Protein Sulfhydryl Groups Research Articles

The Molecular Basis for the Mode of Action of Bicyclomycin

Bicyclomycin (1) is a clinically useful antibiotic exhibiting activity against a broad spectrum of Gram-negative bacteria and against the Gram-positive bacterium, Micrococcus luteus. Bicyclomycin has been used to treat diarrhea in humans and bacterial diarrhea in calves and pigs and is marketed by Fujisawa (Osaka, Japan) under the trade name Bicozamycin. The structure of 1 is unique among antibiotics, and our studies document that its mechanism of action is novel. Early mechanistic proposals suggested that 1 reacted with nucleophiles (e.g., a protein sulfhydryl group) necessary for the remodeling the peptidoglycan assembly within the bacterial cell wall. We, however, showed that 1 targeted the rho transcription termination factor in Escherichia coli. The rho protein is integral to the expression of many gene products in E. coli and other Gram-negative bacteria, and without rho the cell losses viability. Rho is a member of the RecA-type ATPase class of enzymes that use nucleotide contacts to couple oligonucleotide translocation to ATP hydrolysis. Bicyclomycin is the only known selective inhibitor of rho. In this article, we integrate the evidence obtained from bicyclomycin structure-activity studies, site-directed mutagenesis investigations, bicyclomycin affinity labels, and biochemical and biophysical measurements with recent X-ray crystallographic images of the bicyclomycin-rho complex to define the rho antibiotic binding site and to document the pathway for rho inhibition by 1. Together, the structural and functional studies demonstrate how 1, a modest rho inhibitor, can disrupt the rho molecular machinery thereby leading to a catastrophic effect caused by the untimely overproduction of proteins not normally expressed constitutively, thus leading to a toxic effect on the cells.

Effect of glutathione depletion on apoptosis induced by thiram in Chinese hamster fibroblasts

Fungicide thiram, which is also known as an inducer of allergic contact dermatitis (ACD), was used as a model compound of thiuram chemicals, and its cellular effects were investigated in cultured Chinese hamster V79 cells. The level of intracellular reduced glutathione (GSH), protein sulfhydryl (PSH) groups, protein carbonyls (PC), membrane lipid peroxidation reflected by enhanced thiobarbituric acid reactive substrates (TBARS) production, as well as apoptotic effect were determined. The apoptosis induction was determined by assessing DNA fragmentation by TUNEL, annexin V binding, and caspases activation assays, using fluorescent microscope or flow cytometry, respectively. The concentrations of thiram required to induce cellular GSH depletion (by 40–50%), protein, and membrane lipid peroxidation (2-fold, and 1.7-fold, respectively), as well as to induce apoptosis in V79 Chinese hamster fibroblasts without causing necrosis through cytotoxic effects were between 50–100 μM. To investigate the role of decreased GSH content in the toxicity of thiram, GSH level was modified prior to exposure. Pretreatment of V79 cells with N-acetyl- l-cysteine (NAC), a GSH biosynthesis precursor, prevented GSH decrease, PC and TBARS production, as well as caspases activation induced by thiram exposure. On the other hand, thiram effects were enhanced by the previous depletion of cellular GSH by l-buthionine-(S,R)-sulfoximine (BSO).

Optimization and Applications of CDAP Labeling for the Assignment of Cysteines

The aim of the study is to provide a methodology for assigning unpaired cysteine residues in proteins formulated in a variety of different conditions to identify structural heterogeneity as a potential cause for protein degradation. 1-Cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) was employed for cyanylating free cysteines in proteins and peptides. Subsequent basic cleavage of the peptide bond at the N-terminal side of the cyanylated cysteines provided direct information about their location. CDAP was successfully employed to a wide variety of labeling conditions. CDAP was reactive between pH 2.0 and 8.0 with a maximum labeling efficiency at pH 5.0. Its reactivity was not affected by excipients, salt or denaturant. Storing CDAP in an organic solvent increased its intrinsic stability. It was demonstrated that CDAP can be employed as a thiol-directed probe to investigate structural heterogeneity of proteins by examining the accessibility of unpaired cysteine residues. CDAP is a unique cysteine-labeling reagent because it is reactive under acidic conditions. This provides an advantage over other sulfhydryl labeling reagents as it avoids potential thiol-disulfide exchange. Optimization of the cyanylation reaction allowed the utilization of CDAP as a thiol-directed probe to investigate accessibility of sulfhydryl groups in proteins under various formulation conditions to monitor structural heterogeneity.

Dietary lipoic acid supplementation attenuates hypertension in Dahl salt sensitive rats

There is strong evidence that excess dietary salt (NaCl) is a major factor contributing to the development of hypertension. Salt sensitive humans and rats develop hypertension even on a normal salt diet. Salt sensitivity is associated with glucose intolerance and insulin resistance in both humans and animal models, including Dahl salt sensitive (DSS) rats. In insulin resistance, impaired glucose metabolism leads to elevated endogenous aldehydes. These aldehydes bind sulfhydryl groups of membrane proteins, altering calcium channels, increasing cytosolic free calcium ([Ca2+]i) and blood pressure. Treatment with lipoic acid, an endogenous sulfur-containing fatty acid, normalizes insulin resistance and lowers tissue aldehyde conjugates, cytosolic [Ca2+]i, and blood pressure in spontaneously hypertensive rats (SHR). The objective of this study was to investigate the effects of a normal salt diet on tissue aldehyde conjugates, cytosolic [Ca2+]i and blood pressure in DSS rats and to determine whether lipoic acid supplementation prevents the increase in blood pressure and biochemical changes. Starting at 7 weeks of age, DSS rats were divided into three groups of six animals each and treated for 6 weeks with diets as follows: DSS-low salt, 0.4% NaCl; DSS-normal salt, 0.7% NaCl, and; DSS-normal salt + lipoic acid, 0.7% NaCl + lipoic acid 500 mg/kg feed. At completion, animals in the normal salt group had elevated systolic blood pressure, cytosolic [Ca2+]i and tissue aldehyde conjugates as compared to the low salt group. They also showed smooth muscle cell hyperplasia in small arteries and arterioles of the kidney. Dietary lipoic acid supplementation attenuated the increase in systolic blood pressure and associated biochemical and histopathological changes.

S-Nitrosation versus S-Glutathionylation of Protein Sulfhydryl Groups by S-Nitrosoglutathione

S-Nitrosation of protein sulfhydryl groups is an established response to oxidative/nitrosative stress. The transient nature and reversibility of S-nitrosation, as well as its specificity, render this posttranslational modification an attractive mechanism of regulation of protein function and signal transduction, in analogy to S-glutathionylation. Several feasible mechanisms for protein S-nitrosation have been proposed, including transnitrosation by S-nitrosothiols, such as S-nitrosoglutathione (GSNO), where the nitrosonium moiety is directly transferred from one thiol to another. The reaction between GSNO and protein sulfhydryls can also produce a mixed disulfide by S-glutathionylation, which involves the nucleophilic attack of the sulfur of GSNO by the protein thiolate anion. In this study, we have investigated the possible occurrence of S-glutathionylation during reaction of GSNO with papain, creatine phosphokinase, glyceraldehyde-3-phosphate dehydrogenase, alcohol dehydrogenase, bovine serum albumin, and actin. Our results show that papain, creatine phosphokinase, and glyceraldehyde-3-phosphate dehydrogenase were significantly both S-nitrosated and S-glutathionylated by GSNO, whereas alcohol dehydrogenase, bovine serum albumin, and actin appeared nearly only S-nitrosated. The susceptibility of the modified proteins to denitrosation and deglutathionylation by reduced glutathione was also investigated.

Cooling and Freezing of Boar Spermatozoa: Supplementation of the Freezing Media With Reduced Glutathione Preserves Sperm Function

In this study, we evaluated the effects of glutathione (L-gamma-glutamyl-L-cysteinylglycine; GSH) supplementation of the freezing extender on semen parameters during the cooling (2 hours at 5 degrees C) and freezing phases of the cryopreservation process to compensate for the decrease in GSH content observed during sperm freezing. To fully address these questions, we incorporated a new set of functional sperm tests. These included tests of mitochondrial function, inducibility of the acrosome reaction, in vitro penetration (IVP) of oocytes, changes in sulfhydryl group content in membrane proteins, and capacitation status. The main findings emerging from this study were that the addition of GSH to the freezing media resulted in 1) an improvement in percent motility (%MOT) and motion parameters of thawed spermatozoa, as measured by both microscopic analysis and computer-assisted semen analysis (CASA); 2) a higher number of total viable spermatozoa; 3) a higher number of noncapacitated viable spermatozoa; and 4) a decrease in the number of spermatozoa with changes in the sulfhydryl groups in membrane proteins. This protective effect on sperm function was more pronounced with 1 mM of GSH than with 5 mM of GSH.

Antioxidants in the treatment of hypertension

Hypertension is a major health problem worldwide. Individuals with hypertension are at an increased risk for stroke, heart disease, and kidney failure. Although the etiology of essential hypertension has a genetic component, lifestyle factors such as diet play an important role. Insulin resistance and glucose intolerance are common features of hypertension in humans and in animal models. Altered glucose metabolism leads to an increased production of the reactive aldehyde, methylglyoxal. Methylglyoxal binds sulfhydryl and amino groups of proteins forming conjugates/advanced glycation end products (AGEs). This alters protein structure and function and can affect vascular calcium channels, enzymes, and tissue proteins leading to increased oxidative stress. These alterations impair endothelial function leading to an increase in intracellular free calcium, peripheral vascular resistance, and hypertension. Supplementation with antioxidants, including vitamin C, E, or B6, thiols such as lipoic acid and cysteine, and the quinone enzyme Q10, have been shown to lower blood pressure in animal models and humans with essential hypertension. The Dietary Approaches to Stop Hypertension (DASH) studies demonstrated that a well-balanced diet, rich in these nutrients, was effective in lowering blood pressure. These antioxidants may achieve their antihypertensive effects by reducing aldehyde conjugate/AGE formation and oxidative stress, by improving insulin resistance and endothelial function, or by normalizing calcium channels and peripheral vascular resistance. In essential hypertension, deficiencies of antioxidants may exist or a higher than normal amount may be required to correct metabolic abnormalities. Dietary supplementation with antioxidants may be a beneficial, inexpensive, first-line alternate treatment modality for hypertension.

Metals, Toxicity and Oxidative Stress

Metal-induced toxicity and carcinogenicity, with an emphasis on the generation and role of reactive oxygen and nitrogen species, is reviewed. Metal-mediated formation of free radicals causes various modifications to DNA bases, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis. Lipid peroxides, formed by the attack of radicals on polyunsaturated fatty acid residues of phospholipids, can further react with redox metals finally producing mutagenic and carcinogenic malondialdehyde, 4-hydroxynonenal and other exocyclic DNA adducts (etheno and/or propano adducts). Whilst iron (Fe), copper (Cu), chromium (Cr), vanadium (V) and cobalt (Co) undergo redox-cycling reactions, for a second group of metals, mercury (Hg), cadmium (Cd) and nickel (Ni), the primary route for their toxicity is depletion of glutathione and bonding to sulfhydryl groups of proteins. Arsenic (As) is thought to bind directly to critical thiols, however, other mechanisms, involving formation of hydrogen peroxide under physiological conditions, have been proposed. The unifying factor in determining toxicity and carcinogenicity for all these metals is the generation of reactive oxygen and nitrogen species. Common mechanisms involving the Fenton reaction, generation of the superoxide radical and the hydroxyl radical appear to be involved for iron, copper, chromium, vanadium and cobalt primarily associated with mitochondria, microsomes and peroxisomes. However, a recent discovery that the upper limit of "free pools" of copper is far less than a single atom per cell casts serious doubt on the in vivo role of copper in Fenton-like generation of free radicals. Nitric oxide (NO) seems to be involved in arsenite-induced DNA damage and pyrimidine excision inhibition. Various studies have confirmed that metals activate signalling pathways and the carcinogenic effect of metals has been related to activation of mainly redox-sensitive transcription factors, involving NF-kappaB, AP-1 and p53. Antioxidants (both enzymatic and non-enzymatic) provide protection against deleterious metal-mediated free radical attacks. Vitamin E and melatonin can prevent the majority of metal-mediated (iron, copper, cadmium) damage both in vitro systems and in metal-loaded animals. Toxicity studies involving chromium have shown that the protective effect of vitamin E against lipid peroxidation may be associated rather with the level of non-enzymatic antioxidants than the activity of enzymatic antioxidants. However, a very recent epidemiological study has shown that a daily intake of vitamin E of more than 400 IU increases the risk of death and should be avoided. While previous studies have proposed a deleterious pro-oxidant effect of vitamin C (ascorbate) in the presence of iron (or copper), recent results have shown that even in the presence of redox-active iron (or copper) and hydrogen peroxide, ascorbate acts as an antioxidant that prevents lipid peroxidation and does not promote protein oxidation in humans in vitro. Experimental results have also shown a link between vanadium and oxidative stress in the etiology of diabetes. The impact of zinc (Zn) on the immune system, the ability of zinc to act as an antioxidant in order to reduce oxidative stress and the neuroprotective and neurodegenerative role of zinc (and copper) in the etiology of Alzheimer's disease is also discussed. This review summarizes recent findings in the metal-induced formation of free radicals and the role of oxidative stress in the carcinogenicity and toxicity of metals.

Serum Acetyl Cholinesterase as a Biomarker of Arsenic Induced Neurotoxicity in Sprague-Dawley Rats

Arsenic is an environmental toxicant, and one of the major mechanisms by which it exerts its toxic effect is through an impairment of cellular respiration by inhibition of various mitochondrial enzymes, and the uncoupling of oxidative phosphorylation. Most toxicity of arsenic results from its ability to interact with sulfhydryl groups of proteins and enzymes, and to substitute phosphorus in a variety of biochemical reactions. Most toxicity of arsenic results from its ability to interact with sulfhydryl groups of proteins and enzymes, and to substitute phosphorus in a variety of biochemical reactions. Recent studies have pointed out that arsenic toxicity is associated with the formation of reactive oxygen species, which may cause severe injury/damage to the nervous system. The main objective of this study was to conduct biochemical analysis to determine the effect of arsenic trioxide on the activity of acetyl cholinesterase; a critical important nervous system enzyme that hydrolyzes the neurotransmitter acetylcholine. Four groups of six male rats each weighing an average 60 +/- 2 g were used in this study. Arsenic trioxide was intraperitoneally administered to the rats at the doses of 5, 10, 15, 20mg/kg body weight (BW), one dose per 24 hour given for five days. A control group was also made of 6 animals injected with distilled water without chemical. Following anaesthesia, blood specimens were immediately collected using heparinized syringes, and acetyl cholinesterase detection and quantification were performed in serum samples by spectrophotometry. Arsenic trioxide exposure significantly decreased the activity of cholinesterase in the Sprague-Dawley rats. Acetyl cholinesterase activities of 6895 +/- 822, 5697 +/- 468, 5069 +/- 624, 4054 +/- 980, and 3158 +/- 648 U/L were recorded for 0, 5, 10, 15, and 20 mg/kg, respectively; indicating a gradual decrease in acetyl cholinesterase activity with increasing doses of arsenic. These findings indicate that acetyl cholinesterase is a candidate biomarker for arsenic-induced neurotoxicity in Sprague-Dawley rats.

The solid-state molecular structure of the S-nitroso derivative of l-cysteine ethyl ester hydrochloride

Nitrosation of protein sulfhydryl groups to form thionitrites (S-nitrosothiols) has been reported to be important in the biochemistry of nitric oxide. Such S-nitrosation of protein thiol residues has been shown to alter the function of some proteins. In this brief communication, we report the X-ray crystal structure of S-nitroso-L-cysteine ethyl ester hydrochloride. Two rotamers with respect to the NCCS moiety are present in the crystal: the major rotamer is in the gauche+ conformation, and the minor rotamer is in the rare anti (trans, antiperiplanar) conformation for a cysteinyl compound. Importantly, the CSNO groups for both rotamers are in the syn (cis, synperiplanar) form. To the best of our knowledge, this is the first reported high-resolution solid-state structure of an S-nitroso derivative of a cysteine or cysteinyl-containing compound.

Dietary Vitamin E Supplementation Attenuates Hypertension in Dahl Salt-Sensitive Rats

There is strong evidence that excess dietary salt (NaCl) is a major factor contributing to the development of hypertension. Salt-sensitive humans and rats develop hypertension even on a normal-salt diet. Salt sensitivity is associated with glucose intolerance and insulin resistance in both humans and animal models, including Dahl salt-sensitive (DSS) rats. In insulin resistance, impaired glucose metabolism leads to elevated endogenous aldehydes that bind sulfhydryl groups of membrane proteins, altering calcium channels, and increasing cytosolic free calcium ([Ca2+]i) and blood pressure. Vitamin E lowers tissue aldehyde conjugates, cytosolic [Ca2+]i, and blood pressure in spontaneously hypertensive rats and fructose-induced hypertensive Wistar Kyoto rats, models of insulin resistance. This study investigated the effect of a normal-salt diet on tissue aldehyde conjugates, cytosolic [Ca2+]i, and blood pressure in DSS rats and the effect of vitamin E supplementation on blood pressure and associated biochemical changes in these animals. Seven-week-old DSS rats were divided into 3 groups of 6 animals each and treated for 6 weeks with diets as follows: low-salt (0.4% NaCl); normal-salt (0.7% NaCl) and normal salt (0.7% NaCl) plus vitamin E (34 mg/kg feed). At completion, animals in the normal-salt group had significantly elevated systolic blood pressure, cytosolic [Ca2+]i, and tissue aldehyde conjugates compared with the low-salt group. They also showed smooth muscle cell hyperplasia in small arteries and arterioles of the kidney. Dietary vitamin E supplementation significantly attenuated the increase in systolic blood pressure and associated biochemical and histopathologic changes.

Synthesis and characterization of a novel reagent containing dansyl group, which specifically alkylates sulfhydryl group: An example of application for protein chemistry

We have synthesized a novel reagent containing dansyl group, iodoacethyl dansylcadaverine (IADC), which specifically alkylates sulfhydryl groups. The carboxyl group of iodoacetic acid was activated with dicyclohexylcarbodiimide and was condensed with amino group of dansylcadaverine. Purity and chemical structure of IADC was confirmed with mass spectrometry (MS) and NMR. IADC alkylated GSH but not GSSG, which was confirmed by MS. The reactivity of IADC with proteins was also investigated with Western blotting using anti-dansyl antibody. IADC reacted only with sulfhydryl-containing proteins. The specificity of the interaction of IADC with sulfhydryl groups in proteins was confirmed by adding excessive amount of a well-known sulfhydryl-specific reagent, 5, 5′-dithiobis(2-nitrobenzoic acid), which led to a complete inhibition. To show the usefulness of IADC, the cysteines in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from chicken muscle were modified with this reagent, and GAPDH was then digested by lysyl endopeptidase. The peptides generated from digestion of IADC-incorporated GAPDH were applied to an anti-dansyl immunoaffinity column. The peptide fragments bound and eluted from the column were separated by HPLC, and the amino acid sequence of each peptide was analyzed, and peptide was identified as the one containing a Cys residue(s). These data showed that IADC is a useful reagent to specifically identify the positions of a Cys residue(s) in proteins.

Age-related nuclear cataract—oxidation is the key

Age is by far the biggest risk factor for cataract, and it is sometimes assumed that cataract is simply an amplification of this aging process. This appears not to be the case, since the lens changes associated with aging and cataract are distinct. Oxidation is the hallmark of age-related nuclear (ARN) cataract. Loss of protein sulfhydryl groups, and the oxidation of methionine residues, are progressive and increase as the cataract worsens until >90% of cysteine and half the methionine residues are oxidised in the most advanced form. By contrast, there may be no significant oxidation of proteins in the centre of the lens with advancing age, even past age 80. The key factor in preventing oxidation seems to be the concentration of nuclear glutathione (GSH). Provided that nuclear GSH levels can be maintained above 2 m m, it appears that significant protein oxidation and posttranslational modification by reactive small molecules, such as ascorbate or UV filter degradation products, is not observed. Adequate coupling of the metabolically‐active cortex, the source of antioxidants such as GSH, to the quiescent nucleus, is crucial especially since it would appear that the cortex remains viable in old lenses, and even possibly in ARN cataract lenses. Therefore it is vital to understand the reason for the onset of the lens barrier. This barrier, which becomes apparent in middle age, acts to impede the flow of small molecules between the cortex and the nucleus. The barrier, rather than nuclear compaction (which is not observed in human lenses), may contribute to the lowered concentration of GSH in the lens nucleus after middle age. By extending the residence time within the lens centre, the barrier also facilitates the decomposition of intrinsically unstable metabolites and may exacerbate the formation of H 2O 2 in the nucleus. This hypothesis, which is based on the generation of reactive oxygen species and reactive molecules within the nucleus itself, shifts the focus away from theories for cataract that postulated a primary role for oxidants generated outside of the lens. Unfortunately, due to marked variability in the lenses of different species, there appears at present to be no ideal animal model system for studying human ARN cataract.

Role of sulfhydryl groups in band 3 in the inhibition of phosphate transport across erythrocyte membrane in visceral leishmaniasis

Membrane destabilization in erythrocytes plays an important role in the premature hemolysis and development of anemia during visceral leishmaniasis (VL). Marked degradation of the anion channel protein band 3 is likely to allow modulation of anion flux across the red cell membrane in infected animals. The present study describes the effect of structural modification of band 3 on phosphate transport in VL using 31P NMR. The result showed progressive decrease in the rate and extent of phosphate transport during the post-infection period. Interdependence between the intracellular ionic levels seems to be a determining factor in the regulation of anion transport across the erythrocyte membrane in control and infected conditions. Infection-induced alteration in band 3 made the active sites of transport more susceptible to binding with amino reactive agents. Inhibition of transport by oxidation of band 3 and subsequent reversal by reduction using dithiothreitol suggests the contribution of sulfhydryl group in the regulation of anion exchange across the membrane. Quantitation of sulfhydryl groups in the anion channel protein showed the inhibition to be closely related to the decrease of sulfhydryl groups in the infected hamsters. Downregulation of phosphate transport during leishmanial infection may be ascribed to the sulfhydryl modification of band 3 resulting in the impaired functioning of this protein under the diseased condition.

Mechanisms of 4-hydroxynonenal-induced neuronal microtubule dysfunction

We have previously demonstrated that neuronal microtubules are exquisitely sensitive to the lipid peroxidation product 4-hydroxynonenal (HNE). The mechanism, however, by which HNE disrupts the microtubules, is not known. Sulfhydryl groups of protein-cysteines constitute main targets of HNE. Indeed, HNE is mainly detoxified by conjugation to glutathione (GSH), a reaction that leads to depletion of cellular GSH. GSH maintains protein sulfhydryl groups in the reduced form and has been implicated in the regulation of cytoskeletal function. Here, we assess what role depletion of cellular GSH plays in the HNE-induced microtubule disruption. We demonstrate that HNE and its intracellularly activated tri-ester analog, HNE(Ac) 3, cause substantial GSH depletion in Neuro2A cells. However, other compounds inducing GSH depletion had no effect on the microtubule network. Therefore, HNE-induced depletion of cellular GSH does not contribute to the HNE-induced microtubule disruption. We previously demonstrated that another main cellular target of HNE is tubulin, the core protein of microtubules containing abundant cysteines. The functional relevance of this adduction, however, had not been evaluated. Here, we demonstrate that exposure of Neuro 2A cells to HNE or HNE(Ac) 3 results in the inhibition of cytosolic taxol-induced tubulin polymerization. These and our previous observations strongly support the hypothesis that HNE-adduction to tubulin is the primary mechanism involved in the HNE-induced loss of the highly dynamic neuronal microtubule network.

85 ADDITION OF REDUCED GLUTATHIONE TO FREEZING MEDIUM IMPROVED THE SPERM FUNCTIONALITY OF THAWED BOAR SPERMATOZOA

The processes of cooling and freezing/thawing produce physical and chemical stresses on the sperm membrane that are associated with the oxidative stress and reactive oxygen species (ROS) generation that reduce sperm viability and fertilizing ability. It is known that the process of freezing is associated with a significant reduction of the GSH content in boar sperm (Gadea et al. 2004 Theriogenology 62, 690–701). The addition of antioxidants to freezing medium could prevent the formation of ROS and improve the seminal parameters. The aim of these experiments was to investigate the effects of the addition of reduced glutathione (GSH) to freezing extenders on capacitation status and changes in the sulfhydryl groups of proteins on the sperm surface. Ejaculate-rich fractions from three mature boars were frozen by classic methodology (Westendorf et al. 1975 Dtsch. Tierarztl Wochenschr. 82, 393–395) using lactose/egg-yolk extender with 0 mM (group 0), 1 mM (group 1), or 5 mM (group 5) GSH. To detect increase in plasma membrane lipid packing disorder, sperm samples were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Reprod. Dev. 45, 378–391). Cells were classified as low M540 (viable, noncapacitated), high M540 (viable, capacitated), or Yo-Pro-1 positive (dead sperm) using flow cytometry. The sulfhydryl status of proteins from spermatozoa surface was evaluated with a fluorescent stain 5-iodoacetamidofluoresceine (5-IAF). The addition of GSH to the freezing medium had a positive influence on the parameters studied, increasing the proportion of viable noncapacitated spermatozoa and reducing the number of dead with a similar number of viable capacitated (Table 1). The proportion of spermatozoa stained by 5-IAF was significantly lower when GSH was added. In conclusion, we can assume that the addition of reduced glutathione to the freezing medium had a protective effect on spermatozoa functionality. Table 1. Capacitation status and changes in sulfhydryl groups of proteins (5-IAF staining) after thawing of frozen boar spermatozoa This work was supported by AGL- 2003-03144.

Colorimetric Analysis of Protein Sulfhydyl Groups in Milk: Applications and Processing Effects

Methods for protein sulfhydryl (SH) group analysis in food systems have been largely overlooked. Nevertheless, changes in SH group concentration affect both physical and nutritional characteristics of high protein foods and ingredients. Food scientists and technologists require improved understanding of protein SH chemistry in order to design processes that minimize loss of thiol groups. This article surveys colorimetric methods for food protein SH group analysis with applications to fluid milk and dried milk powder. Most colorimetric assays (chloromeribenzoate, pyridine disulfide, Nitrobenzo-2-oxa-1,3-diazole, papain reactivation assay, etc.) were found to be inferior to the Ellman method based on the use of 5,5′dithio (bis-2 nitro benzoic acid). Techniques for SH group analysis in fluid milk and dried milk powder are described, along with typical results, their interpretations, and current research related to processing effects and the role of milk SH content on a wider range of technological issues, such as development of cooked flavors, fouling and cleaning of plate heat exchanges, protein-protein interactions, and the storage stability. Finally, a number of areas requiring further research are presented.

Design and synthesis of site directed maleimide bifunctional chelators for technetium and rhenium

A new family of heterobifunctional linkers (L1-L9) containing a terminus consisting of a tridentate donor set for coordination of the {M(CO)(3)}(+) core (M = Tc, Re), and a thiol reactive maleimide group has been prepared conveniently and in high yield under Mitsunobu reaction conditions by the coupling of an appropriate alcohol derivative with maleimide. The rhenium complexes [Re(CO)(3)(Lx)]Br (x= 1-9) were prepared in good yields from the reactions of the ligands and (NEt(4))(2)[Re(CO)(3)Br(3)] in refluxing methanol. The ligands and their Re complexes were characterized by (1)H and (13)C NMR, IR, and ESI-MS. Ligand L4 and [Re(CO)(3)(L5)]Br have been structurally characterized by X-ray crystallography. Photoexcitation of solutions of the complexes [Re(CO)(3)(Lx)]Br (x= 4-6) gives rise to intense and prolonged luminescence at room temperature (fluorescence lifetimes of ca. 16 micros). The ligands and their Re complexes react smoothly at the maleimide linker with sulfhydryl groups of peptides and proteins at room temperature in phosphate-buffered saline (PBS, pH 7.4) to form stable thioether bioconjugates. The photoluminescence properties of the labeled conjugates are similar to those of the parent complexes, but with even longer lifetimes. The ligands can also be labeled at room temperature with (99m)Tc to give chemically robust complexes. The corresponding hydrazinonicotinamide derivative N-[5-(6'-hydrazinopyridine-3'-carbonyl)aminopentyl]maleimide (L10) was also prepared. While coupling of L10 to cysteine ethylester and synthesis of the rhenium derivative [ReCl(3)(HYNIC-maleimide)(2)] were successfully accomplished, attempts to couple [ReCl(3)(HYNIC-maleimide)(2)] to glutathione or BSA yielded intractable mixtures.

Biochemical and Proteomics Approaches to Characterize Topoisomerase IIα Cysteines and DNA as Targets Responsible for Cisplatin-Induced Inhibition of Topoisomerase IIα

Cisplatin was shown to strongly inhibit the decatenation and relaxation activity of isolated human DNA topoisomerase IIalpha. This inhibition was not accompanied by stabilization of a covalent topoisomerase IIalpha-DNA intermediate. Pretreatment of kinetoplast plasmid DNA (kDNA) or pBR322 DNA with submicromolar concentrations of cisplatin quickly rendered these substrates incompetent in the topoisomerase IIalpha catalytic assay. Cisplatin nearly equally inhibited growth of a parental K562 and an etoposide-resistant K/VP.5 cell line that contained decreased topoisomerase IIalpha levels, a result consistent with isolated enzyme experiments demonstrating that cisplatin was not a topoisomerase IIalpha poison. Because cisplatin is known to react with protein sulfhydryl groups, the 13 cysteine groups in the topoisomerase IIalpha monomer were evaluated by mass spectrometry to determine which cysteines were free and disulfide-bonded to identify possible sites of cisplatin adduction. High-pressure liquid chromatography-matrix-assisted laser desorption ionization mass spectrometry showed that topoisomerase IIalpha contained at least five free cysteines (170, 216, 300, 392, and 405) and two disulfide-bonded cysteine pairs (427-455 and 997-1008). Cysteine 733 was also disulfide-bonded, but its partner cysteine could not be identified. Cisplatin antagonized the formation of a fluorescence adduct between topoisomerase IIalpha and the sulfhydryl-reactive maleimide reagent 10-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-9-methoxy-3-oxo-3H-naphtho[2,1-b]pyran-2-carboxylic acid methyl ester (ThioGlo-1). Dithiothreitol, which was shown by spectrophotometry to react rapidly with cisplatin (6-min half-time), diminished the capacity of cisplatin to interfere with ThioGlo-1 binding to topoisomerase IIalpha. The results of this study suggest that cisplatin may exert some of its cell growth inhibitory and antitumor activity by inhibition of topoisomerase IIalpha through reaction with critical enzyme sulfhydryl groups and/or by forming DNA adducts that render the DNA substrate refractory to topoisomerase IIalpha.

Measurement of methylglyoxal in rat tissues by electrospray ionization mass spectrometry and liquid chromatography

Introduction: Increased methylglyoxal formation due to insulin resistance has been implicated in the development of essential hypertension and in type-2 diabetic complications in animal models. Methylglyoxal is a highly reactive aldehyde, which binds sulfhydryl and amino groups of membrane proteins forming conjugates, advanced glycation end products (AGEs), which alter membrane function, leading to increased blood pressure and oxidative stress. We have shown elevated aldehyde conjugates in tissues of hypertensive rats which may be formed primarily from methylglyoxal. Our objective was to develop a specific method to measure methylglyoxal in rat tissues. Method: This method involves preparation of plasma, blood and tissue homogenates, solid phase extraction of methylglyoxal, derivitization using o-phenylenediamine, further purification of derivatized products by solid phase extraction and quantification by electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS). Results: Methylglyoxal was highest in aorta followed by heart, liver, kidney and blood in that order in Sprague–Dawley rats. Levels of methylglyoxal in plasma were about an order of magnitude lower than that in tissues, but above the concentration used for the lowest calibration standard. Discussion: We have successfully developed an ESI/LC/MS method for quantification of methylglyoxal in rat tissues. The high selectivity of this method offers an advantage over other methods based on fluorescence. This method will allow the evaluation of methylglyoxal in essential hypertension and type-2 diabetes.