ascorbate stabilization at the cell surface. K-562 cells stabilized ascorbate and decreased the steady-state levels of the semiascorbyl radical. The ability of cells to reduce ascorbyl free radical was inhibited by the quinone analogs capsaicin and chloroquine and stimulated by supplementing cells with coenzyme Q10. Purified plasma membranes also reduced ascorbyl free radical in the presence of NADH. Free-radical reduction was not observed in quinone-depleted plasma membranes, but restored after its reconstitution with coenzyme Q10. Addition of reduced coenzyme Q10 to depleted membranes allowed them to reduce the signal of the ascorbyl free radical without NADH incubation and the addition of an extra amount of purified plasma membrane quinone reductase further stimulated this activity. Reduction was abolished by treatment with the reductase inhibitor p-hydroximercuribenzoate and by blocking surface glycoconjugates with the lectin wheat germ agglutinin, which supports the participation of transmembrane electron flow. The activity showed saturation kinetics by NADH and coenzyme Q, but not by the ascorbyl free radical in the range of concentrations used. Our results support that reduction of ascorbyl free radicals at the cell surface involves coenzyme Q reduction by NADH and the membrane-mediated reduction of ascorbyl free radical.

Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part 4: hyperbolic activation of rat liver microsomal NADPH-cytochrome C reductase by the novel acetylator 7,8-diacetoxy-4-methylcoumarin.

The effect of 7,8-diacetoxy-4-methylcoumarin (DAMC) has been studied on hepatic NADPH cytochrome C reductase-- an enzyme participating in the microsomal electron transport. The preincubation of liver microsomes with DAMC resulted in a time-dependent activation of NADPH cytochrome C reductase. The catalytic activity of the enzyme enhanced nearly 600% by 25 microM concentration of DAMC after 10 min of preincubation. The action of DAMC on the reductase resulted in enhanced v(max) while Km remained constant. A plot of 1/v(max) as a function of DAMC concentration resulted in a non-linear, but rectangular hyperbola indicative of hyperbolic activation. DAMC was also proved to be effective in significantly enhancing the activity of NADPH cytochrome C reductase in vivo. 7,8-Dihydroxy-4-methylcoumarin (DHMC), the deacetylated product of DAMC failed to irreversibly activate the enzyme. The activation effect of DAMC upon the enzyme was abolished by p-hydroxymercury benzoate. The role of a transacetylase in transferring the acetyl group of DAMC to the amino acid(s) of the active site of NADPH cytochrome C reductase causing irreversible enzyme activation is enunciated.

Chemistry of the "molecular trap" of protease-catalyzed splicing reaction of complementary segments of alpha-subunit of hemoglobin A.

The complementary fragments of human Hb alpha, alpha1-30, and alpha31-141 are spliced together by V8 protease in the presence of 30% n-propanol to generate the full-length molecule (Hb alpha-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb alpha is facilitated by the organic cosolvent induced alpha-helical conformation of product acting as the "molecular trap" of the splicing reaction. The segments alpha24-30 and alpha31-40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) alpha24-40 has been manipulated by engineering the amino acid replacements to the positions alpha27 and alpha31 to delineate the structural basis of the molecular trap. The location of Glu27 and Arg31 residues in the contiguous segment alpha24-40 (as well as in other larger segments) is ideal to generate (i, i + 4) side-chain carboxylate-guanidino interaction in its alpha-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at alpha27 and alpha31 facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The gamma-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an alpha-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu30-Arg31 peptide bond. The second structural aspect is a site-specific event, an i, i + 4 side-chain interaction in the alpha-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven "molecular traps" of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.

Crystallization and stabilization of MB-1, a de novo designed protein for optimized feeding technology.

Milk Bundle-1 is a de novo protein that was designed for application in agriculture. It has a high content of selected essential amino acids, and is intended to adopt an alpha-helical bundle fold. Crystallization experiments with MB-1 have been carried out on the ground and in reduced gravity on board Columbia orbiter during mission STS-80. Rather small crystals were obtained (< 0.05 mm) in both environments. Among other factors, the lack of stability of purified MB-1 has been detrimental to crystal growth. We report here on our progress with regard to optimizing crystal growth conditions, protein purification and protein stability. The first MB-1 mutant we present (MB-1-His) contains a poly-histidine tail, allowing the use of metal affinity chromatography for purification. MB-1-His has been found to keep its original mass for a month at room temperature, a spectacular improvement over MB-1. The other mutant (MB-1-Cys) was engineered to carry a cysteine residue on a solvent exposed face. The exposed cysteine binds readily to p-HMB, and allows for dimerization of MB-1-Cys. The dimer was found to be twice as stable as MB-1 during proteolytic degradation studies.

Bilitranslocase can exist in two metastable forms with different affinities for the substrates--evidence from cysteine and arginine modification.

Bilitranslocase is an organic anion carrier involved in bilirubin and phthalein uptake by the liver. In rat liver plasma membranes, its function is assayed by recording the electrogenic sulfobromophthalein movement. This has been found to be inhibited by both cysteine-specific and arginine-specific reagents. Inhibition is both partial and it occurs to the same extent, i.e. approximately 50%. The effects are not additive. Here we describe the mechanism underlying the above observations. It is concluded that bilitranslocase occurs in two possible states, featured by high and low affinity for the substrates (for sulfobromophthalein, Km = 5 microM and 37 microM, respectively). Cysteine- or arginine-reactive reagents, by reacting selectively with the low-affinity form, entrap it and shift the equilibrium between the two forms, so that, at completion, only the low-affinity form is present. The substrate concentration in the standard transport assay is 39 microM, a value at which the modified low-affinity form operates in the range of half-maximal velocity. This explains both the apparent half-inhibition measured after the chemical treatments and the lack of additivity. In addition, the substrates are shown to enhance the rate of conversion from the low-affinity to the high-affinity form of the translocator, thus favouring its high-affinity form under physiological conditions.

Site and significance of cysteine residues in xylose reductase from Neurospora crassa as deduced by fluorescence studies.

Inactivation of xylose reductase (XR) by p-hydroxy-mercury benzoate (PHMB) was found to be biphasic with second-order rate constants of 80 and 6 M-1s-1 for the fast (kf) and slow (ks) phase respectively. Spectroscopic studies indicated that the inactivation was due to modification of one Cys residue per molecule of XR and not due to subsequent disruption of the quaternary structure. The binding of NADPH to XR (Kd 0.9 microM) was depressed on modification of the enzyme by PHMB (Kd 2.3 microM). The dependence of PHMB induced inactivation of XR in the presence of alcohols and varying temperature revealed that the Cys residue is situated in a hydrophobic microenvironment and is not involved in hydrogen bonding. Our present investigation using o-phthalaldehyde (OPTA) as the chemical initiator for fluorescent chemo-affinity labeling and double inhibition studies indicates that Cys residues involved in the reaction with PHMB (SHI) and OPTA (SHII) are distinctly different. Experimental evidence presented here serves to implicate that SHI located in a hydrophobic microenvironment at the high affinity NADPH binding site of XR plays a role in the binding of the coenzyme to XR, whereas SHII serves to maintain the conformation of the active site essential for catalysis by interacting with the NH2 group of an essential lysine residue.

Mercuration of vanillyl-alcohol oxidase from Penicillium simplicissimum generates inactive dimers.

Vanillyl-alcohol oxidase (EC 1.1.3.7) from Penicillium simplicissimum was modified with p-mercuribenzoate. One cysteine residue reacts rapidly without loss of enzyme activity. Three sulfhydryl groups then react in an 'all or none process' involving enzyme inactivation and dissociation of the octamer into dimers. The inactivation reaction is slowed down in the presence of the competitive inhibitor isoeugenol and fully reversible by treatment of the modified enzyme with dithiothreitol. Vanillyl-alcohol oxidase is more rapidly inactivated at low enzyme concentrations and protected from mercuration by antichaotropic salts. It is proposed that subunit dissociation accounts for the observed sensitivity of vanillyl-alcohol oxidase crystals towards mercury compounds.

Raman spectroscopy of calf lens gamma-II crystallin: direct evidence for the formation of mixed disulfide bonds with 2-mercaptoethanol and glutathione.

This study presents Raman spectra of calf lens gamma-II crystallin and its reaction products with reduced glutathione, 2-mercaptoethanol and p-hydroxymercuribenzoate. The absence of a disulfide vibration in gamma-III crystallin (both in aqueous solution and in lyophilized state) indicates that the seven thiol groups in this protein are resistant to air oxidation, and are capable of maintaining their reduced state in the absence of added reducing agents during isolation. However, treatment of the protein with low molecular weight thiols such as glutathione and 2-mercaptoethanol results in mixed disulfide bonds. We have detected, for the first time, the S--S bond stretching vibration from the mixed disulfides at 510 cm-1, which is very similar to the 508 cm-1 reported for the inter/intramolecular disulfide bonds in intact mouse lenses (Yu, N.-T., DeNagel, D.C., Pruett, P.L. and Kuck, J.F.R., Jr. (1985). Proc. Natl. Acad. Sci. U.S.A., 82, 7965-8). Upon titration with five equivalents of p-hydroxymercuribenzoate, a strong Raman line was detected at 345 cm-1, which is tentatively attributed to the Hg--S stretching vibration of the mercaptide complex. The S--H vibration region (2500-2700 cm-1) exhibits two resolved peaks at 2562 and 2580 cm-1 with an intensity ratio of 2:5. Both reactive surface thiol groups and buried cysteines give rise to the S--H vibration at 2580 cm-1.

Site-specific substitutions of the Tyr-165 residue in the catalytic chain of aspartate transcarbamoylase promotes a T-state preference in the holoenzyme.

The amino acid residue Tyr-165C of aspartate transcarbamoylase (EC 2.1.3.2) of Escherichia coli has been proposed to be involved in the transition from the T-state to the R-state upon binding of the bisubstrate analogue N-(phosphonacetyl)-L-aspartate. Site-specific mutagenesis has been used to substitute phenylalanine for tyrosine, thus maintaining the aromatic R-group but removing the charged hydroxyl moiety. This mutation dramatically altered the aspartate requirements for the holoenzyme but did not substantially affect the homotropic or heterotropic characteristics of the oligomer. The aspartate requirements for half-maximal saturation increased from 5.5 mM at pH 7.0 for the native holoenzyme to approximately 90 mM in the mutant enzyme. Nonetheless, estimates of the kinetic cooperativity index remained similar (Hill coefficients: Tyr-165C, n = 2.1; Phe-165C, n = 2.5). CTP inhibited both enzymes approximately 70% and ATP activated approximately 40% at the aspartate concentrations required for half-maximal saturation (5 and 90 mM, respectively). The maximal velocity of the mutant holoenzyme is almost identical to that of the wild-type enzyme. The phenylalanine substitution does not affect the stability of the holoenzyme to heat or mercurials, and the Vmax of the catalytic trimer was 444% greater than that of the holoenzyme. Upon dissociation of the wild-type native enzyme into catalytic trimers, the Vmax increased 450%. The Km for aspartate in the separated catalytic trimer is approximately 2-fold higher than for the native catalytic trimer (16.5 versus 8 mM at pH 7.0). It is clear from the data that although Tyr-165C is not directly involved in the active site of the enzyme, it does play a pivotal role in catalytic transitions of the holoenzyme. In addition, the homotropic and heterotropic characteristics of the enzyme do not seem to be altered by the substitution of phenylalanine for Tyr-165C in the E. coli aspartate transcarbamoylase, although other substitutions have been reported (Robey, E. H., and Schachman, H. K. (1984) J. Biol. Chem. 259, 11180-11183) which show more complex effects.

On the formation of a dicloxacillin-p-hydroxymercuribenzoate suicide complex mediated by beta-lactamase I from Bacillus cereus.

p-Hydroxymercuribenzoate is a non-competitive inhibitor of beta-lactamase I from Bacillus cereus and also, after preliminary preincubation, an inactivator of the enzyme. Submitted to the simultaneous action of PCMB plus dicloxacillin, the enzyme completely loses its activity. Extensive dialysis can restore the enzymatic activity only if preincubation had been carried out with either PCMB or dicloxacillin but not if both inhibitors had been simultaneously present. Mercaptoethanol protects the enzyme from the action of PCMB, but not from the severe inactivation caused by dicloxacillin-PCMB mixtures. All these data suggest the formation of a complex between PCMB and the acyl-enzyme intermediate generated upon hydrolysis of the beta-lactam bond of dicloxacillin.

The reaction of metallothionein with mercuribenzoate. A dialysis and 113Cd-n.m.r. study.

Reaction of rat liver cadmium-metallothionein-II(Cd-MT-II) with p-hydroxymercuribenzoate(pHOHgBzO-) causes displacement of bound Cd. When pHOHgBzO- -induced displacement of 109Cd is observed after dialysis of the reaction mixture, the stoichiometry is consistent with stepwise displacement of tetraco-ordinate Cd atoms by non-random entry of reagent into the polynuclear clusters. 113Cd n.m.r. allows direct observation of the effects on bound Cd of stepwise titration of 113Cd-MT-II with pHOHgBzO-. The first equivalent reduces all resonances approximately equally. Subsequently differential reactivity of the protein thiolates towards the reagent gives rise to differential decreases in the 113Cd signal intensities. Resonances previously attributed to a three-metal cluster are lost before those arising from the four-metal cluster. These results are interpreted in terms of current models of the MT structure. They are distinct from the results of reaction of MT with 5,5'-dithiobis-(2-nitrobenzoic acid), which distinguishes between only two classes of thiolates, terminal and bridging. Such different patterns of reactivity of the protein thiolates may underlie a biological activity of this protein.

Conformation of cross-linked aspartate transcarbamoylase.

We have previously shown that aspartate transcarbamylase loses its substrate cooperativity after modification with a cross-linking reagent. Depending on the presence or absence of substrate analogues during cross-linking, the derivatives resemble the relaxed (R) or taut (T) state, respectively. In the present study, we attempt to characterize the conformation of these derivatives and the effects of ligands. The putative T-state derivative was similar to the native enzyme in its reactivity towards p-hydroxymercuribenzoate and in the increase of reactivity upon addition of succinate. However, unlike the native enzyme it was not activated by succinate at low substrate concentrations. On the other hand, the putative R-state derivative showed greatly enhanced reactivity which was not substantially increased by succinate. In the presence of urea, the native enzyme and the two cross-linked derivatives all resembled the R state. Thus at low substrate concentrations urea activated both the native enzyme and the t-state derivative. In contrast, the effect of urea on the R state derivative is mainly inhibitory. The above results show that the R state has been definitely stabilized whereas the T-state derivative retains some conformational flexibility. Our observations also indicate that the conformational change induced by succinate has two distinct components of which only one is allowed in the T-state derivative.

Hyperglycemia following p-hydroxymercuribenzoate administration to mice.

p-Hydroxymercuribenzoate (PMB) administration to fed mice induced transient hyperglycemia of a few hours' duration, and an increase in mitochondrial volume and pyroantimonate precipitation in the cytoplasmic ground substance of the pancreatic islet B-cells, whereas the secretory granules were unaffected. No significant blood glucose elevation or any obvious structural alterations were observed in starved mice treated with PMB. The serum inorganic phosphate concentration was unaffected in fed mice but decreased in starved mice 10 min after PMB injection, and increased in both fed and starved mice 2 h following PMB treatment, whereas the hydrogen ion concentration was increased in both fed and straved mice 10 min after PMB administration. Mitochondrial and ionic alterations, and possible inhibited insulin release may play a role in the development of hyperglycemia in PMB-treated mice.

Functionally important arginine residues of aspartate transcarbamylase.

The reaction of phenylglyoxal with aspartate transcarbamylase and its isolated catalytic subunit results in complete loss of enzymatic activity (Kantrowitz, E. R., and Lipscomb, W. N. (1976) J. Biol. Chem. 251, 2688-2695). If N-(phosphonacetyl)-L-aspartate is used to protect the active site, we find that phenylglyoxal causes destruction of the enzyme's susceptibility to activation by ATP and inhibition by CTP. Furthermore, CTP only minimally protects the regulatory site from reaction with this reagent. The modified enzyme still binds CTP although with reduced affinity. After reaction with phenylglyoxal, the native enzyme shows reduced cooperativity. The hybrid with modified regulatory subunits and native catalytic subunits exhibits slight heterotropic or homotropic properties, while the reverse hybrid, with modified catalytic subunits and native regulatory subunits, shows much reduced homotropic properties but practically normal heterotropic interactions. The decrease in the ability of CTP to inhibit the enzyme correlates with the loss of 2 arginine residues/regulatory chain (Mr = 17,000). Under these reaction conditions, 1 arginine residue is also modified on each catalytic chain (Mr = 33,000). Reaction rate studies of p-hydroxymercuribenzoate, with the liganded and unliganded modified enzyme suggest that the reaction with phenylglyoxal locks the enzyme into the liganded conformation. The conformational state of the regulatory subunit is implicated as having a critical role in the expression of the enzyme's heterotropic and homotropic properties.

Acceleration of tetramer formation by the binding of inositol hexaphosphate to hemoglobin dimers.

The aggregation of deoxyhemoglobin dimers was studied by dropping the pH of a dilute solution of deoxyhemoglobin originally at high pH. In the presence of inositol hexaphosphate, a sharp increase in the rate of dimer association was observed. At higher concentrations of the phosphate, the rate decreased to a value close to that seen in the absence of phosphate. These observations require that inositol hexaphosphate binds to deoxyhemoglobin dimers. The dependence of the aggregation rate on phosphate concentration occurs because the reaction of a dimer containing bound phosphate with a phosphate-free dimer is 30 to 50 times faster than either the association of phosphate-free dimers or the association of dimers both containing bound phosphate.