Dynamic Thiol/Disulphide Homeostasis in Patients With Fibromyalgia.
ABSTRACT: Objectives:This study aims to investigate dynamic thiol/disulphide homeostasis in patients with fibromyalgia syndrome (FMS). Patients and methods:Fifty female patients with FMS (mean age 40.5±7.2 years; range 21 to 55 years) and 40 healthy female controls (mean age 39±9.4 years, range 22 to 55 years) were included in the study. Pain visual analog scale, tender points, Fibromyalgia Impact Questionnaire, and Beck Depression Inventory were evaluated. Age, body mass index (BMI), and symptom durations were also recorded. Native thiol, disulphide and total thiol levels were measured with a novel automated method. Results:Serum disulphide levels were 14.7±3.4 ?mol/L and 22.2±3.6 ?mol/L in the FMS and control groups, respectively (p<0.001). Native thiol levels were 452.1±33.8 ?mol/L and 433.5±37.6 ?mol/L in the FMS and control groups, (p=0.015), while total thiol levels were 481.7±35.6 ?mol/L and 477.5±38.9 ?mol/L in the FMS and control groups, respectively (p=0.593). In the FMS group, disulphide/native thiol percent ratios and disulphide/ total thiol percent ratios were statistically significantly lower and native/total thiol percent ratios were statistically significantly higher than those of the control group. There were no correlations between serum thiol/disulphide profiles and pain scores & clinical variables in patients with FMS. Conclusion:Because of the decreased disulphide and increased native thiol levels, the thiol/disulphide balance has shifted to the reductive side. This metabolic disturbance may have a role in the pathogenesis of FMS.
Project description:The thiol groups of beta-lactoglobulins A and B have been cyanylated using [13C]KCN. The samples of [cyanato-13C]-cyanylated-beta-lactoglobulins A and B which we prepared had signals at 109.7 p.p.m. and 114.4 p.p.m. We conclude that the thiocyanate carbon having a chemical shift of 109.7 p.p.m. is in an apolar environment similar to a cyclohexane solvent, whereas the thiocyanate carbon having a chemical shift of 114.4 p.p.m. is in a polar environment similar to water. The signals with chemical shifts of 109.7 p.p.m. are assigned to the thiocyanate carbons of the native [cyanato-13C]cyanylated-beta-lactoglobulins A and B. We deduce that the signal at 114.4 p.p.m. is due to an irreversibly denatured/unfolded species produced by alkaline denaturation, which is caused by intramolecular thiol/disulphide exchange occurring during our cyanylation procedure. We propose that Cys-119 is cyanylated in the irreversibly denatured species and Cys-121 is cyanylated in the native [cyanato-13C]cyanylated-beta-lactoglobulins A and B. We suggest that the same intramolecular thiol-disulphide exchange reactions occurred when McKenzie and co-workers [McKenzie, Ralston and Shaw (1972) Biochemistry 11, 4539-4547] alkylated beta-lactoglobulins with iodoacetamide. Therefore the one mol of thiol/mol of monomer in the native beta-lactoglobulins is due to the thiol of Cys-121 and is not due to an equimolar mixture of Cys-119 and Cys-121 as they suggested.
Project description:Objectives:This study aims to investigate the relationship of serum insulin-like growth factor 1 (IGF-1) level with obstructive sleep apnea, the Pittsburgh sleep quality index (PSQI), age, body mass index, and fibromyalgia impact questionnaire (FIQ) in fibromyalgia syndrome (FMS) patients. Patients and methods:A total of 105 female patients (mean age 41.8±9.0 years; range 26 to 55 years) with fibromyalgia (FMS group) who were diagnosed according to 2010 American College of Rheumatology criteria, and 51 female patients (mean age 39.9±10.8 years; range 24 to 54 years) with mechanical low back pain (MLBP) (control group) were included in the study. Age, smoking, educational status, tender point number, body mass index, duration of disease, FIQ, PSQI, and polysomnographic assessment of both FMS and control groups were recorded. Results:Sleep disorder was detected in 88 patients in FMS group and 15 patients in control group (p<0.05). The PSQI score was higher in the FMS group compared to the control group (9.9±4.6 vs. 5.7±3.5). The FIQ score was higher in the FMS group compared to the control group (53.4±17.4 vs. 26.4±13.9; p<0.05). The serum IGF-1 level of FMS group was significantly lower than that of the control group (140.6±49.5 ng/mL vs. 177.2±58.5 ng/mL; p<0.05). In the FMS group, an examination of the correlation between serum IGF-1 level with the age, body mass index, obstructive sleep apnea syndrome, FIQ, and PSQI revealed a negative correlation between serum IGF-1 with the age and PSQI. The obstructive sleep apnea syndrome ratios of study groups were comparable with regard to the frequencies of mild, moderate, and severe obstructive sleep apnea syndrome. Conclusion:In FMS patients, serum IGF-1 levels may decrease due to age and PSQI; however, this may not be related to the severity of obstructive sleep apnea.
Project description:AIM:To determine the frequency of ferritin deficiency in individuals with fibromyalgia syndrome (FMS) and to evaluate the association of ferritin level with depression, anxiety, sleep quality, and physical functioning. METHODS:This cross-sectional study, conducted from 2016 to 2017, compared the frequency of ferritin deficiency between 100 non-anemic fibromyalgia patients and 100 non-anemic individuals without FMS. Serum ferritin level of <30 ng/mL indicated iron deficiency. FMS patients filled out demographic questionnaire, Fibromyalgia Impact Questionnaire, Beck Anxiety Inventory, Beck Depression Inventory, and Pittsburg Sleep Quality Index. RESULTS:Median serum ferritin level was 20.95 ng/mL. A total of 64% of patients and 42% of controls had iron deficiency. Beck Anxiety Inventory, Beck Depression Inventory, and Pittsburgh Sleep Quality Index scores were not associated with ferritin levels. FMS patients with poor sleep quality had significantly higher Beck Depression Inventory, Beck Anxiety Inventory, and Fibromyalgia Impact Questionnaire scores (P<0.05). In individuals with poor sleep quality, lower ferritin levels also correlated with higher Beck Depression Inventory scores (r=-0.277, P<0.05). Sleep quality was not significantly associated with age, body mass index, duration of diagnosis, and serum ferritin levels. CONCLUSIONS:Patients with fibromyalgia syndrome have a rather high prevalence of non-anemic iron deficiency. No associations were found between serum ferritin level and anxiety, depression, sleep quality, and physical functioning.
Project description:The number and the reactivity of accessible thiol groups of the Folch-Pi apoprotein and proteolipid (50% of myelin proteins) were studied, by using a specific thiol-disulphide interchange reaction, in connection with the known solubility of this protein in organic and aqueous solvents. The high reactivity of 2,2'-dipyridyl disulphide towards thiol groups leads to the titration of 4.8 mol of SH groups/mol of protein (Mr 30000) in alkaline and acidic chloroform/methanol (2:1, v/v). Unlike previous findings, this value was consistently found from batch to batch and remained stable with time. In the proteolipid 1 mol of SH groups/mol was not accessible as compared with the apoprotein. In aqueous solvents, a similar number of 4.4 mol of SH groups/mol was also found. For the first time, kinetic studies carried out in chloroform/methanol discriminated between two classes of thiol groups. The reaction of 2 mol of SH groups/mol was characterized by apparent second-order rate constants whose values were 5-10-fold higher than those of the other class. Kinetic studies and cyanylation experiments in aqueous solvents also indicated the high reactivity of these thiol groups with Ellman's reagent. Together with kinetic results, studies on the stoichiometry of the interchange reaction of equimolar solutions of protein and disulphide indicate that these highly reactive thiol groups are near to each other in the amino acid sequence. The location of the thiol groups at the boundary between hydrophilic and hydrophobic domains of the Folch-Pi protein is suggested in connection with their possible structural and biological significance.
Project description:Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.
Project description:A heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate, was synthesized. Its N-hydroxysuccinimide ester group reacts with amino groups and the 2-pyridyl disulphide structure reacts with aliphatic thiols. A new thiolation procedure for proteins is based on this reagent. The procedure involves two steps. First, 2-pyridyl disulphide structures are introduced into the protein by the reaction of some of its amino groups with the N-hydroxysuccinimide ester sie of the reagent. The protein-bound 2-pyridyl disulphide structures are then reduced with dithiothreitol. This reaction can be carried out without concomitant reduction of native disulphide bonds. The technique has been used for the introduction of thiol groups de novo into ribonuclease, gamma-globulin, alpha-amylase and horseradish peroxidase. N-Succinimidyl 3-(2-pyridyldithio)propionate can also be used for the preparation of protein-protein conjugates. This application is based on the fact that protein-2-pyridyl disulphide derivatives (formed from the reaction of non-thiol proteins with the reagent) react with thiol-containing proteins (with native thiols or thiolated by, for example, the method described above) via thiol-disulphide exchange to form disulphide-linked protein-protein conjugates. This conjugation technique has been used for the preparation of an alpha-amylase-urease, a ribonuclease-albumin and a peroxidase-rabbit anti-(human transferrin) antibody conjugate. The disulphide bridges between the protein molecules can easily be split by reduction or by thiol-disulphide exchange. Thus conjugation is reversible. This has been demonstrated by scission of the ribonuclease-albumin and the alpha-amylase-urease conjugate into their components with dithiothreitol. N-Succinimidyl 3-(2-pyridyldithio)propionate has been prepared in crystalline form, in which state (if protected against humidity) it is stable on storage at room temperature (23 degrees C).
Project description:The activity of ferredoxin: NADP+ reductase (FNR) was found to decline to approximately 20% maximal levels with little or no loss in enzyme levels when cultures of the cyanobacterium Anabaena variabilis were maintained in the stationary phase of growth. Re-activation of enzyme activity occurred when cells were diluted into either fresh or re-utilized media and illuminated. This reversible de-activation/re-activation process was found, in vivo, to be dependent on the intensity of light illuminating the cells. The de-activated form of FNR was purified to homogeneity and exhibited the same molecular mass, isoelectric-focusing pattern and N-terminal amino acid sequence as the native form. Both de-activated and native FNR preparations each exhibited three reactive thiol groups on denaturation in urea; however, the rate of reaction with Ellman's reagent was much faster with the de-activated form than with the native form. Both preparations contain a single disulphide bond. Upon reduction of the disulphide bond in either form of the enzyme, the five reactive thiol groups exhibited identical reactivities in the presence of urea. Steady-state kinetic analysis of the de-activated form showed a marked increase in Km values for NADPH in diaphorase assays and an increase in Km for ferredoxin in the ferredoxin-mediated reduction of cytochrome c. No significant difference in kcat. was observed in comparison of the de-activated with the native form in any of the above assays; however, the de-activated form did exhibit a lower kcat. value in the transhydrogenase assay. The de-activated form of FNR bound ferredoxin with a 16-fold lower affinity than the native enzyme. These data suggest that the de-activation of FNR in vivo in response to low light intensity involves an alteration in protein structure, possibly via an intramolecular thiol disulphide interchange, which influences the interaction of the enzyme with its substrates.
Project description:The disulphide-coupled refolding of recombinant prochymosin from Escherichia coli inclusion bodies was investigated. Prochymosin solubilized from inclusion bodies is endowed with free thiol groups and disulphide bonds. This partially reduced form undergoes renaturation more efficiently than the fully reduced form, suggesting that some native structural elements existing in inclusion bodies and remaining after denaturation function as nuclei to initiate correct refolding. This assumption is supported by the finding that in the solubilized prochymosin molecule the cysteine residues located in the N-terminal domain of the protein are not incorrectly paired with the other cysteines in the C-terminal domain. Addition of GSH/GSSG into the refolding system facilitates disulphide rearrangement and thus enhances renaturation, especially for the fully reduced prochymosin. Based on the results described in this and previous papers [Tang, Zhang and Yang (1994) Biochem. J. 301, 17-20], a model to depict the refolding process of prochymosin is proposed. Briefly, the refolding process of prochymosin consists of two stages: the formation and rearrangement of disulphide bonds occurs at the first stage in a pH11 buffer, whereas the formation and adjustment of tertiary structure leading to the native conformation takes place at the second stage at pH8. The pH11 conditions help polypeptides to refold in such a way as to favour the formation of native disulphide bonds. Disulphide rearrangement, the rate-limiting step during refolding, can be achieved by thiol/disulphide exchange initiated by free thiol groups present in the prochymosin polypeptide, GSH/GSSG or protein disulphide isomerase.
Project description:1. The report by Robyt et al. (1971) that the 2-nitro-5-mercaptobenzoate dianion (Nbs(2-)) produced by reaction of papain with the 5,5'-dithiobis-(2-nitrobenzoate) dianion (Nbs(2) (2-); Ellman's reagent) cleaves the three disulphide bonds in papain is shown to be incorrect. 2. When partially active papain containing approx. 0.4 mol of thiol/mol of protein is incubated with excess of Nbs(2) (2-) at pH8, Nbs(2) (2-) reacts with the protein in an amount stoicheiometric with the cysteinyl thiol group of papain to produce Nbs(2-) in an amount stoicheiometric with the original papain cysteinyl thiol group, and the catalytically inactive mixed disulphide, papain-Nbs(-). 3. Papain catalyses the hydrolysis of Nbs(2) (2-) at pH10.5 probably by nucleophilic catalysis involving the enzyme's thiol group. 4. These results cast very serious doubts on the claim by Robyt et al. (1971) to have established a new general method for the determination of cystinyl disulphide residues in proteins.
Project description:Many extracellular globular proteins have evolved to possess disulphide bonds in their native conformations, which aids in thermodynamic stabilisation. However, disulphide bond breakage by heating leads to irreversible protein denaturation through disulphide-thiol exchange reactions. In this study, we demonstrate that methanethiosulphonate (MTS) specifically suppresses the heat-induced disulphide-thiol exchange reaction, thus improving the heat-resistance of proteins. In the presence of MTS, small globular proteins that contain disulphides can spontaneously refold from heat-denatured states, maintaining wild-type disulphide pairing. Because the disulphide-thiol exchange reaction is triggered by the generation of catalytic amounts of perthiol or thiol, rapid and specific perthiol/thiol protection by MTS reagents prevents irreversible denaturation. Combining MTS reagents with another additive that suppresses chemical modifications, glycinamide, further enhanced protein stabilisation. In the presence of these additives, reliable remnant activities were observed even after autoclaving. However, immunoglobulin G and biotin-binding protein, which are both composed of tetrameric quaternary structures, failed to refold from heat-denatured states, presumably due to chaperon requirements. Elucidation of the chemical modifications involved in irreversible thermoinactivation is useful for the development of preservation buffers with optimum constitutions for specific proteins. In addition, the impact of disulphide bond breakage on the thermoinactivation of proteins can be evaluated using MTS reagents.