Decrease In SH Groups Research Articles

Effect of pulsed electric fields on the activity of food-grade papain in a continuous system

Papain is an enzyme widely studied due to its multiple applications in foods, brewery, and the pharmaceutical industry, among others. Food-grade papain was treated with pulsed electric field technology to evaluate possible changes on its residual activity (RA), relative protein concentration (RPC), sulfhydryl group content, and structural changes. Three factors were evaluated: electric field (10 and 13 kV/cm), number of pulses (48–432), and flow rate (0.2, 0.4 and 0.6 L/min). Maximal inactivation and decrease in SH groups of papain (64 and 20% respectively) were obtained when a PEF process with 13 kV/cm, 288 pulses and 0.2 L/min was used. The maximal decrease in RPC (18%) was attained when 10 kV/cm, 0.4 L/min, and 144 pulses were applied on the enzyme. Important changes in papain secondary and tertiary structures as a result of the PEF treatments were noticed. PEF technology can be considered then as an effective process to reduce the activity of papain.

Age-related Oxidative Modifications of Proteins and Lipids in Rat Brain

Oxidants have been shown to play a major role in ageing and ageing-related neurodegenerative diseases. In the present study, we investigated the effect of ageing on oxidative damage to lipids and proteins in brain homogenate, mitochondria and synaptosomes of adult (6-month-old), old (15-month-old), and senescent (26-month-old) Wistar rats. There was a significant increase in thiobarbituric acid-reactive substances and conjugated dienes in homogenates, which indicate increased lipid peroxidation (LPO). Oxidative modifications of homogenate proteins were demonstrated by a loss of sulfhydryl content, accumulation of dityrosines and formation of protein conjugates with LPO-end products. Increase in protein conjugates with LPO-end products and a decrease in SH groups were observed also in mitochondria and synaptosomes, but dityrosine content was elevated only in synaptosomes. Protein surface hydrophobicity, measured by fluorescent probe 1-anilino-8-naphthalenesulfonate (ANS), was increased only in homogenate. These results suggest that besides mitochondria and synaptosomes other cellular compartments are oxidatively modified during brain ageing.

ALBUMINS FROM THE BEAN PHASEOLUS VULGARIS: EFFECTS OF HEAT TREATMENT

Bean albumins heated at different temperature and time intervals (60C to 135C, 1 to 30 min) suffered a reduction of in vitro digestibility, proportional to the intensity of treatment. The electrophoretic (SDS-PAGE) and chromatographic (gel filtration) profiles showed the formation of high molecular weight protein aggregates, involving disulfide bonds. More intense heat treatments caused formation of bonds not broken by β-mercaptoethanol or SDS, resulting in aggregates resistant toproteolysis. Determination of available lysine showed that these residues were not implied in possible crosslinking reactions, and heated albumins showed the same amino acid profile as the control. The decrease of SH groups upon heating followed a first order kinetics, with an activation energy of 76.9kJ/mol, and an increasing k value ranging from 0.0012 at 60C to 0.267 at 135C. DSC thermograms of albumins showed two peaks, with maximum denaturation temperature of 87C and 98C, and total enthalpy of denaturation of 7.7 J/g.

Ataxia telangiectasia heterozygotes and patients display increased fluidity and decrease in contents of sulfhydryl groups in red blood cell membranes.

In red blood cell membranes of ataxia telangiectasia mutated (ATM) homozygotes and heterozygotes, decreased values of the corrected fluorescence anisotropy and the anisotropy parameter were found, indicating increased fluidity and decreased microviscosity, respectively. These changes in membranes were accompanied by a decrease in SH-groups and an increase in malondialdehyde (MDA) contents. The content of MDA both in homozygotes and in heterozygotes exceeded roughly threefold the respective control values. Decreased content of GSH in red blood cells was found only in ATM homozygotes. The change most specific for the ATM gene appears to be the increase in fluidity, since only this parameter displays the proportionally greater changes in ATM homozygotes compared to ATM heterozygotes. The observations presented here may indicate that the ATM gene is expressed in precursors of red blood cells and deficiency of normal AT gene function may produce the changes which persist in circulating cells.

Modification of the xanthine-converting enzyme of perfused rat heart during ischemia and oxidative stress

The reversible and irreversible conversion of xanthine dehydrogenase to xanthine oxidase during ischemia/reperfusion and oxidative stress induced by hydrogen peroxide or diamide and its relationship with glutathione and protein SH groups were studied. The direct spectrophotometric measurement of the various forms of the xanthine-converting enzyme indicates that, in the fresh rat heart or after normoxic perfusion, there always is a basal level of 80% xanthine dehydrogenase and 20% of xanthine oxidase (15% irreversible and 5% reversible) that could contribute to the background production of free radicals. There is no significant increase of irreversible xanthine oxidase during ischemia nor during reperfusion. After global ischemia the reversible oxidase shows almost no increase while, when ischemia is followed by reperfusion, there is a limited increase (less than 9%) of the reversible xanthine oxidase. In the latter conditions there is a decrease of glutathione and of SH groups of about 70% and 25%, respectively. Perfusion for 1 h with oxidizing agents like hydrogen peroxide (60 μM) or diamide (100 μM) determines a marked conversion of xanthine dehydrogenase to reversible xanthine oxidase of about 40% and 60%, respectively; this oxidase activity partially reconverts to the dehydrogenase after withdrawing the oxidizing agents from the perfusion medium. The level of irreversible xanthine oxidase remains unchanged in all the conditions tested. Both hydrogen peroxide and diamide induce a strong decrease in SH groups and depletion of glutathione. The xanthine dehydrogenase → xanthine oxidase conversion thus appears to be sensitive to the redox state of thiol groups.

Spectroscopic studies on the mixed disulfide formation of lens crystallin with glutathione

Mixed disulfide was formed through thiol-disulfide exchange reaction of lens crystallin with oxidized glutathione (GSSG). The reaction was monitored by isoelectric focusing (IEF) and DTNB [5,5'-dithiobis(2-nitrobenzoic acid)] assay. The effects on protein conformation were studied by circular dichroism (CD) and fluorescence. The DTNB shows 22% and 49% decrease of SH groups after the exchange reaction in alpha-crystallin and gamma-crystallin, respectively. The exchange reaction was further shown by an acidic shifting in IEF pattern. The near ultraviolet CD shows a slight decrease in the GSSG-treated crystallins. The fluorescence measurements of the SH specific probe IANBD, 4-(N-iodoacetoxy)ethyl-N-methylamino-7-nitrobenz-2-oxa-1,3-diazole, indicate that the surface SH groups were oxidized in the GSSG-treated samples. The labeling with amine selectively reactive probe FITC, fluorescein-5-isothiocyanate, indicates an increase of amine reactivity with mixed disulfide formation. Polarization measurements show that bound FITC probes are in a less rigid structure in the mixed disulfide rich crystallin. All these results point out that the formation of mixed disulfide partially unfolds protein.

Consequences of cadmium toxicity in rat hepatocytes: Effects of cadmium on the glutathione-peroxidase system

Cadmium (Cd; 10–100 μM) produced in isolated hepatocytes the formation of thiobarbituric acid-reactants (lipid peroxidation; LPO) and a decrease in SH groups in a time- and concentration-dependent manner. However, glutathione peroxidase (GSH-Px) was not affected indicating that LPO does not originate from GSH-Px injury. In contrast, in presence of Cd a time- and concentration-dependent decrease of glucose-6-phosphate dehydrogenase (G6PDH) and, most strongly, of glutathione reductase (GSSG-R), both members of the GSH-Px system, was observed. These effects could not be inhibited by ( + )-cyanidanol-3, a compound known as inhibitor of LPO. Therefore, our data show that (l)Cd-induced LPO is not a function of GSH-Px injury and (2) inhibition of GSSG-R and G6PDH-activity is not a function of LPO. They also indicate that Cd-induced depletion of cellular SH groups are associated with toxic effects of Cd on GSSG-R and G6PDH different from LPO-induction.

Incorporation of 35S-cystamine into rat spleen nuclear proteins and its influence on the SH level and on chromatin nucleolytic degradation

1. 1. Incorporation of 345S-cystamine in vitro to rat spleen nuclei, chromatin, histones and non-histone proteins, was investigated. Whole nuclei incorporate labelled disulphide quicker than chromatin. Among chromatin proteins, the highest cystamine. level was found in non-histone proteins. 2. 2. The process of incorporation of cystamine into nuclei was accompanied by a decrease in SH groups of chromatin was well as in the whole bulk of nuclear SH. It is supposed that cystamine suppresses enzymatic formation of SH groups in the nucleus. 3. 3. Cystamine completely inhibits the process of chromatin degradation by endogenic nucleases, which may be the result of either inhibition of the respective enzyme or disulphide modification of chromatin.

Studies on Reduction of Moya (aging) Period in Kori-tofu Making Process

To make kori-tofu, fresh tofu is frozen and then kept -1--3°C in a storage room for about 3 weeks. During this period called Moya, original tofu changed into unique structure like sponge but it is extremely time consuming. Standing on the viewpoint that ionized SH groups can promote protein denaturation by freezing and cause the formation of spongy structure, present paper deals with practical trials to reduce Moya period.Blowing of nitrogen gas into soymilk before heating caused the increase of SH groups and promoted to form spongy structure, while blowing of oxygen gas caused the decrease of SH groups and prevented the formation of spongy structure. Addition of 5×10-4M sodium suifite to soymilk before coagulation also promoted the increase of SH groups and also the formation of spongy structure, consequently, Moya period can be reduced. But this adding amounts is limited by food regulation.It may be concluded that to reduce Moya period and prepare Kori-tofu in good quality, it is important to make tofu with a care for preventing soymilk from decrease of SH groups.

MOLECULAR PROPERTIES OF POST‐MORTEM MUSCLE: EFFECT OF SULFHYDRYL REAGENTS AND POST‐MORTEM STORAGE ON CHANGES IN MYOSIN B

Studies to determine the relationship of SH groups to certain changes of the myofibrillar proteins of post‐mortem muscle were carried out with myosin B from at‐death and post‐mortem stored rabbit skeletal muscle (2° C and 25° C for 3 days) and with SH reagent modified myosin B from at‐death and post‐mortem stored muscle. Quantitative SH analysis, ATPase activity, turbidity rate and analytical ultracentrifugation were employed to determine the changes in myosin B associated with changes in SH groups. Post‐mortem storage of muscle at 2°C for 3 days had no effect on SH content of myosin B; a decrease in SH groups, however, was observed in myosin B from muscle stored at 25°C for 3 days and for iodoacetamide (IAA) and N‐ethylmaleimide (NEM) modified myosin B. ATPase activity was inhibited by reacting myosin B with enough NEM or IAA to block all SH groups. Dialysis of myosin B from at‐death and post‐mortem muscle against MCE to restore SH groups resulted in partial reversal of Mg++ and EGTA‐activated ATPase of myosin B from post‐mortem muscle and a less rapid rate of turbidity development. These results suggest that the state and nature of SH groups are partly involved in the actin‐myosin interaction of post‐mortem muscle; other constituents, however, in addition to SH groups are evidently modified and, in some instances, irreversibly modified, under certain post‐mortem storage conditions.