A sensitive gel filtration method for determination of protein sulfhydryl groups with 14C-chloromercuribenzoate

A gel filtration method employing ¹⁴C-chloromercuribenzoic acid is described for the quantitative determination of sulfhydryl groups in microgram quantities of protein. The method has been applied to several native proteins, hemoglobin, monoamine oxidase, and yeast cytochrome c. In all cases values in close agreement with known literature values were obtained. Horse heart cytochrome c and lysozyme, which have no sulfhydryl groups, did not bind the mercurial reagent. Modifications of the method are described for determining the sulfhydryl content of denatured proteins in the presence of sodium lauryl sulfate. The precision of the method was found to be compatible with known methods for determining the sulfhydryl composition of proteins.     

Staining of free sulfhydryl groups of proteins after separation by isoelectric focusing of Bio-Rad standards and lens crystallins.

In this study, we propose a new staining method for free sulfhydryl groups of proteins after having separated native samples by thin-layer isoelectric focusing (IEF) in absence of detergents. A comparison was made between proteins stained purple for free sulfhydryl groups (SH) and proteins stained blue by Coomassie blue (CB). A stainability factor, F = %SH/%CB, was calculated for each protein. The Bio-Rad IEF standards containing seven marker proteins for pH scale determination were stained purple, in the same way as they were designed for CB staining. To prove the validity of the currently proposed staining method for a defined protein system such as the eye lens crystallins, these proteins were also stained after IEF as described above. The factor F was calculated for all alpha-, beta-, and gamma-crystallin components that stained in both methods. We discovered that alpha-crystallins contained comparatively high amounts of free SH groups, while some beta- and gamma-crystallin components also contained considerable amounts of free SH groups. The SH staining procedure with 2,2'-dihydroxy-6,6'-dinaphthyl disulfide applied after IEF appeared to be useful, specific, and reproducible.     

Abundance of protein-bound sulfhydryl and bisulfide groups at chromosomal nucleolus organizing regions

Silver stainability of the chromosomal nucleolus organizing regions that contain the structural genes for ribosomal RNA can be abolished by proteolytic and oxidative treatments. Histone extraction has no effect. This indicates that reducing groups of non-histone chromosomal proteins are responsible for silver staining. Treatment with fluorescent sulfhydryl and disulfide specific reagents followed by silver staining demonstrates coincidence of silver dots and brightly fluorescent spots at the short arms of human acrocentric chromosomes where ribosomal RNA-genes are located. After treatment with cupric sulfite reagent in the presence of urea fluorescence and silver staining was no longer possible. Silver staining has been reported to be associated with ribosomal RNA-gene activity. Acrocentric chromosomes that are negative in silver staining also lack the brightly fluorescent spots. Therefore, we conclude that an abundance of protein-bound sulfhydryl and disulfide groups occur at nucleolar organizing regions with active genes. Differentially fluorescing spots could not be observed after staining with fluorescamine. So, either the sulfhydryl reagents used in this study are much more sensitive than fluorescamine to study protein distributions in cytological preparations, or our observations point to a local accumulation of some specific protein(s) rich in sulfhydryls. The presence of many sulfhydryl and disulfide groups at the nucleolus organizing regions seems suggestive of a great flexibility of protein(s) by transition of sulfhydryl groups to disulfide bridges and vice versa at these highly active regions of the genome.     

Effects of freezing and hardening on the sulfhydryl groups of protein fractions from cabbage leaves

Disc electrophoresis was used to separate water soluble proteins from hardy, non-hardy, and frost killed cabbage (Brassica oleracea var. capitata) leaves. Amidoschwarz staining failed to reveal any new bands as a result of hardening although the relative amounts of proteins in individual bands changed. Sulfhydryl groups in the protein bands were stained with 2,2-dihydroxy-6,6-dinaphthyl disulfide and labeled with ¹⁴C p-chloromercuribenzoate. Significant decreases in the sulfhydryl content of the total water soluble protein were found during hardening and as a result of frost death. The decrease during hardening was paralleled by a significant increase in the water soluble protein. There was a significant increase in the sulfhydryl content per unit high molecular weight protein but a decrease in the sulfhydryl content per total protein as a result of frost death. This was interpreted as evidence for intermolecular disulfide bond formation during freezing.     

Involvement of sulfhydryl groups of chromosomal proteins in sister chromatid differentiation

The fluorescence of human lymphocyte chromosomes stained with sulfhydryl group-specific fluorochromes is markedly enhanced by a mild near-ultraviolet irradiation pretreatment, indicating breakage of protein disulfide bonds. When metaphase preparations of cells cultured in the presence of BrdU during two cell cycles are irradiated and subsequently stained with the sulfhydryl group-specific fluorescent reagents used in this study, a differential fluorescence of sister chromatids is observed. After staining with the DNA-specific fluorochrome DAPI an opposite pattern of lateral differentiation appears. It can be concluded that the chromatid containing bifilarly BrdU-substituted DNA has a higher content of sulfhydryl groups than the chromatid containing unifilarly BrdU-substituted DNA. This implies a more pronounced effect of breakage of disulfide bonds in the chromatid with the higher degree of BrdU-substitution. BrdU-containing chromosomes pretreated with the mild near-ultraviolet irradiation procedure used by us, do not show any differentiation of sister chromatids after Feulgen staining. Using sulfhydryl group-specific reagents, differential fluorescence of sister chromatids could still be induced by irradiation with near-ultraviolet light after the complete removal of DNA from the chromosomes by incubation with DNase I. Thus, the protein effect of irradiation of BrdU-containing chromosomes takes place independently of what occurs to DNA.Our results indicate that subsequent to the primary alteration of chromatin structure caused by the incorporation of BrdU into DNA, breakage of disulfide bonds of chromosomal proteins might play an important role in bringing about differential staining of sister chromatids, at least for those procedures that use irradiation as a pretreatment or prolonged illumination during microscopic examination.     

Dimethylformamide interferes with Coomassie dye staining of proteins on blue native gel electrophoresis

Blue native gel electrophoresis (BN–PAGE) is used extensively for characterization of mitochondrial respiratory complexes and uses the binding of Coomassie brilliant blue G-250 to visualize proteins. Oxidative modification of sulfhydryl groups of such proteins can be evaluated by labeling with iodoacetamide conjugated to biotin (BIAM) and detected with streptavidin peroxidase on Western blots following BN–PAGE. However, dissolving BIAM in dimethylformamide, a recommended solvent, reduces Coomassie blue G staining to proteins during BN–PAGE. This interference is prevented by dissolving BIAM in dimethyl sulfoxide. Precautions in the use of the dye for protein staining subsequent to BIAM labeling are discussed.     

Food Processing Characteristics of Soybean Proteins

Relation between sulfhydryl groups in soybean proteins and the physical properties of tofu was studied. Changes in the amount of sulfhydryl groups by heating or treatment with urea were more rapid in 11S protein as compared with 7S protein. Moreover, by changing the amount of sulfhydryl groups in proteins by N-ethylmaleimide, 2-mercapto-ethanol and dithiothreitol, the physical properties of tofu from 11S protein were more significantly effected than that from 7S protein. Namely, tofu-gel from 11S. protein got harder and stronger as the amount of sulfhydryl groups increased.

The results may suggest that tofu prepared from IIS protein has more disulfide bonds in its gel than that from 7S protein.     

A colorimetric assay specific for gamma-carboxyglutamic acid-containing proteins: its utility in protein purification procedures

A colorimetric method for the detection of gamma-carboxyglutamic acid (Gla)-containing proteins after reaction with 4-diazobenzenesulfonic acid is presented. Proteins can be visualized after electroblotting from polyacrylamide gels onto membrane supports, after dot-blotting onto membranes, or in solution as a red colored product with an absorbance maximum at 530 nm. The method is specific since other proteins without gamma-carboxyglutamic acid do not form a red color. The presence of other proteins does not inhibit or affect color production by gamma-carboxyglutamic acid-containing proteins. Application of the method for staining a Western blot of a crude extract of bone resulted in staining of only the gamma-carboxyglutamic acid-containing proteins. The usefulness of the method was verified when a second gamma-carboxyglutamic acid-containing protein, prothrombin, also resulted in red color production. A linear color response is seen up to 17 microM for the gamma-carboxyglutamic acid-containing protein bone Gla protein and up to 27 microM for the amino acid. The detection limit is down to 1 microgram of bone Gla protein or 0.17 nmol of the protein on electroblots or dot blots. The simplicity of the method allows rapid screening for gamma-carboxyglutamic acid-containing proteins or allows monitoring of purifications of these proteins in chromatographic or electrophoretic separations.     

Inhibition by brefeldin A of protein secretion from the apical cell surface of Madin-Darby canine kidney cells

The effect of brefeldin A (BFA) on total and polarized protein secretion was examined in MDCK cells. Increasing concentrations of BFA have increasingly inhibitory effects on total protein secretion. The total protein secretion was essentially unaffected by BFA at 0.5 microgram/ml. When the BFA concentration was increased to 10 and 30 micrograms/ml, the total protein secretion was reduced to about 70 and 25%, respectively, of the control level. Consistent with this effect on total protein secretion, the Golgi structure as revealed by C6-NBD-ceramide (a fluorescent ceramide analog) staining was essentially unaltered by 0.5 microgram/ml BFA, while 10 and 30 micrograms/ml BFA significantly dispersed the Golgi apparatus. When the polarity of protein secretion was examined, it was found that the ratio of proteins secreted from the apical to those from the basolateral surface was reduced from 1.5-2.0 to 0.4-0.7 by all three BFA concentrations. Furthermore, several proteins which are preferentially released from the apical surface were found to be released without apparent surface polarity, while several other proteins which were preferentially released from the basolateral surface were unaffected. This study suggests that BFA, at 0.5 microgram/ml, can selectively inhibit protein secretion from the apical surface without affecting total protein secretion. The inhibition of apical secretion results in enhanced protein secretion from the basolateral surface.     

Biochemical and immunological identification of human neutrophil elastase on nitrocellulose membranes.

Human neutrophil elastase (HNE) was analyzed for protein(s), antibody staining and activity staining, on lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis followed by Western blotting. The HNE activity, which was identified with N-acetyl-D,L-alanine alpha-naphthyl ester as substrate, was well preserved in the presence of 0.1% LDS at 4 C during electrophoresis. As little as 0.1 microgram HNE was required for the activity staining. The HNE appeared to be three peptides having a major band at mass ratio 27,000, a second major band at mass ratio 28,000 with a minor protein band at mass ratio 29,000. On transfer to nitrocellulose, the mass ratio 28,000 band displayed poor immunoreactivity. This was the second most dense band with highest enzymatic staining. This procedure is a useful method and analytical tool to determine the correlation of enzymatically active proteins, subunits and immunoreactive protein(s) of elastase from various sources, including neutrophils.     

A method for detecting proteins immobilized on nitrocellulose membranes by in situ derivatization with fluorescein isothiocyanate

A method for the fluorescent staining of proteins on nitrocellulose filters is described. The single step procedure uses a 100 microgram/ml solution of fluorescein isothiocyanate in sodium carbonate buffer, pH 9.5. The proteins are visible under uv light within 30 s and the staining reaction is virtually complete after 10 min. The method can detect a minimum of 50 ng protein/band providing a sensitivity similar to that obtained with anionic dye stains. The method is suitable for blots prepared from both isoelectric focusing gels and sodium dodecyl sulfate-polyacrylamide gels. The fluorescently labeled proteins can be probed using immunochemical techniques with the retention of fluorescence. The method can therefore be used to accurately locate antigens among a number of proteins and allows the sensitive and rapid detection of marker proteins directly on the blot.     

Determination of protein by Coomassie dye-binding in agarose gels

A variation of the Coomassie dye-binding assay for proteins is described. Protein samples were pipetted to the surface of agarose plates in uniformly sized spots and stained with Coomassie Blue G-250. The bound dye was determined by densitometric scanning using double wavelength and flying spot facilities. The response curves were linear in an about 10-fold concentration range with a lower detection limit of 0.5 microgram. No background correction was necessary because unbound dye and most substances known to interfere with other protein assays were removed during the staining and destaining of the agarose gels. Membrane proteins could be analyzed since the samples were applied as solutions in 1% sodium dodecyl sulfate.     

Detection and quantification of free sulfhydryls in monoclonal antibodies using maleimide labeling and mass spectrometry

The detection of free sulfhydryls in proteins can reveal incomplete disulfide bond formation, indicate cysteine residues available for conjugation, and offer insights into protein stability and structure. Traditional spectroscopic methods of free sulfhydryl detection, such as Ellman’s reagent, generally require a relatively large amount of sample, preventing their use for the analysis of biotherapeutics early in the development cycle. These spectroscopic methods also cannot accurately determine the location of the free sulfhydryl, further limiting their utility. Mass spectrometry was used to detect free sulfhydryl residues in intact proteins after labeling with Maleimide-PEG₂-Biotin. As little as 2% cysteine residues with free sulfhydryls (0.02 mol SH per mol protein) could be detected by this method. Following reduction, the free sulfhydryl abundance on antibody heavy and light chains could be measured. To determine free sulfhydryl location at peptide-level resolution, free sulfhydryls and cysteines involved in disulfide bonds were differentially labeled with N-ethylmaleimide and d₅-N-ethylmaleimide, respectively. Following enzymatic digestion and nanoLC-MS, the abundance of free sulfhydryls at individual cysteine residues was quantified down to 2%. The method was optimized to avoid non-specific labeling, disulfide bond scrambling, and maleimide exchange and hydrolysis. This new workflow for free sulfhydryl analysis was used to measure the abundance and location of free sulfhydryls in 3 commercially available monoclonal antibody standards (NIST Monoclonal Antibody Reference Material (NIST), SILu?Lite SigmaMAb Universal Antibody Standard (Sigma-Aldrich) and Intact mAb Mass Check Standard (Waters)) and 1 small protein standard (β-Lactoglobulin A).     

Purification and characterization of protein carboxyl methyltransferase from Torpedo ocellata electric organ

Posttranslational modification of proteins by the enzyme protein carboxyl methyltransferase (PCM) has been associated with a variety of cellular functions. A prerequisite for the understanding of cellular mechanisms associated with PCM is the characterization of purified PCMs from different tissues. We describe here the purification and characterization of PCM from the electric organ of Torpedo ocellata. The enzyme was purified to homogeneity by ion-exchange chromatography and ammonium sulfate precipitation, followed by chromatography on Sephadex G-100 and hydroxylapatite columns. When visualized by silver staining, the 700-fold-purified PCM exhibited a single band on sodium dodecyl sulfate-polyacrylamide gels, corresponding to a polypeptide of Mr 29,000. The molecular weight of the nondenatured enzyme (as determined by rechromatography on Sephadex G-100 column) was also 29,000, suggesting that the enzyme is a monomer. Two isoelectric forms of PCM (pI = 6.1 and pI = 6.4) were detected in the purified enzyme preparation. The enzyme methylates various exogenous and endogenous proteins, including the acetylcholine receptor. Of the four different polypeptides of the acetylcholine receptor, the gamma and beta polypeptides were selectively methylated by the purified PCM. Purified Torpedo PCM is highly sensitive to sulfhydryl reagents. The competitive inhibitor of PCM S-adenosyl-L-homocysteine (AdoHcy) protected the enzyme from inactivation by sulfhydryl reagents, suggesting the existence of a cysteine residue at the active site of the enzyme. The purified PCM has a low affinity toward DEAE-cellulose and toward AdoHcy-agarose. This property, as well as the relatively high molecular weight and the marked sensitivity to sulfhydryl reagents, distinguishes between the electric organ PCM and analogous enzymes of mammalian tissues.     

Silver staining of extensively glycosylated proteins on sodium dodecyl sulfate-polyacrylamide gels: enhancement by carbohydrate-binding dyes.

Two methods are described for detecting less than 1 microgram of highly glycosylated proteins, such as mucins, on sodium dodecyl sulfate-polyacrylamide gels. They combine commonly employed periodic acid-Schiff (PAS) and Alcian blue dyes with silver stain. Carbohydrate prestaining renders mucins more cationic and favors greater complexation with ionic silver. Comparisons of different mucin samples stained either with PAS-silver or alcian blue-silver indicate differential staining between the two techniques. Such differences may, in part, be due to an affinity of Alcian blue for sulfated glycoproteins. These two staining protocols when used in conjunction with silver staining alone are particularly valuable for assessing sample purity and for detecting contaminating proteins during mucin purification protocols.     

Introduction of sulfhydryl groups into proteins at carboxyl sites

A two-step procedure for introduction of sulfhydryl groups at protein carboxyl groups is described. The resultant proteins contain 2-aminoethanethiol residues bound by amide linkages to the protein carboxyl groups. First an amide bond is formed between a carboxyl group of the protein and one of the amino groups of cystamine. Then the disulfide bond is reduced with dithiothreitol, yielding the amide of 2-aminoethanethiol. This procedure was used to incorporate sulfhydryl groups into carbonic anhydrase and adrenocorticotropic hormone. The effect of carbodiimide concentration and pH of the coupling reaction on stoichiometry of sulfhydryl group incorporation was examined. The method was used to prepare bovine carbonic anhydrase containing up to nine sulfhydryl groups per molecule with no loss of enzymatic activity and biologically active adrenocorticotropic hormone containing one sulfhydryl group per molecule.     

晶状体生化的研究——亚硒酸钠诱发大鼠白内障晶状体中巯基、二硫键及维生素C的含量

我们测定了正常及亚硒酸钠诱发的白内障大鼠晶状体中非蛋白质巯基、蛋白质巯基、蛋白质结合巯基和维生素C的含量,发现随着白内障的进展非蛋白质巯基及蛋白质巯基均减少,蛋白质结合巯基在核混浊时增加,而在整个晶状体混浊时下降到与正常对照组相近,在白内障形成过程中二硫交联的蛋白质含量明显增加,而维生素C含量似乎无明显变化。     

Models of protein modification in Tris-glycine and neutral pH Bis-Tris gels during electrophoresis: effect of gel pH

The pH of conventional Tris-glycine SDS-PAGE gels during a run is determined to be 9.5, in contrast to Bis-Tris-Mes gels where the pH is 7.2. Concentrations of free acrylamide are determined to be less than 10mM in commercial gels of both types, and it is found that of the major components in these gels, only glycine and protein amine or sulfhydryl functions are likely to react with residual acrylamide during the time frame of typical separations. The addition of acrylamide to sulfhydryl groups on proteins is modeled using glutathione and cysteine at acrylamide concentrations found in the commercial gels. Rate constants are determined for these reactions as well as for reaction with glycine at the pH that proteins will encounter in these gel types. The half-life for glutathione sulfhydryl at 10mM acrylamide and pH 7.2 is more than 4h at room temperature. Rates are significantly lower in Bis-Tris-Mes gels than in Tris-glycine gels, reducing the risk of adventitious protein modification. Commercial Bis-Tris-Mes gels provide a sample reduction buffer at pH 8.5 versus the conventional pH 6.8 of Tris-glycine gels. It is shown that significantly less protein degradation occurs during sample preparation at the higher pH used with Bis-Tris gels.     

Redox control of changes in protein sulfhydryl levels during human sperm capacitation

Capacitation, the series of transformations that spermatozoa undergo to become fertile, is regulated by reactive oxygen species (ROS) and associated with an increase in the sulfhydryl content of Triton-soluble proteins. Our aims were to determine the fate of sulfhydryl groups in Triton-soluble proteins from capacitating human spermatozoa using two-dimensional (2D) gel electrophoresis, to evaluate the role of ROS in the changes observed, and to correlate the time course of the changes with that of the sperm generation of O(2)(*)(-). Triton-soluble proteins of control and capacitating human spermatozoa were labeled with 3-(N-maleimidylpropionyl) biocytin, separated by 2D gel electrophoresis, and probed with horseradish peroxidase-conjugated streptavidin. The sulfhydryl content of 10 out of the 14 proteins studied (pI: 4-7) was modified by the induction of capacitation, and the increases (by 200-400%, five proteins) and decreases (by 45-95%, five proteins) were prevented by superoxide dismutase and/or catalase. The alterations in protein sulfhydryl content occurred within 5-15 min but were reversed within 30-120 min. Three capacitation inducers triggered similar modifications. Therefore, human sperm capacitation is associated with rapid and reversible changes in protein sulfhydryl groups that appear to be redox regulated. The number of proteins affected, the types, and the kinetics of changes emphasize the complexity of sperm capacitation.     

Homology between egg white sulfhydryl oxidase and quiescin Q6 defines a new class of flavin-linked sulfhydryl oxidases.

The flavin-dependent sulfhydryl oxidase from chicken egg white catalyzes the oxidation of sulfhydryl groups to disulfides with the reduction of oxygen to hydrogen peroxide. Reduced proteins are the preferred thiol substrates of this secreted enzyme. The egg white oxidase shows an average 64% identity (from randomly distributed peptides comprising more than 30% of the protein sequence) to a human protein, Quiescin Q6, involved in growth regulation. Q6 is strongly expressed when fibroblasts enter reversible quiescence (Coppock, D. L., Cina-Poppe, D., Gilleran, S. (1998) Genomics 54, 460-468). A peptide antibody against Q6 cross-reacts with both the egg white enzyme and a flavin-linked sulfhydryl oxidase isolated from bovine semen. Sequence analyses show that the egg white oxidase joins human Q6, bone-derived growth factor, GEC-3 from guinea pig, and homologs found in a range of multicellular organisms as a member of a new protein family. These proteins are formed from the fusion of thioredoxin and ERV motifs. In contrast, the flavin-linked sulfhydryl oxidase from Aspergillus niger is related to the pyridine nucleotide-dependent disulfide oxidoreductases, and shows no detectable sequence similarity to this newly recognized protein family.