Sensitive quantitative analysis of disulfide bonds in polypeptides and proteins

A sensitive quantitative method has been developed to determine the number of disulfide bonds in peptides and proteins. The disulfide bonds of several peptides and proteins were cleaved quantitatively by excess sodium sulfite at pH 9.5 and room temperature. Guanidine thiocyanate (2 M) was added to the protein solutions in order to denature them and thereby make the disulfide bonds accessible. The reaction with sulfite leads to a thiosulfonate and a free sulfhydryl group; the concentration of the latter was determined by reaction with disodium 2-nitro-5-thiosulfobenzoate (NTSB) in the presence of excess sodium sulfite. The synthesis, purification, and characterization of NTSB are described. The assay is rapid, requiring 3-5 min for oligopeptides and 20 min for proteins, and is as sensitive and quantitative as the sulfhydryl group assay employing 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent). It can be used for the analysis of as little as 10(8) mol of disulfide bonds, with an error of +/- 3%.     

The N-hydroxysuccinimide ester of Boc-[S-(3-nitro-2-pyridinesulfenyl)]-cysteine: a heterobifunctional cross-linking agent

Synthetic cysteine-containing peptides were unidirectionally conjugated to albumin via disulfide bonds using the S-(3-nitro-2-pyridinesulfenyl) derivative of cysteine. This method employs the N-hydroxysuccinimide ester of Boc-[S-(3-nitro-2-pyridinesulfenyl)]-cysteine, a protected amino acid derivative used in peptide synthesis, as a heterobifunctional cross-linking agent. The disulfide bonds in the conjugates are formed by the reaction of free thiols with S-(3-nitro-2-pyridinesulfenyl) groups. Bovine albumin was conjugated in this manner to several synthetic peptides derived from human fibrin. Amino acid analysis of these conjugates demonstrated incorporations of from 6 to 11 peptide molecules per molecule of protein.     

Interchain and intrachain crosslinking of actin thiols by a bifunctional thiol reagent.

From 1,9-nonylenedithiol and Ellman's Reagent the bifunctional asymmetric disulfide n-nonylene-1,9-bis-[5-dithio-(2-nitrobenzoic acid)] (NBDN) was prepared. By monovalent reaction with cysteine-374 the crosslinker could be introduced into monomeric actin, with release of one equivalent of yellow 2-nitro-5-thiobenzoate (NTB). From the monovalent actin derivative we prepared a crosslinked actin dimer (Cys-374-Cys-374') as well as a monomer with a crosslink between Cys-374 and Cys-10. Neither crosslinked actin species was able to polymerize the crosslinked monomer even in the presence of phalloidin. The crosslinked monomer polymerized on the addition of dithiothreitol, thus providing the first unpolymerizable actin species whose polymerizability can be restored under mild conditions. We suggest the use of NBDN as a thiol-specific crosslinker that reacts under spectrophotometric control and can be removed by the addition of thiols.     

一种方便快速检测蒜素的方法

基于硝基硫代苯甲酸 ( 2 nitro 5 thiobenzoate,NTB)与硫代亚磺酸脂 (Thiosulfinates)反应 ,对蒜素 (allicin)进行检测。一分子allicin与两分子NTB进行反应 ,形成NTB二硫键衍生物 ,通过吸光值 (A₄₁₂ )的测定来计算蒜素 (allicin)含量。     

Measurement of biological disulfides by postcolumn sulfitolysis following separation by HPLC

We developed a method to measure disulfides which is applicable to biological fluids. It consisted of two parts. First, certain thiols and disulfides were separated by HPLC. Second, the eluted materials were submitted to postcolumn reaction with 2-nitro-5-thiosulfobenzoate in the presence of sulfite. The resultant yellow product, 2-nitro-5-thiobenzoate, was measured by its absorbance at 412 nm. We determined the elution characteristics of the thiols and disulfides derived from cysteine, glutathione, alpha-mercaptopropionylglycine (Thiola), and cysteamine. Penicillamine and its disulfide did not react. Cystine in the urine of 22 cystinuric patients, measured by this method, was compared with results obtained by automatic amino acid analysis.     

A method for the controlled cleavage of disulfide bonds in proteins in the absence of denaturants

A simple method was developed for the controlled cleavage of protein disulfide bonds and the simultaneous blockage of the free sulfhydryl groups in the absence of a denaturant. The disulfide bonds of bovine serum albumin were cleaved unsymmetrically at pH 7.0 using 0.1 M sulfite in 0.1 M phosphate buffer and the free sulfhydryl groups formed were sulfonated in an oxidation-reduction cycle using molecular oxygen and 400 microM cupric sulfate as a catalyst. The reaction was affected by cupric ion concentration, sulfite concentration, reaction pH and temperature. The standardized method was successfully used to cleave the disulfide bonds of other proteins pepsin, trypsin, and chymotrypsin. The method is reliable and can be used for achieving progressive cleavage of disulfide bonds in proteins without employing a denaturant.     

A spectrophotometric assay for allicin,alliin, and alliinase (alliin lyase) with a chromogenic thiol: reaction of 4-mercaptopyridine with thiosulfinates

Allicin (diallylthiosulfinate) is the best known active compound of garlic. It is generated upon the interaction of the nonprotein amino acid alliin with the enzyme alliinase (alliin lyase, EC 4.4.1.4). Previously, we described a simple spectrophotometric assay for the determination of allicin and alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin. This reagent is not commercially available and must be synthesized. In this paper we describe the quantitative analysis of alliin and allicin, as well as of alliinase activity with 4-mercaptopyridine (4-MP), a commercially available chromogenic thiol. The assay is based on the reaction of 4-MP (lambda(max)=324nm) with the activated disulfide bond of thiosulfinates -S(O)-S-, forming the mixed disulfide, 4-allylmercaptothiopyridine, which has no absorbance at this region. The structure of 4-allylmercaptothiopyridine was confirmed by mass spectrometry. The method was used for the determination of alliin and allicin concentrations in their pure form as well as of alliin and total thiosulfinates concentrations in crude garlic preparations and garlic-derived products, at micromolar concentrations. The 4-MP assay is an easy, sensitive, fast, noncostly, and highly efficient throughput assay of allicin, alliin, and alliinase in garlic preparations.     

Enzymic and immunochemical properties of lysozyme. IX. Conformation and immunochemistry of derivatives succinylated at certain lysine residues.

Succinylation of lysozyme in the presence of 7 molar excess of [1,4-14C2]-succinic anhydride gave a reaction product which showed at least six components by disc electrophoresis. Chromatography on CM-cellulose enabled the isolation of six homogeneous derivatives. The derivatives were succinylated at the following locations: derivative I, lysines-1 (alpha- and epsilon-NH2), -13, -97 and -116 and the OH group at position 43 (or 36 or 40); derivative II, lysines-1 (alpha- and epsilon-NH2), -13, -96, -116; derivative III, lysines-1 (alpha-and epsilon-NH2), -13, -97, -116; derivative IV, lysines-1 (alpha-NH2), -33, -96 and -116; derivative V, lysines-1 (alpha-NH2), -33 and -96; derivative VI, lysines-33 and -116. Conformational changes were detectable in derivative I by ORD and CD measurements and by accessibility of the disulfide bonds to reduction. On the other hand, the other five succinyl derivatives showed no conformational changes by ORD and CD measurements. However, their disulfide bonds were slightly more accessible to reduction than lysozyme, with the increase being somewhat higher in derivatives I, II and III. Enzymic activity measurements showed that only derivative VI possessed some (10%) enzymic activity. Immunochemical studies with antisera to lysozyme showed that the reactivity of each of the derivatives was lower than the homologous reaction. Correlation of the extent of decrease in immunochemical reaction with the locations of modification and with the results of conformational analysis, led to the conclusion that lysines 33, 96 and 116 are part of antigenic reactive regions in lysozyme. The modification results are also discussed in relation to the three-dimensional structure of lysozyme in solution.     

Reversible blocking of half-cystine residues of proteins and an irreversible specific deamidation of asparagine-67 of S-sulforibonuclease under mild conditions

For use in protein-folding studies, a rapid procedure for the preparation of octa-S-sulforibonuclease A (SO3-RNase A) with 2-nitro-5-(sulfothio)benzoate is described. The modification is specific for thiols and disulfide bonds. The modified protein was characterized and found to be enzymatically inactive and predominantly conformationally disordered. In the absence of thiols, the modified sulfhydryl groups were found to be stable over the pH range of 2-9. However, when the modified protein is incubated at neutral to slightly alkaline conditions for prolonged periods of time or at elevated temperatures, it undergoes a further (irreversible) modification that decreases its net charge at pH 8.0. Evidence is presented that demonstrates that this additional modification is due to the specific deamidation of asparagine-67. When incubated with an excess of reduced and oxidized glutathiones for 24 h at pH 8.2 and 25 degrees C, the reversible sulfo blocking group was removed, and essentially quantitative (94%) native enzymatic activity was regenerated from both SO3-RNase A and its deamidated derivative (SO3-RNase B). Although the two fully active refolded species differ in their elution behavior on ion-exchange chromatography, they are indistinguishable by many other methods. The significance of this finding for studies of the folding of RNase A is discussed.     

A sensitive method for localization of disulfide containing peptides in column effluents

A sensitive method is reported to localize cystine-containing peptides in column effluents. The method depends on reduction of disulfide bonds by sodium borohydride in guanidine followed by destruction of excess of sodium borohydride by acid and acetone. The liberated sulfhydryl groups are quantitated by 5,5′-dithiobis(2-nitrobenzoic acid).     

Species variability in plasma S-sulfonate levels during and following sulfite administration.

It has been shown that S-sulfonate compounds (R-S-SO-3) are produced by the action of sulfite on reactive disulfide bonds [4,5]. Plasma S-sulfonate production was determined as a function of sulfite ingestion and intraperitoneal injection in rats, mice and rhesus monkeys. The tendency of these species and of the rabbit [8] to produce S-sulfonates in plasma was related to the availability of sulfite and of reactive disulfide bonds and to the stability of plasma protein S-sulfonates. The rhesus monkey and the rabbit accumulated plasma S-sulfonates much more readily than did the rat, while the mouse produced little, if any, under the same test conditions. Plasma protein S-sulfonate fractions in the rat and rhesus monkey were characterized by half-lives of approximately 4 and 8 days respectively. The sensitivity and precision of the analytical method for plasma protein S-sulfonate were improved by incorporation of 35S into the outer sulfur atom of the S-sulfonate moiety (R-S-35SO-3).     

Analyses of intramolecular disulfide bonds in proteins by polyacrylamide gel electrophoresis following two-step alkylation

A method that makes use of polyacrylamide gel electrophoresis was developed for the analysis of intramolecular disulfide bonds in proteins. Proteins with different numbers of cleaved disulfide bonds are alkylated with iodoacetic acid or iodoacetamide as the first step. The disulfide bonds remaining were reduced by excess dithiothreitol, and the newly generated free sulfhydryl groups were alkylated with the reagent not yet used (iodoacetamide, iodoacetic acid, or vinyl-pyridine) as the second step. This treatment made it possible for lysozyme (Mr, 14,000; 4 disulfides), the N-terminal half-molecule of conalbumin (Mr, 36,000; 6 disulfides), the C-terminal half-molecule of conalbumin (Mr, 40,000; 9 disulfides), and whole conalbumin (Mr, 78,000; 15 disulfides) to be separated by acid-urea polyacrylamide gel electrophoresis into distinct bands depending on the number of disulfide bonds cleaved. The method allowed us to determine the total number of disulfide bonds in native proteins and to assess the cleaved levels of disulfide bonds in partially reduced proteins. Two-step alkylation used in combination with radioautography was especially useful for the analysis of disulfide bonds in proteins synthesized in complex biological systems.     

Site-specific fragmentation and modification of albumin by sulfite in the presence of metal ions or peroxidase/H2O2: role of sulfate radical.

Exposure of albumin to sulfite in the presence of Co(II) or peroxidase/H2O2 caused site-specific fragmentation, which was not due to cleavage of methionyl nor tryptophanyl peptide bonds. The reaction of GlyPro with sulfite in the presence of Co(II) or peroxidase/H2O2 led to Gly liberation, suggesting the oxidative cleavage of protein at Pro residues. Sulfite plus Co(II) induced bityrosine production, Trp loss and a new Trp-derived fluorescence. ESR-spin trapping method provided evidence for the formation of sulfate radical (SO4.-) during Co(II)-catalyzed autoxidation of sulfite. The order of reactivity with SO4.- seemed to be Trp greater than GlyPro greater than GlyGly approximately Gly approximately Pro. The results suggest that SO4.- plays an important role in fragmentation and modification of albumin.     

2-Nitro-5-Thiosulfobenzoic Acid as a Novel Inhibitor Specific for Deoxyribonuclease I

Bovine pancreatic deoxyribonuclease I (bpDNase I) contains four cysteine residues forming two disulfide bonds. Though there are no free sulfhydryl groups, incubation of bpDNase I with 2-nitro-5-thiosulfobenzoic acid (NTSB) in the presence of Ca(2+) or Mg(2+) at pH 7.5 results in inactivation of the enzyme. Amino acid analysis shows that NTSB-treated bpDNase I still contains all 4 half-cystine residues. The only amino acid residues having reduced values are threonine and serine, indicating that these may be the reaction sites for NTSB. Plasmid scission assay and circular dichroism analysis reveal the structural integrity of the inactivated enzyme. Treatment of bpDNase I with NTSB does not result in fragmentation, as demonstrated by SDS-PAGE analysis. NTSB binds bpDNase I through covalent modification, since dialysis and gel filtration can not reverse the inactivation reaction. However, after dilution into an acid buffer of pH 4.7, the inactivated enzyme regains about 40% of its initial activity, suggesting a reversible inactivation by acid treatment. NTSB does not inactivate DNase II, ribonuclease, chymotrypsin and lysozyme, while it effectively inactivates rat parotid DNase I. These results strongly suggest that NTSB can be considered as a novel inhibitor specific for DNase I.     

Template-directed polymerization of oligoadenylates using cyanogen bromide

Cyanogen bromide (BrCN) condensed oligoadenylates [oligo(A)] on a poly(uridylic acid) [poly(U)] template in an aqueous solution. Imidazole and divalent metal ions such as Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Mg2+, and Fe2+ were required for the condensation. Chain length of oligo(A) and reaction temperature affected the coupling yield. Hexaadenylate [(pA)6] was converted to (pA)12, (pA)18, (pA)24, (pA)30, (pA)36, (pA)42, and (pA)48 in a 68% overall yield for 20 h at 25 degrees C. The coupling yield increased with increase in the poly(U) concentration. Five- to sevenfold molar excess of uridylyl residues of poly(U) to adenylyl residues of oligo(A) gave the best yield (68%). Metal ions affected the formation of linkage isomers of the phosphate bonds: The 2',5'- and 3',5'-phosphodiester bonds were predominant in the presence of Co2+, Zn2+, and Ni2+ and the 5',5'-pyrophosphate bond was predominant in the presence of Mn2+. In particular, Ni2+ gave the highest ratio of the 3',5'-phosphodiester bond (30%). N-Cyanoimidazole (1), N,N'-iminodiimidazole (2), and N-carboxamidoimidazole (3) were formed in a reaction of imidazole with BrCN in an aqueous solution. 1 and 2 had much the same condensing activity for the polymerization of adenylates as BrCN. A reaction pathway was proposed in which 1 and 2 are not only intermediates for the production of 3 but also the true condensing agent in the coupling reaction of oligo(A). Phosphorimidazolide derivative was detected in a reaction of 5'-AMP with either 1 or 2. The condensation would proceed by way of N-cyanoimidazole-phosphate adduct, the phosphorimidazolide derivative, or both.     

Identification of plasma proteins containing sulfite-reactive disulfide bonds

Plasma protein S-sulfonate compounds (RS-SO-3) have previously been shown to form, presumably by sulfitolysis of disulfide bonds, as a result of exposure to sulfite. In the investigations reported here, we identify two proteins in rabbit plasma, namely albumin and plasma fibronectin, which contain reactive sites for S-sulfonate formation. Separation and identification of these proteins following in vitro and in vivo exposure to sulfite was accomplished primarily by column chromatographic and electrophoretic techniques. In addition, the structure of presumed S-sulfonate groups was confirmed by the identification of cysteinyl-S-sulfonate residues in protein hydrolysates generated by enzymatic digestion. The molar ratio of RS-SO-3 in both albumin and plasma fibronectin was less than one. Data from our experiments suggest that the mixed disulfide site of non- mercaptalbumin is the reactive site for S-sulfonate formation. The site(s) of formation within the plasma fibronectin molecule was not investigated. The possible physiological significance of disulfide sulfitolysis of albumin and plasma fibronectin is discussed.     

Chip-based gel electrophoresis method for the quantification of half-antibody species in IgG4 and their by- and degradation products

The inter-heavy-chain disulfide bonds of the IgG4 subclass can be described as being at equilibrium with the intra-chain disulfide bonds. This means that a fraction of IgG4 has noncovalently linked heavy chains (half-antibody). The percentage of half-antibodies produced depends upon the expression system used. Nondenaturing assays fail to separate the half-antibodies from the native form because two half-molecules are held together by noncovalent forces. The pharmaceutical industry needs a reliable denaturing assay for checking batch-to-batch consistency. Until now sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been the standard method used to detect the presence of half-antibodies. However, this technique is laborious and cannot be automated. Furthermore, cumbersome densitometric measurements are necessary for quantification. To overcome these disadvantages a chip-based gel electrophoresis method was investigated. In the nonreduced format the separation profile is compared with that from SDS-PAGE. The limit of quantification as a percentage of the amount applied, repeatability, reproducibility, and linearity are compared with those of SDS-PAGE. The amounts of half-antibody and of by- and degradation products were determined for several batches by using area percentage and by external calibration with IgG4 as a reference standard. Both methods allow avoidance of error introduction for the quantification as is the case by application of myosin as reference concentration. Both sets of results are compared with each other and with the results from SDS-PAGE. In the reduced format it is noted that the reduction of the inter-heavy-chain disulfide bridges proceeds faster than the reduction of the heavy-light-chain bonds. Therefore optimized conditions are necessary to obtain a complete reduction.     

Na_2SO_3对不同状态CF_1－ATPase活力的促进作用

Ｎａ２ＳＯ３对ＣＦ１－ＡＴＰａｓｅ活力的促进作用与酶所处状态有关。ＣＦ０降低ＣＦ１对Ｎａ２ＳＯ３的亲和力和Ｎａ２ＳＯ３促进的最大反应速率。在Ｎａ２ＳＯ３作用下,膜上ＣＦ１－ＡＴＰａｓｅ的活化能高于游离的。膜上和游离ＣＦ１－ＡＴＰａｓｅ的γ亚基上二硫键的还原可以提高Ｎａ２ＳＯ３对酶活力的促进作用。Ｎａ２ＳＯ３对甲醇活化的ＣＦ１－ＡＴＰａｓｅ活力的促进作用只有在甲醇活化的亚适浓度下才能充分表现出来。Ｎａ２ＳＯ３对Ｍｇ２＋抑制的解除作用因ＣＦ１－ＡＴＰａｓｅ处于不同活化状态而不同。     

Preparation of Boc-[S-(3-nitro-2-pyridinesulfenyl)]-cysteine and its use for unsymmetrical disulfide bond formation

The 3-nitro-2-pyridinesulfenyl (Npys) derivative of cysteine was prepared and used to facilitate the formation of an unsymmetrical disulfide bond. Since this derivative is stable in trifluoroacetic acid:CH2 Cl2 (1:1) and anhydrous hydrogen fluoride, Boc-Cys(Npys) could be used directly in solid phase synthesis of the 14-peptide acetyl-Cys(Npys)-Gly-Glu-Gln-Gln-His-His-Pro-Gly-Gly-Gly-Ala-Lys-G ln-Ala-amide. Reaction of this peptide with the free thiol of another peptide, acetyl-Gly-Glu-Gln-His-His-Pro-Gly-Gly-Gly-Ala-Lys-Gln-Cys-amide, gave a single product containing an unsymmetrical disulfide bond. The amino acid composition of this product and HPLC analysis of its dithiothreitol reduction products were consistent with the desired heterodimer. As evidenced by HPLC, the mixed disulfide forms rapidly at alkaline pH and usefully over a wide pH range in aqueous buffers.     

The renaturation of reduced polyalanyl-chymotrypsinogen and chymotrypsinogen.

Chymotrypsinogen has been successfully renatured in solution, after reduction of its 5 disulfide bonds in 6 M guanidine-HCl. This has been made possible by the study of the renaturation of a model derivative, polyalanyl-chymotrypsinogen. The reduced derivative is shown to refold and reoxodize spontaneously, with a 30-40% yield, into molecules which are monomeric and fully susceptible to activation by trypsin. Chymotrypsinogen can also be renatured but only in the presence of reagents allowing disulfide interchange and of moderate concentrations of guanidine-HCl or urea. These results illustrate how the kinetic trapping of incorrectly folded molecules by wrong S-S bonds and aggregation can be overcome, thus allowing the correct refolding of the protein.