Molecular ageing in bovine plasma albumin I. Exposure of disulfide bonds in the N-B transition (author's transl)

An accessibility of the disulfide bonds of defatted SH-blocked bovine plasma albumin (BPA) to reduction by dithiothreitol was studied at 4 degree C. Upon reduction SH-blocked BPA acquired one SH per mole in the pH range from 4.0 to 6.0. Above pH 6.5 (the pH range of the N-B transition) an increasing number of the disulfide bonds became susceptible to reduction by dithiothreitol. Chemicals, such as KC1, fatty acid, sodium dodecylsulfate which suppress the structural fluctuation of BPA in the alkaline region decreased the accessibility of the disulfide bonds to reduction. The conversion of buried disulfide bonds into exposed ones is interpreted as being due to conformational changes in the N-B transition (pH 7.0 approximately 9.0).     

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.     

Kinetics and mechanism of the synthesis of a novel protein-based plastic using subcritical water

We investigated the intermolecular mechanism and kinetics of the synthesis of a novel biodegradable protein-based plastic from bovine serum albumin under subcritical water conditions using batch reactors. The reaction mechanism could be viewed as a chain reaction stabilized by the formation of intermolecular disulfide bonds. The kinetic analysis was based on non-steady-state kinetics using a theoretical model developed in one of our previous works. The activation energy and pre-exponential factor were found to be 7.2 kJ/mol and 0.9 s-1, respectively. These low values signify that the reaction is relatively temperature-insensitive with some diffusion limitation.     

Oxygen effect in the radiolysis of proteins. Part 2. Bovine serum albumin

Radiolysis of bovine serum albumin under aerobic and anaerobic conditions was studied by SDS-polyacrylamide gel electrophoresis. After Coomassie Blue or Fast Green staining quantitative evaluations give information about the degradation processes of the protein. Under nitrogen the main reaction is the aggregation caused by covalent cross-links, which includes only a small portion of intermolecular S-S bridges. Under air the radiolysis leads to peptide chain scission, which is not a random process, but yields specific protein fragments. A mechanism for this fragmentation reaction is suggested. The radiation-induced broadening of the serum albumin peak is interpreted as being a result of intramolecular disulfide exchange. In contrast to lactate dehydrogenase the degradation of serum albumin is enhanced by oxygen, probably because of its low tryptophan content.     

Role of disulfide bonds in the attachment and function of large, external, transformation-sensitive glycoprotein at the cell surface

Reduction of disulfide linkages by dithiothreitol removes LETS (large, external, transformation-sensitive) protein from the cell surface. This process is dependent upon the concentration of dithiothreitol and the time and temperature of reaction. At 0 degrees C the release of LETS protein by dithiothreitol is completely blocked, but this is apparently not due to a requirement for metabolic energy. At this temperature, reduction of LETS protein is incomplete. These results suggest that intact disulfide bonds are involved in the retention of this protein on the cell surface. Furthermore, reduction of purified LETS protein interferes with its ability to confer flattened morphology and increased adhesivity when added to transformed cells. It appears, therefore, that disulfide bonds are functionally important at the cell surface.     

Fluorescent labeling of cysteinyl residues to facilitate electrophoretic isolation of proteins suitable for amino-terminal sequence analysis

A protein labeling procedure which enables detection of subpicomole quantities of proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gels is described. Proteins are rendered fluorescent by reduction of disulfide bonds with dithiothreitol followed by alkylation with 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulfonic acid (5-I-AEDANS) or 5-iodoacetamido-fluorescein. Labeling is performed prior to electrophoresis, thus eliminating the need for staining with dyes and destaining after electrophoresis. As little as 375 fmol (25 ng) of prelabeled bovine serum albumin can be readily visualized after electrophoresis. Bands are still visible after electrophoretic transfer to nitrocellulose. Simultaneous labeling of proteins in complex mixtures is possible using this technique. This includes cysteine containing proteins of disrupted Newcastle disease virus. The magnitudes of the molecular weight increases which occur upon labeling reflect the cysteine contents of proteins. The mode of chemical modification for the prelabeling procedure was chosen because of its compatibility with analytical techniques, such as amino acid analysis, peptide mapping, or sequence analysis, which may be applied to the protein after electroelution from SDS-acrylamide gels. It replaces the need for reduction and carboxymethylation prior to these analytical procedures. Protein-sequence analysis of prelabeled bovine serum albumin, including samples electroeluted from SDS-acrylamide gels, has justified the choice of this method to facilitate isolation of proteins for sequence analysis. Equivalent sequence data were obtained with reduced bovine serum albumin S-alkylated with iodoacetic acid or 5-I-AEDANS.     

Tryptophanyl fluorescence of sodium dodecyl sulfate treated and 2-mercaptoethanol reduced proteins: a simple assay for tryptophan

Relative tryptophanyl fluorescence intensities of eleven different proteins (bovine liver glutamate dehydrogenase, bovine pancreas trypsin and α-chymotrypsinogen, egg white lysozyme, ovalbumin, bovine serum albumin and γ-globulin, bovine heart and rabbit muscle lactate dehydrogenases, rabbit muscle glyceraldehyde-3-phosphate dehydrogenase, and yeast alcohol dehydrogenase) were evaluated as a function of the physical state of the protein, i.e., native, denatured with intact disulfide bonds, and denatured with reduced disulfide bonds.     

Kinetics of formation of hydrophobic regions during refolding of bovine serum albumin

The rate of formation of hydrophobic regions during refolding of bovine serum albumin was studied using 1-anilinonaphthalene-8-sulfonate as the hydrophobic fluorescent probe. The refolding of serum albumin exhibited a sigmoidal behavior. The exhibition of a lag phase followed by a faster kinetic phase suggested that the refolding is a cooperative, sequential process. Refolding under reducing conditions almost completely inhibited the regeneration of hydrophobic binding regions, suggesting that the formation of disulfide bonds plays an important role in the refolding of serum albumin. The rate and the extent of refolding was apparently maximum at about 20 degrees; at 37 degrees the extent of refolding was very low compared to that at the other temperatures studied. Based on the results, the mechanism of albumin refolding is interpreted in terms of domain structures and interdomain interactions.     

Radiolytic reduction of protein and nonprotein disulfides in the presence of formate: a chain reaction

We have reported recently that the disulfide groups in bovine serum albumin can be reduced by a radiolytic chain reaction which occurs in deoxygenated solutions containing formate ions. This reaction, which involves the reduction of disulfide groups by hydrated electrons and carbon dioxide radical anions, has now been studied in greater detail and compared with an analogous reaction in small, disulfide containing molecules over a range of pH values and substrate concentrations. A two-step reaction is proposed to account for the reduction of disulfides in reactions which can have chain lengths of 20 or more. Thiols produced by the disulfide reduction are stable to the conditions of the reaction. For example, a biological assay showed that the integrity of glutathione was maintained even at radiation doses much larger than those required to achieve complete reduction of glutathione disulfide. It was found that the extent of disulfide reduction could easily be controlled by varying the radiation dose delivered to the solutions. Radiolytic reduction is a very useful way of reducing protein and low molecular weight disulfides without the use of excess quantities of reagents such as dithiothreitol. In many cases, the reaction solutions could be used directly for subsequent reactions and this may be of considerable value in modifying the structure of hormones, enzymes, membrane receptors, and other disulfide containing proteins. If ammonium formate is used, freeze drying is an effective way to remove the formate salt, should this be required.     

Effects of disulfide bonds on compactness of protein molecules revealed by volume, compressibility, and expansibility changes during reduction

To elucidate the effects of disulfide bonds on the compactness of protein molecules, the partial specific volume (v(o)) and coefficients of adiabatic compressibility (beta(s)(o)) and thermal expansibility (alpha) of five globular proteins (ovalbumin, beta-lactoglobulin, lysozyme, ribonuclease A, and bovine serum albumin) were measured in aqueous solutions with pH values of 7 and 2 at 25 degrees C when their disulfide bonds were totally reduced by carboxamidomethylation. Circular dichroism and fluorescence spectra show that the secondary and tertiary structures are partly disrupted by reduction, depending on the number of disulfide bonds in the proteins and the pH of the medium. The conformational changes are accompanied by decreases in v(o) and beta(s)(o) and by an increase in alpha, indicating that reduction decreases the internal cavity and increases surface hydration. The beta(s)(o) values of native or oxidized proteins decrease, and the effects of reduction on the volumetric parameters become more significant as the number of disulfide bonds increases and as they are formed over a larger distance in the primary structure. These results demonstrate that disulfide bonds play an important role, mainly via entropic forces, in the three-dimensional structure and compactness of protein molecules.     

Preparation and partial characterization of iron-sulfur, iron-selenium, and iron-tellurium complexes of bovine serum albumin.

An artificial Fe-S* protein was prepared by the reaction of bovine serum albumin with FeSO4 and Na2S or with a synthetic Fe-S*-1,4-butanenedithiol complex. These improved methods enabled us to characterize the derivatives from serum albumin. The Fe-S* albumin complex has about 20 iron ions and 14 labile sulfur atoms per molecule of the protein, whose absorption spectrum closely resembled that of 2Fe-2S* proteins. Its electron paramagnetic resonance spectrum exhibited signals different from those of ferredoxins. The addition of p-chloromercuriphenylsulfonate quenched the optical absorption in the visible region as well as the electron paramagnetic resonance signals. These properties of the albumin-iron complex are similar to those of iron-sulfur dithiothreitol and mercaptoethanol complexes, suggesting that the albumin-iron complex has one or more protein ligands besides sulfur lignads. Presumably, the oxygen atom of the tyrosine residue, or other hydroxyamino acids participates in the complex formation. In this context, the albumin polypeptide appears to be incapable of forming an iron-sulfur cluster identical to those of ferredoxins. Yet, from the albumin-iron derivative, the extrusion of the iron-sulfur core with benzenethiol provided products similar to those from ferredoxins. The iron-selenium and iron-tellurium derivatives of the bovine serum albumin were prepared and partially characterized by optical absorption and electron paramagnetic resonsnace spectroscopies. These results imply that both selenium and tellurium can be incorporated into the protein molecule as the respective labile components.     

Renaturation of reduced ribonuclease A with a microsphere-induced refolding system

We intended to refold reduced ribonuclease A (RNase A) using polymeric microspheres. Polymeric microspheres were allowed to react with dithiothreitol (DTT) to immobilize the disulfide and thiol moieties on their surface. The fully reduced RNase A was added to the dispersion of the modified microspheres. Protein refolding and renaturation were estimated by the change in the number of disulfide bonds of RNase A and the recovery of the enzymatic activity, respectively. Without microspheres, the activity gradually recovered with the increase in the number of disulfide bonds. However, the formation of disulfide bonds of reduced RNase A was accelerated by adding the modified microspheres, and the rate of renaturation was increased depending on the amount of charged DTT and the reaction time of the immobilization. These results indicate that modified microspheres significantly catalyze the recovery of active RNase A from the reduced form. The protein adsorption data demonstrated that the disulfide moieties of the modified microspheres react with the thiol moieties of the reduced RNase A to form a mixed disulfide. The thiol/disulfide exchange reaction can possibly proceed at the microsphere/protein interface, resulting in the formation of a correct three-dimensional structure.     

Cross-linking of cardiac gap junction connexons by thiol/disulfide exchanges

Summary SDS-polyacrylamide gel electrophoresis and immunoblotting were used to investigate inter- and intramolecular disulfide bonds to connexin 43 (the cardiac gap junctional protein) in isolated rat heart gap junctions and in whole heart fractions. In gap junctions isolated in the absence of alkylating agent, connexin 43 molecules are cross-linked by disulfide bonds. The use of iodoacetamide (100mm) for the first steps of isolation procedure prevents the formation of these artifactual linkages. Investigation of connexin 43 in whole heart fractions by means of antibodies confirms the results obtained with isolated gap junctions; that is, connexin 43 molecules are not interconnected with disulfide bridges. In whole heart fractions treated with alkylating agents, a 38 kD protein, immunologically related to connexin 43, and containing intramolecular disulfide bonds is detected. It is hypothesized that this protein might be a folded form of connexin 43, a precursory form of the molecules embedded in the gap junctions.The abbreviations used are BSA bovine serum albumin - EDTA ethylene diamine tetra-acetic acid - IAA iodoacetamide - NEM N-ethylmaleimide - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsfonyl fluoride - SDS sodium dodecyl sulfate - Tris trishydroxymethyl-aminomethane     

Reductive unfolding of serum albumins uncovered by Raman spectroscopy

The reductive unfolding of bovine serum albumin (BSA) and human serum albumin (HSA) induced by dithiothreitol (DTT) is investigated using Raman spectroscopy. The resolution of the S-S Raman band into both protein and oxidized DTT contributions provides a reliable basis for directly monitoring the S-S bridge exchange reaction. The related changes in the protein secondary structure are identified by analyzing the protein amide I Raman band. For the reduction of one S-S bridge of BSA, a mean Gibbs free energy of -7 kJ mol(-1) is derived by studying the reaction equilibrium. The corresponding value for the HSA S-S bridge reduction is -2 kJ mol(-1). The reaction kinetics observed via the S-S or amide I Raman bands are identical giving a reaction rate constant of (1.02 +/- 0.11) M(-1) s(-1) for BSA. The contribution of the conformational Gibbs free energy to the overall Gibbs free energy of reaction is further estimated by combining experimental data with ab initio calculations.     

Selenium detoxification of heavy metals: a possible mechanism for the blood plasma

Among the activities of the essential trace element selenium is the ability to reduce the toxicity of heavy metal ions like cadmium(II) and mercury(II). Detoxification often depends on the metabolic reduction of selenium to hydrogen selenide; the mechanism generally advanced to explain such selenium/metal interactions is that selenide combines with heavy metal ions to give a metal selenide which is metabolically inert. However, this hypothesis does not consider circumstances where selenide is quickly removed by other reactions. Given the ease with which selenide is oxidized, such conditions are likely to occur in the blood plasma, an environmental rich in oxidizing agents and a site for many selenium/metal interactions. Using polarography to monitor both selenide and cadmium, we have found that selenide reacts rapidly in vitro with the disulfide bonds present in bovine serum albumin in preference to forming cadmium selenide. We hypothesize that a similar reaction occurs in the blood plasma with the disulfide bonds of plasma proteins to generate thiol groups on the protein involved, and that these newly formed thiols are responsible for the observed reduction of metal toxicity through the ability to chelate heavy metal ions.     

On the molecular mechanism of maize phosphoenolpyruvate carboxylase activation by thiol compounds

Incubation of purified phosphoenolpyruvate carboxylase from Zea mays L. leaves with dithiothreitol resulted in an almost 2-fold increase in the enzymic activity. The activated enzyme showed the same affinity for its substrates and the same sensitivity with respect to malate and oxalacetate inhibition. The activation induced by dithiothreitol was reversed by diamide, an oxidant of vicinal dithiols, suggesting that the redox state of disulfide bonds of the enzyme may be important in the expression of the maximal catalytic activity.

Titration of thiol groups before and after activation of maize phosphoenolpyruvate carboxylase by dithiothreitol shows an increase of the accessible groups from 8 to 12 suggesting that the reduction of two disulfide bonds accompanied the activation. The thiols exposed by the treatment with dithiothreitol were available to reagents in nondenatured enzyme and two of them were reoxidized to a disulfide bond by diamide. It is concluded that the mechanism of phosphoenolpyruvate carboxylase activation by dithiothreitol involves the net reduction of two disulfide bonds in the enzyme.

     

Selective reduction of the disulfide bonds of ovine placental lactogen

Reduction and carbamidomethylation of two of the three disulfide bridges of ovine placental lactogen was accomplished by the use of 20-fold molar excess of dithiothreitol over protein disulfide content. The derivative retained its binding capacity to somatogenic as well as lactogenic rat liver receptors, although the latter was somewhat diminished. The two disulfide bonds exposed to the reducing agent are those located near the carboxy- and amino-terminus, while the larger loop remained intact after reduction. This behaviour is similar to that of bovine growth hormone, where the larger loop was also more resistant to reduction.     

Polymerization of Several Proteins by Ca2+-Independent Transglutaminase Derived from Microorganisms

α_s1-Casein and soybean globulins were polymerized and gelatinized by Ca²⁺-independent transglutaminase that was isolated from the culture filtrate of a microorganism thought to belong to Streptoverticillium sp. of actinomycetes. This enzyme polymerized such albumins as bovine serum albumin, human serum albumin and conalbumin in the presence of dithiothreitol. Rabbit myosin was polymerized by the present emzyme but actin was not. An RP-HPLC analysis after enzymic digestion of the polymerized a_sl -casein showed existence of the £-(y-Glu)Lys bond. Thus, it was confirmed that the polymerization was formed by a catalytic reaction of the transglutaminase.     

Binding of secretory component to dimers of immunoglobulin A in vitro. Mechanism of the covalent bond formation.

A complex between secretory component and an immunoglobulin A (IgA) myeloma dimer has been studied in vitro as a model to elucidate the mechanism of the formation of disulfide bonds during assembly in vivo of secretory immunoglobin A. A small amount of free thiol groups, totally about 0.4 groups per mole of protein, were shown to be present on both the heavy and light chains of the IgA dimer, but not on its J-chain, while no such groups could be demonstrated on free secretory component. The SH-groups on IgA most likely exist as a result of incomplete oxidation of some intra-or interchain disulfide bonds of the molecule, analogous to what has been suggested for IgG. Several types of evidence indicated that the disulfide bonds between secretory component and IgA are formed after the noncovalent association of the two proteins by a sulfhydryl group-disulfide bond exchange reaction, in which the small amount of free sulfhydryl groups on the IgA dimer initiate the reaction by reducing a reactive disulfide bond on secretory component. This exchange reaction, which thus proceeds by the mechanism of so-called disulfide interchange reactions, requires certain conformational features of one or both of the proteins and leads to the formation of presumably two new interchain disulfide bonds between secretory component and IgA. The reaction does not progress to completion, however, but ends in an equilibrium so that a small proportion of the secretory component molecules always are unattached by disulfide bonds.     

The role of disulfide bond reduction during mammalian sperm nuclear decondensation in vivo

These studies were designed to test the hypothesis that sperm nuclear decondensation and male pronuclear formation during hamster fertilization depend upon the ability of the fertilized oocyte to reduce sperm nuclear disulfide bonds. In a first series of experiments, treatment of mature oocytes with the sulfhydryl blocking agent iodoacetamide or the glutathione oxidant diamide caused a dose-dependent inhibition of decondensation in microinjected sperm nuclei. Inhibition of decondensation was not observed, however, when sperm nuclei were treated in vitro with dithiothreitol (DTT) to reduce disulfide bonds prior to their microinjection. In a second series of experiments, germinal vesicle (GV)-intact oocytes and pronuclear eggs, in which mature, disulfide-rich sperm nuclei do not decondense, were found to support the decondensation of disulfide-poor DTT-treated sperm nuclei or testicular spermatid nuclei. The decondensed sperm nuclei were not, however, transformed into male pronuclei. The results of these studies suggest: (1) that sperm nuclear decondensation in the hamster requires disulfide bond reduction, (2) that GV-intact oocytes and pronuclear eggs lack sufficient reducing power to effect sperm nuclear decondensation, and (3) that disulfide bond reduction is required but not sufficient for pronuclear formation.