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1.
Cys34 in domain I of the three-domain serum protein albumin is the binding site for a wide variety of biologically and clinically important small molecules, provides antioxidant activity, and constitutes the largest portion of free thiol in blood. Analysis of X-ray structures of albumin reveals that the loop containing Tyr84 occurs in multiple conformations. In structures where the loop is well defined, there appears to be an H-bond between the OH of Tyr84 and the sulfur of Cys34. We show that the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) with Tyr84Phe mutant albumin is approximately four times faster than with the wild-type protein between pH 6 and pH 8. In contrast, the His39Leu mutant reacts with DTNB more slowly than the wild-type protein at pH < 8, but at a similar rate at pH 8. Above pH 8 there is a dramatic increase in reactivity for the Tyr84Phe mutant. We also report (1)H NMR studies of disulfide interchange reactions with cysteine. The tethering of the two loops containing Tyr84 and Cys34 not only appears to control the redox potential and accessibility of Cys34, but also triggers the transmission of information about the state of Cys34 throughout domain I, and to the domainI/II interface. 相似文献
2.
Preparation and characterization of bovine albumin isoforms 总被引:1,自引:0,他引:1
Gabaldón M 《International journal of biological macromolecules》2002,30(5):43-267
Albumin undergoes changes in conformation and isomerizations by disulfide interchange of unknown biological significance. The aim of this study was to prepare and characterize albumin isoforms, which were stable under near physiological conditions. Modified albumins were obtained by urea denaturation and renaturation, and by aging at low ionic strength and alkaline pH in the presence of cysteine. We describe a cathodic electrophoresis technique, which allows the separation of albumin isoforms with greater positive charge. Differences between native and modified albumins were analyzed by new criteria based on the reactivity of the thiol and histidyl residues and on the susceptibility of the disulfide bonds to sulfitolysis. Modified albumins had, (i) a more cationic component which disappears by sulfitolysis of the disulfide bonds or by incubation with a glutathione redox system; (ii) higher reactivities of the free thiol group and of the histidyl residues, and; (iii) decreased fluorescence. These differences were not observed when processes were carried out on albumin with the thiol group blocked by iodacetic acid, but reappeared with the addition of cysteine. Renatured and aged albumins differed in the nature of the cationic component. Generation of albumin isoforms is dependent on the presence of a free thiol group and seems to involve thiol disulfide interchanges. 相似文献
3.
Studies on fatty-acid-binding proteins. The purification of rat liver fatty-acid-binding protein and the role of cysteine-69 in fatty acid binding. 总被引:1,自引:1,他引:0
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D C Wilton 《The Biochemical journal》1989,261(1):273-276
1. A new, simple and high-yield procedure is described for the purification of hepatic fatty-acid-binding protein from rat liver using naphthylaminodecyl-agarose as an affinity column. 2. Cysteine-69 is shown to react slowly, but quantitatively, with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), indicating that the thiol group is free, but may be buried within the protein. 3. Fatty acids do not affect the DTNB reactivity of this cysteine residue; however, cysteine reactivity is enhanced in the presence of haem and oleoyl-CoA. 4. Fatty-acid-binding protein that has been modified with DTNB is still able to bind the fluorescent fatty acid 11-(dansylamino)undecanoic acid, indicating that cysteine-69 may be remote from the fatty-acid-binding site. 相似文献
4.
The interaction of several serum albumins with chelated (iminodiacetate, IDA) and immobilized (agarose-IDA) metal ions, Co2+, Ni2+, Cu2+ and Zn2+, was studied. There was no retention of human, bovine, porcine, murine and avian albumins on IDA-Zn(II) and IDA-Co(II) columns. However, all albumins studied, i.e., those of: man, cow, pig, dog, rabbit, rat, mouse, chicken and pigeon were retained on IDA-Cu(II) columns, and all except dog albumin were retained also on IDA-Ni(II). The recognition of albumins by chelated and immobilized transition metals seems to be related to an affinity for the imidazole side chains. It is postulated that one to three imidazoles is involved in this interaction, under the employed experimental conditions (pH 7.0; 1 M sodium chloride). There is no evidence for any significant contribution of tryptophan or cysteine (Cys 34) residues to the chromatographic event. The retention of defatted albumin and albumin oligomers (human), on IDA-Cu(II) columns was not significantly different from that of non-defatted albumin or albumin monomer, respectively. 相似文献
5.
Okazaki T Okudaira N Ishii N Yotsuyanagi H Nagai T Tokudome S Fujioka T Takahashi S 《Zoological science》2008,25(2):172-177
We measured the antioxidant activity of human, rat, bovine, rabbit, and guinea pig albumins against the superoxide, hydroxyl, and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radicals. The albumins of different animal species did not differ in antioxidant activity against superoxide. Human and rat albumins exhibited antioxidant activity against hydroxyl radicals, but bovine, rabbit, and guinea pig albumins showed weaker antioxidant activity than human and rat albumins. Human, rat, rabbit, and guinea pig albumins, but not bovine albumin, exhibited strong antioxidant activity against DPPH radicals. Human and rat albumins with strong antioxidant activity against hydroxyl radicals contained methionine-123 in domain 1, but bovine, rabbit, and guinea pig albumins did not. Rat, rabbit, and guinea pig albumins with strong antioxidant activity against DPPH radicals had methionine-264 in domain 2. Human albumin did not have methionine-264, but methionine-298 and methionine-329 in domain 2. Bovine albumin, with the weakest antioxidant activity against DPPH radicals, contained no methionine residues in domain 2. These results suggest that methionine residues in domain 1 or 2 influence the antioxidant activity of albumin. 相似文献
6.
Bovine albumin was phosphorylated by both cAMP-dependent protein kinase and casein kinase I to a significant extent. Other albumins were also tested and it was found that the extent of phosphorylation varied with the species of origin of the albumin, but was between 1 and 3 mol phosphate per mole albumin for the cAMP-dependent protein kinase-catalyzed reactions. The phosphorylation occurred at and above pH 7.5 and required the presence of thiol reagents. Phosphoamino acid analyses of bovine albumin showed that it was phosphorylated on at least two serine residues. The phosphorylation could not be demonstrated in vivo. 相似文献
7.
In this study, we synthesized a zwitterionic DTNB derivative, 5-(2-aminoethyl)-dithio-2-nitrobenzoate (ADNB), and characterized its reactions with several cationic, anionic, and neutral thiols. Reactions with ADNB, unlike those with DTNB, are relatively insensitive to electrostatic environments and ionic strengths. At relatively low ionic strength, rate ratios,kADNB/kDTNB, varied from 0.22 for reactions with low-molecular-weight cationic thiols to 3.0 for those with low-molecular-weight anionic thiols. AkADNB/kDTNBratio of 200 for Cys-34 of BSA appears to reflect a very anionic environment.kADNB/kDTNBratios of 6 and 1, respectively, for canine and equine serum albumins, which have Glu-82 → Asp and Glu-82 → Ala substitutions suggest Glu-82 is the most important anionic residues affecting the reactivity of Cys-34 in BSA.kADNB/kDTNBratios appear to be useful for characterizing electrostatic environments of thiol groups in proteins. 相似文献
8.
Two classes of sulfhydryl groups in histidine decarboxylase from Lactobacillus 30 a can be differentiated by their reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Five cysteinyl residues (class I) of the native enzyme are titrated by DTNB as the pH of the reaction medium is increased from 6.5 to 7.5; the pH-rate profile for their reaction is described by a pKa of 9.2. An additional five thiol groups (class II) are titrated only when denaturing agents are added above neutral pH. Histidine decarboxylase is completely inactivated by DTNB in a kinetically second-order process (Kapp = 660 +/- 20 M-1 min-1 at pH 7.6 and 25 degrees C) which occurs coincident with and at the same rate as modification of the five class-I SH groups of the enzyme, i.e., one thiol group per pyruvoyl prosthetic group. The competitive inhibitors, histamine and imidazole, markedly enhanced the reactivity of these cysteinyl residues toward DTNB; this enhancement is accompanied by a concomitant increase in the rate of inactivation. A single SH group in each of the five catalytic units of histidine decarboxylase is thus implicated as being critical for the expression of enzymatic activity. 相似文献
9.
Torres MJ Turell L Botti H Antmann L Carballal S Ferrer-Sueta G Radi R Alvarez B 《Archives of biochemistry and biophysics》2012,521(1-2):102-110
The single cysteine residue of human serum albumin (HSA-SH) is the most abundant plasma thiol. HSA transports fatty acids (FA), a cargo that increases under conditions of diabetes, exercise or adrenergic stimulation. The stearic acid-HSA (5/1) complex reacted sixfold faster than FA-free HSA at pH 7.4 with the disulfide 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and twofold faster with hydrogen peroxide and peroxynitrite. The apparent pK(a) of HSA-SH decreased from 7.9±0.1 to 7.4±0.1. Exposure to H(2)O(2) (2mM, 5min, 37°C) yielded 0.29±0.04mol of sulfenic acid (HSA-SOH) per mole of FA-bound HSA. The reactivity of HSA-SOH with low molecular weight thiols increased ~threefold in the presence of FA. The enhanced reactivity of the albumin thiol at neutral pH upon FA binding can be rationalized by considering that the corresponding conformational changes that increase thiol exposure both increase the availability of the thiolate due to a lower apparent pK(a) and also loosen steric constraints for reactions. Since situations that increase circulating FA are associated with oxidative stress, this increased reactivity of HSA-SH could assist in oxidant removal. 相似文献
10.
Cyst(e)ine residues of bovine white-matter proteolipid proteins. Role of disulphides in proteolipid conformation.
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P I Oteiza A M Adamo P A Aloise A C Paladini A A Paladini E F Soto 《The Biochemical journal》1987,245(2):507-513
Cyst(e)ine residues of bovine white-matter proteolipid proteins were characterized in a highly purified preparation. From a total of 10.6 cyst(e)ine residues/molecule of protein, as determined by performic acid oxidation, 2.5-3 thiol groups were freely accessible to iodoacetamide, iodoacetic acid and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), when the proteins were solubilized in chloroform/methanol (C/M) (2:1, v/v). The presence of lipids had no effect on thiol-group exposure. One thiol group available to DTNB in C/M could not be detected when proteolipids were solubilized in the more polar solvent n-butanol. In a C/M solution of purified proteolipid proteins, SDS did not increase the number of reactive thiol groups, but the cleavage of one disulphide bridge made it possible to alkylate six more groups. C.d. and fluorescence studies showed that rupture of this disulphide bond changed the protein conformation, which was reflected in partial loss of helical structure and in a greater exposure to the solvent of at least one tryptophan residue. Cyst(e)ine residues were also characterized in the different components [PLP (principal proteolipid protein), DM20 and LMW (low-Mr proteins)] of the proteolipid preparation. Although the numbers of cyst(e)ine residues in PLP and DM20 were similar, in LMW fewer residues were alkylated under four different experimental conditions. The differences, however, are not simply related to differences in Mr. 相似文献
11.
Cysteine (Cys) residues often play critical roles in proteins, for example, in the formation of structural disulfide bonds, metal binding, targeting proteins to the membranes, and various catalytic functions. However, the structural determinants for various Cys functions are not clear. Thiol oxidoreductases, which are enzymes containing catalytic redox-active Cys residues, have been extensively studied, but even for these proteins there is little understanding of what distinguishes their catalytic redox Cys from other Cys functions. Herein, we characterized thiol oxidoreductases at a structural level and developed an algorithm that can recognize these enzymes by (i) analyzing amino acid and secondary structure composition of the active site and its similarity to known active sites containing redox Cys and (ii) calculating accessibility, active site location, and reactivity of Cys. For proteins with known or modeled structures, this method can identify proteins with catalytic Cys residues and distinguish thiol oxidoreductases from the enzymes containing other catalytic Cys types. Furthermore, by applying this procedure to Saccharomyces cerevisiae proteins containing conserved Cys, we could identify the majority of known yeast thiol oxidoreductases. This study provides insights into the structural properties of catalytic redox-active Cys and should further help to recognize thiol oxidoreductases in protein sequence and structure databases. 相似文献
12.
P A Nygren C Ljungquist H Tr?mborg K Nustad M Uhlén 《European journal of biochemistry》1990,193(1):143-148
The interaction of the serum albumin binding domain from streptococcal protein G to serum albumins isolated from different species was investigated. The highest affinity to protein G was found for serum albumins from rat, man and mouse. A medium binding was found for serum albumin from rabbit, cow, hen and horse, while little or no binding was found for ovalbumin and serum albumin from sheep. The interaction between human serum albumin and protein G showed rapid binding kinetics at the temperatures 7, 22 and 37 degrees C. Furthermore, the ability of different serum albumins to function as affinity ligands when covalently coupled to a solid support was tested. The results show that protein G derivatives could be eluted at different pH depending on the origin of the serum albumin. It was also possible to elute the streptococcal receptor efficiently from the mouse serum albumin matrix with human serum albumin. Based on these results, a gene fusion system for recovery of sensitive proteins by affinity purification is described, where high yields are obtained under mild elution conditions. 相似文献
13.
Effect of nutrient supply on mungbean protein fractions was studied with respect to four concentration levels (0.0, 25, 50 and 100 mM) each of sulphur, potassium and phosphorus. Whereas amount (mg/g seed meal) of globulins increased under the increasing concentrations of all the three minerals, albumins increased under sulphur and potassium and glutelins under sulphur and phosphorus only. Tryptophan contribution due to albumins and globulins was found to increase while that due to glutelins decreased under increasing supplies of sulphur and phosphorus. However, under higher supply levels of potassium, tryptophan contribution due to all the fractions was found to increase. Methionine contribution due to albumin and globulin fractions increased under sulphur and potassium and that due to glutelins decreased as the concentration levels of potassium and phosphorus increased. 相似文献
14.
Role of thiols, pH and cathepsin D in the lysosomal catabolism of serum albumin. 总被引:3,自引:2,他引:1
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J L Mego 《The Biochemical journal》1984,218(3):775-783
Attempts were made to assess the role of thiols and to determine the cathepsins involved in the degradation of serum albumin in mouse liver and kidney lysosomes. Unlike cysteine or beta-mercaptoethanol, reduced glutathione (GSH) did not stimulate the degradation of formaldehyde-treated albumin in liver lysosomes, suggesting that the tripeptide did not penetrate the membrane. However, GSH was a much more effective stimulant of proteolysis in kidney lysosomes than was cysteine at low concentrations, and the effect was saturable at 1-2 mM concentrations. Thiols did not stimulate proteolysis in lysosomes when the disulphide bonds of albumin were reduced and alkylated, suggesting that the stimulatory effects were solely due to disulphide-bond reduction in protein substrates. Results obtained with thiols and iodoacetamide suggested that albumins denatured by disulphide-bond reduction and alkylation, disulphide-bond reduction without alkylation, or by treatment with 8 M-urea, were all degraded primarily by cathepsin D in lysosomes, but formaldehyde-denatured albumin was attacked by thiol proteinases. These findings correlated well with studies on the degradation of these proteins by rat liver lysosome (tritosome) extracts. Studies with the proteinase inhibitors leupeptin and pepstatin and the stimulatory effects of thiols in these extracts suggested that formaldehyde-denatured albumin was degraded primarily by the thiol proteinases, but that native albumin or albumins denatured by disulphide-bond reduction or by treatment with 8 M-urea were attacked by cathepsin D. Denaturation of serum albumin by any of the methods used caused a shift in the pH optimum of albumin catabolism by tritosome extracts or by purified cathepsin D from approx. 3-4 to 5-6. These results were discussed in terms of a possible mechanism for the catabolic aspect of serum albumin turnover. 相似文献
15.
P M Sokolove 《FEBS letters》1988,234(1):199-202
Induction of Ca2+ release from isolated, preloaded rat heart mitochondria by low concentrations (less than 5 micrM) of adriamycin aglycones, has recently been reported [(1988) Biochem. Pharmacol. 37, 803]. Ca2+ release occurs via a generalized, Ca2+-dependent increase in the permeability of the inner mitochondrial membrane to small molecules. The process is antagonized by dithiothreitol, suggesting thiol involvement. This communication demonstrates modification of mitochondrial sulfhydryl groups, detected as decreased 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) reactivity, by adriamycin aglycones. Ca2+ release and sulfhydryl modification are shown to depend similarly on aglycone concentration and on the C-7 substituent of the anthracycline ring. In addition, DTNB elicits Ca2+ release. It can therefore be proposed that adriamycin aglycones alter mitochondrial membrane permeability by altering mitochondrial thiol status. 相似文献
16.
17.
The metabolites of gold in the urine of rats given the antiarthritic drug aurothiomalate were investigated by gel permeation chromatography, electrophoresis, and chemical studies. Following a single dose of aurtothiomalate, the excreted gold was protein-bound in the high-molecular-weight (greater than or equal to 150,000 dalton) and serum albumin fractions. Electrophoresis confirmed the presence of albumin, but showed that the other proteins present differ from those in normal or in vitro aurothiomalate-incubated rat sera. The pattern of the proteins establishes that the proteinuria was of the glomerular type. The alterations in the gold distribution produced by incubation of the urine with the low-molecular-weight thiol penicillamine and with exogenously added aurothiomalate indicated the existence of a labile equilibrium of gold among protein binding sites in the urine. Incubation of rat and human sera and commercially prepared serum albumins with aurothiomalate increased the electrophoretic mobility of the albumin. The significance of this change in electrophoretic mobility with respect to two models of gold binding by serum albumin is discussed. 相似文献
18.
The reactivity of sulfhydryl groups of bovine cardiac troponin C 总被引:2,自引:0,他引:2
Bovine cardiac troponin C (cTnC) contains 2 cysteine residues, Cys-35 located in the nonfunctional Ca2+-binding loop I and Cys-84 in the N-terminal segment of the central helix. We have studied the reactivity of Cys residues in cTnC with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). The latter compound fluoresces only when reacted with the protein. The reaction with DTNB followed second order kinetics with respect to DTNB, the rate constants being 3.37 s-1 M-1 and 1.82 s-1 M-1 in the presence and absence of Ca2+, respectively. These rates are much slower than the rate of reaction with Cys-98 of skeletal TnC (sTnC) or with the urea-denatured cTnC, indicating that both Cys residues are partly buried within the structure of the protein. The increase in reactivity was induced by binding of Ca2+ to the single low affinity Ca2+ binding site (site II). The fluorescence increase upon reaction of cTnC with CPM in the absence of Ca2+ could be fitted with a single exponential equation indicating that both cysteine residues are equally available to the reagent. The reaction in the presence of Ca2+ was biphasic. Analysis of CNBr fragments of cTnC labeled with CPM under various conditions indicated that in the presence of Ca2+ the reactivity of Cys-84 is increased while that of Cys-35 is slightly decreased. This finding is consistent with the model of Herzberg et al. (Herzberg, O., Moult, J., and James, M. N. G. (1986) J. Biol. Chem. 261, 2638-2644) and the data of Ingraham and Hodges (Ingraham, R. H., and Hodges, R. S. (1988) Biochemistry 27, 5891-5898), suggesting that the Ca2+-induced conformational change in the N-terminal half of TnC involves separation of the helix C from the central helix, thereby increasing the accessibility of Cys-84. The slow overall kinetics, however, indicates that the structure in the vicinity of Cys residues is relatively compact regardless of Ca2+. We interpret the increase in reactivity towards CPM as consistent with a Ca2+-induced exposure of a hydrophobic pocket in the vicinity of Cys-84. 相似文献
19.
The reactivity of chick erythrocyte and calf thymus histone H3 thiol groups toward 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) has been investigated both in the soluble, DNA-free state and in various nucleohistone complexes. We have found that the thiol reactivity of both tetramers and octamers decreases continuously as the ionic strength of the assay is increased, up to and beyond 2.0 M NaCl. Upon association of dimers with tetramers, there is loss of labeling by DTNB at one site, suggesting the existence of allosteric regulation [see also Godfrey, J. E., Eickbush, T. H., & Moudrianakis, E. N. (1980) Biochemistry 19, 1339-1346] of dimer-tetramer interfaces emanating from within the tetramer complex. Comparison of the thiol reactivities of chick and calf tetramers indicates that the thiol groups at amino acid positions 96 and 110 are not chemically equivalent. When the histones are associated with DNA, in either reconstituted complexes, core particles, or long soluble chromatin, the thiol reactivity is greatly diminished, and this "DNA effect" overwhelms any influence of dimers. However, if single-strand nicks are introduced into the DNA backbone of core particles and other chromatin-like complexes by the action of DNase I, the influence of the DNA double helix upon thiol reactivity is reduced, and the effect of dimers can be detected once again. We can therefore conclude that the DNA effect derives from intranucleosomal torsional strain of the continuum of the double helix in equilibrium with coupled protein conformational changes. These observations support the concept that the octamer complex is a dynamic tripartite structure whose properties can be modulated through its interactions with DNA and by changes occurring in the dimer-tetramer interfaces. 相似文献
20.
Accumulation of different protein fractions in chickpea seed harvested from plants grown under four concentration levels (0, 25, 50 and 100 mM) of sulphur, nitrogen, potassium and phosphorus has been studied. Whereas globulins showed an increase with the increasing concentration of all the four minerals, albumins increased under sulphur, nitrogen and potassium, and glutelins increased under potassium and phosphorus. An increase in nitrogen regime led to increased contribution of tryptophan in the seed protein due to albumins and globulins and methionine contribution due to albumins, globulins and glutelins. Under higher supplies of sulphur, relative contribution of tryptophan due to albumins and methionine contribution due to albumins and globulins had increased. 相似文献