Thiol-disulphide Exchange Reactions Research Articles

Vitamin K Epoxide Reductase Complex-Protein Disulphide Isomerase Assemblies in the Thiol-Disulphide Exchange Reactions: Portrayal of Precursor-to-Successor Complexes.

The human Vitamin K Epoxide Reductase Complex (hVKORC1), a key enzyme that converts vitamin K into the form necessary for blood clotting, requires for its activation the reducing equivalents supplied by its redox partner through thiol-disulphide exchange reactions. The functionally related molecular complexes assembled during this process have never been described, except for a proposed de novo model of a 'precursor' complex of hVKORC1 associated with protein disulphide isomerase (PDI). Using numerical approaches (in silico modelling and molecular dynamics simulation), we generated alternative 3D models for each molecular complex bonded either covalently or non-covalently. These models differ in the orientation of the PDI relative to hVKORC1 and in the cysteine residue involved in forming protein-protein disulphide bonds. Based on a comparative analysis of these models' shape, folding, and conformational dynamics, the most probable putative complexes, mimicking the 'precursor', 'intermediate', and 'successor' states, were suggested. In addition, we propose using these complexes to develop the 'allo-network drugs' necessary for treating blood diseases.

Identification of the Primary Factors Determining theSpecificity of Human VKORC1 Recognition by Thioredoxin-Fold Proteins.

Redox (reduction–oxidation) reactions control many important biological processes in all organisms, both prokaryotes and eukaryotes. This reaction is usually accomplished by canonical disulphide-based pathways involving a donor enzyme that reduces the oxidised cysteine residues of a target protein, resulting in the cleavage of its disulphide bonds. Focusing on human vitamin K epoxide reductase (hVKORC1) as a target and on four redoxins (protein disulphide isomerase (PDI), endoplasmic reticulum oxidoreductase (ERp18), thioredoxin-related transmembrane protein 1 (Tmx1) and thioredoxin-related transmembrane protein 4 (Tmx4)) as the most probable reducers of VKORC1, a comparative in-silico analysis that concentrates on the similarity and divergence of redoxins in their sequence, secondary and tertiary structure, dynamics, intraprotein interactions and composition of the surface exposed to the target is provided. Similarly, hVKORC1 is analysed in its native state, where two pairs of cysteine residues are covalently linked, forming two disulphide bridges, as a target for Trx-fold proteins. Such analysis is used to derive the putative recognition/binding sites on each isolated protein, and PDI is suggested as the most probable hVKORC1 partner. By probing the alternative orientation of PDI with respect to hVKORC1, the functionally related noncovalent complex formed by hVKORC1 and PDI was found, which is proposed to be a first precursor to probe thiol–disulphide exchange reactions between PDI and hVKORC1.

The effect of thiol reagents on the denaturation of the whey protein in milk and whey protein concentrate solutions

Adding cysteine (CYS-HCl) to skim milk, whey protein isolate (WPI), or milk/WPI solutions promoted the irreversible denaturation of α-lactalbumin at temperatures between 61 and 70 °C, whereas little denaturation of β-lactoglobulin occurred at these temperatures. Above 70 °C, CYS-HCl promoted the irreversible denaturation of both α-lactalbumin and β-lactoglobulin. A greater effect was observed at higher CYS-HCl concentrations at any given pH, or at higher pH at any given CYS-HCl concentration. Similar effects were observed with glutathione and 2-mercaptoethanol. It is proposed that at temperatures between ∼61 and 70 °C, CYS-HCl initiates thiol-disulphide exchange reactions with the disulphide bonds on unfolded α-lactalbumin, but not with those on the native β-lactoglobulin, thus α-lactalbumin irreversibly aggregates. At higher temperatures, CYS-HCl and the free thiol on unfolded β-lactoglobulin initiates thiol-disulphide exchange reactions with the disulphide bonds on both α-lactalbumin and β-lactoglobulin, rapidly aggregating both proteins via disulphide bonds and non-covalent interactions.

Effect of free cysteine on the denaturation and aggregation of holo α-lactalbumin

α-Lactalbumin (α-LA) is a key commercial whey protein for nutritional purposes. The holo protein (calcium saturated) is considered the most heat stable whey protein, capable of refolding from unfolded states under many conditions. This is due to the absence of free thiols (cysteine residues) that are typically involved in thermal aggregation and thiol–disulphide exchange reactions of other whey proteins. Heating (0–120 min at 90 °C, pH 7.0) holo α-LA generates free thiols through thermal cleavage of disulphide bonds, resulting in aggregates comprising unfolded α-LA species. The addition of free cysteine promotes the formation of soluble aggregates, effectively decreasing the holding time required to reach a particular aggregate size in a dose-dependent manner (0.35–1.4 mm cysteine). Excess cysteine (≥14 mm) causes a destabilisation of α-LA, shown by decreased denaturation temperature and gel formation. These data indicate that low doses of cysteine can be used to control α-LA aggregation.

Effects of Diaceto-Dipropyl-Disulphide on Plasma Sialic Acid and Renal Tissue Thiol Levels in Alloxan Diabetic Rats.

Plasma sialic acid levels are elevated in Diabetes Mellitus (DM) patients with proteinuria. Renal damage is mainly caused by free radicals that are excessively generated in DM. Thiols play an important role in the cellular antioxidative defence mechanisms mainly through thiol-disulphide exchange reaction. Diallyl disulphide, a garlic oil principle component, is known for its anti-diabetic properties. Its structural analogue, Diaceto-Dipropyl Disulphide (DADPDS), is a less toxic and more palatable disulphide and possesses similar anti-diabetic actions. This study was undertaken to assess the usefulness of DADPDS in prevention of de-sialation of Glomerular Basement Membrane (GBM) in alloxan diabetic rats and to assess effect of DADPDS on renal tissue thiol levels. Rats were divided into Normal, Diabetic and DADPDS treated diabetic groups. Diabetes was induced by intraperitoneal injection (IP) of alloxan. DADPDS was fed by gastric intubation. Plasma Sialic acid was determined by Ehrlich's method and renal tissue thiol levels by Nitroprusside reaction method. This study showed a significant decrease (p<0.001) in plasma sialic acid, plasma glucose and renal tissue TBARS levels along with significant increase (p<0.001) in renal tissue thiol levels in DADPDS treated alloxan diabetic rats when compared to diabetic control rats. Hence it may be concluded that DADPDS helps in preventing de-sialation of GBM in alloxan diabetic rats and improves renal tissue antioxidant defence mechanisms, may be through thiol-disulphide exchange reaction and thereby exhibits a possible clinical use in prevention of renal complications like diabetic nephropathy.

Studying the Conformational Equilibrium of the N-Terminal Domain of Dsbd by NMR and Computer Simulation

The N-terminal domain of the membrane protein DsbD (nDsbD) acts as a ‘redox hub’ in the periplasm of Gram-negative bacteria. Reductant, from cytoplasmic thioredoxin, is channelled via a series of thiol-disulphide exchange reactions involving the transmembrane domain of DsbD, the C-terminal domain (cDsbD) and finally nDsbD. nDsbD supplies reductant to crucial biosynthetic pathways. The active-site cysteines of nDsbD are protected from the oxidising periplasm by a loop. This ‘cap’ loop must open when nDsbD associates with its physiological interaction partners. To understand the mechanism of loop opening we have applied NMR relaxation methods alongside molecular dynamics (MD) simulations, probing the dynamic behaviour of the ‘cap’ loop in the oxidised (with C103-C109 disulphide) and reduced (without disulphide) forms of nDsbD. Spin-relaxation experiments demonstrate the ‘cap’ loop is rigid on a ps-ns timescale. 15N relaxation dispersion experiments revealed a slow conformational change in the ‘cap’ loop of oxidised but not reduced nDsbD. The chemical shift differences measured from fits of the nDsbDox data provide insights into its lowly-populated excited state. MD simulations confirm that the loop protects the active site of nDsbDred. It is more flexible in nDsbDox; the disulphide bond induces frustration in the active site, which leads to spontaneous loop opening in some trajectories. The simulations agree well with experimental S2, RDCs and torsion angle predictions from TALOS-N. The differences in the conformational flexibility of oxidised and reduced nDsbD may account for the observed redox-state dependence of its interaction affinities. Once reductant is transferred from cDsbDred to nDsbDox, the complex dissociates, enhancing turnover in the pathway. NMR experiments have revealed the kinetic basis of the redox-state dependent formation of the cDsbD-nDsbD complex. MD simulations identified dynamic encounter complexes in the association of cDsbD with nDsbD.

The effect of the addition of thiol reagents to heated milk on protein interactions and acid gelation properties

Low levels of β-mercaptoethanol (β-ME) or N-ethylmaleimide (NEM) were added to preheated skim milk (SM) and preheated whey protein-enriched skim milk (WPE-SM). Addition of NEM did not affect the heat-induced interactions between the proteins in heated SM and WPE-SM; addition of β-ME reduced most disulphide bonds of κ-casein. Neither NEM nor β-ME affected the distribution of proteins between the colloidal and serum phases. The heated then treated SM and WPE-SM were acidified to form acid gels. Acid gels containing β-ME had higher yield stress and G′ values than those made from control heated milk. In contrast, adding NEM to heated SM and WPE-SM lowered the yield stress values of the acid gels, but affected the final G′ values only slightly. The rheological results suggested that thiol-disulphide exchange reactions occurred during acid gelation. However, the newly formed disulphide bonds influenced only the yield properties, not the G′ values of the gels.

Effect of adding low levels of β-mercaptoethanol on the disulphide bonds of κ-casein and β-lactoglobulin solutions

Low levels of β-mercaptoethanol (up to 0.5%, v/v) were added to κ-casein and β-lactoglobulin solutions (5mgmL−1), mixtures thereof, and milk. The reduction of disulphide bonds was monitored over time for up to 24h, using micro-fluidic electrophoresis, at temperatures ranging from 20 to 50°C. Lower concentrations of β-mercaptoethanol and a shorter reaction time were required to reduce κ-casein to the same extent as β-lactoglobulin. This suggests that the intermolecular disulphide bonds of κ-casein were more accessible than the intra-molecular disulphide bonds of β-lactoglobulin. The reduction of β-lactoglobulin at any β-mercaptoethanol concentration and reaction time was enhanced as the temperature increased up to 50°C. The level of reduced κ-casein increased with increasing temperature during the first 2h; however, as the reaction time was prolonged, the level of monomeric κ-casein decreased. This suggests that monomeric κ-casein can re-form disulphide bonds via either re-oxidation or thiol-disulphide exchange reactions.

A novel disulphide switch mechanism in Ero1α balances ER oxidation in human cells

Oxidative maturation of secretory and membrane proteins in the endoplasmic reticulum (ER) is powered by Ero1 oxidases. To prevent cellular hyperoxidation, Ero1 activity can be regulated by intramolecular disulphide switches. Here, we determine the redox-driven shutdown mechanism of Ero1alpha, the housekeeping Ero1 enzyme in human cells. We show that functional silencing of Ero1alpha in cells arises from the formation of a disulphide bond-identified by mass spectrometry--between the active-site Cys(94) (connected to Cys(99) in the active enzyme) and Cys(131). Competition between substrate thiols and Cys(131) creates a feedback loop where activation of Ero1alpha is linked to the availability of its substrate, reduced protein disulphide isomerase (PDI). Overexpression of Ero1alpha-Cys131Ala or the isoform Ero1beta, which does not have an equivalent disulphide switch, leads to augmented ER oxidation. These data reveal a novel regulatory feedback system where PDI emerges as a central regulator of ER redox homoeostasis.

Effects of garlic (Allium sativum) juice containing allicin on Phytophthora infestans and downy mildew of cucumber caused by Pseudoperonospora cubensis

The volatile antimicrobial substance allicin (diallylthiosulphinate) is produced in garlic when the tissues are damaged and the substrate allicin (S-allyl-l-cysteine sulphoxide) mixes with the enzyme alliin-lyase (E.C.4.4.1.4). Allicin undergoes thiol-disulphide exchange reactions with free thiol groups in proteins and it is thought that this is the basis of its antimicrobial action. At 50 μg ml-1, allicin in garlic juice inhibited the germination of sporangia and cysts and subsequent germ tube growth by Phytophthora infestans both in vitro and in vivo on the leaf surface. Disease severity in P. infestans-infected tomato seedlings was also reduced by spraying leaves with garlic juice containing allicin over the range tested (55–110 μg ml−1) with an effectiveness ranging from approximately 45–100%. Similarly, in growth room experiments at concentrations from 50–1,000 μg ml−1, allicin in garlic juice reduced the severity of cucumber downy mildew caused by Pseudoperonospora cubensis by approximately 50–100%. These results suggest a potential for developing preparations from garlic for use in specialised aspects of organic farming, e.g. for reducing pathogen inoculum potential and perhaps for use under glass in horticulture.

Control of plant diseases by natural products: Allicin from garlic as a case study

This review aims to increase awareness of the potential for developing plant protection strategies based on natural products. Selected examples of commercial successes are given and recent data from our own laboratory using allicin from garlic are presented. The volatile antimicrobial substance allicin (diallylthiosulphinate) is produced in garlic when the tissues are damaged and the substrate alliin (S-allyl-l-cysteine sulphoxide) mixes with the enzyme alliin-lyase (E.C.4.4.1.4). Allicin is readily membrane-permeable and undergoes thiol-disulphide exchange reactions with free thiol groups in proteins. It is thought that these properties are the basis of its antimicrobial action. We tested the effectiveness of garlic juice against a range of plant pathogenic bacteria, fungi and oomycetes in vitro. Allicin effectively controlled seed-borne Alternaria spp. in carrot, Phytophthora leaf blight of tomato and tuber blight of potato as well as Magnaporthe on rice and downy mildew of Arabidopsis. In Arabidopsis the reduction in disease was apparently due to a direct action against the pathogen since no accumulation of salicylic acid (a marker for systemic acquired resistance, SAR) was observed after treatment with garlic extract. We see a potential for developing preparations from garlic for use in organic farming, e.g. for reducing the pathogen inoculum potential in planting material such as seeds and tubers. We have tested various encapsulation formulations in comparison to direct treatment.

Oxidant‐dependent switching between reversible and sacrificial oxidation pathways for Bacillus subtilis OhrR

The Bacillus subtilis OhrR protein functions as a transcriptional repressor of the inducible peroxidase, OhrA. Derepression is mediated by the organic-peroxide selective oxidation of an active site cysteine (C15). In the presence of cumene hydroperoxide (CHP), oxidation of OhrR leads to a sulphenic acid intermediate which reacts to form either a mixed-disulphide or a protein sulphenamide. These inactive forms of OhrR can be reactivated by thiol-disulphide exchange reactions allowing restoration of repression. Here, we demonstrate that linoleic acid hydroperoxide (LHP) is a potent oxidant for OhrR and even low levels lead to overoxidation of OhrR to cysteine sulphinic (and sulphonic) acid derivatives. Kinetic competition experiments indicate that further oxidation of the initial OhrR sulphenate product occurs at least 100-fold more rapidly with LHP than with CHP. Thus, depending on the oxidant, OhrR can be either reversibly oxidized or can instead function as a sacrificial regulator.

Presystemic Metabolism of Orally Administered Peptide Drugs and Strategies to Overcome It

To date, the majority of therapeutic peptides and proteins have to be administered via parenteral routes, which are painful and inconvenient. Consequently, "injectable-to-oral-conversions" are highly on demand. Apart from a poor membrane uptake, however, an extensive presystemic metabolism of orally given peptide drugs is responsible for a comparatively very poor oral bioavailability. This presystemic metabolism in the gastrointestinal tract is based on luminally secreted enzymes (I) including pepsins, trypsin, chymotrypsin, elastase and carboxypeptidase A/B, on brush border membrane bound enzymes (II) including various carboxypeptidases and aminopeptidases and on cytosolic enzymes (III). In addition, thiol-disulphide exchange reactions between orally administered peptide drugs and sulfhydryl bearing components of the gastrointestinal juice are responsible for a presystemic metabolism. Strategies to avoid a presystemic metabolism in the gastrointestinal tract are on the one hand based on chemical modifications of peptide drugs in order to make them more stable towards an enzymatic attack. On the other hand various formulation techniques are applied in order to protect therapeutic peptides, being incorporated in appropriate carrier systems. They include liposomes, nano-/microparticles and matrix tablets comprising various auxiliary agents such as enzyme inhibitors and multifunctional polymers. Within this review an overview about "the enemy's strength" and the current strategies to avoid a presystemic metabolism of orally administered peptides is provided.

Characterisation of the thiol–disulphide chemistry of desmopressin by LC, μ-LC, LC-ESI-MS and Maldi-Tof

To date, the majority of therapeutic peptides and proteins have to be administered via parenteral routes, which are painful and inconvenient. In order to gain sufficient high blood concentrations after oral application, various barriers in the gastrointestinal tract have to be overcome. Apart from a poor membrane uptake and intense enzymatic degradation, this study will demonstrate that thiol-disulphide reactions are an underestimated essential part of the presystemic metabolism. Glutathione, integrative part of the antioxidant defence system in the gastrointestinal tract, may play an important role in the inactivation of orally given peptides and proteins. In order to verify this hypothesis, desmopressin which bears a single disulphide bond was used as model peptide drug. Desmopressin was incubated with GSH in various concentrations, and the extent of thiol/disulphide exchange reactions between the peptide drug and GSH was investigated in dependence on pH and ratio of reactants determined as a function of time via HPLC, LC-MS and Maldi-Tof-MS analyses. Results showed that desmopressin is degraded by 1% reduced glutathione at pH 4 and pH 5.5. In presence of 0.01%, 0.1% and 1% of reduced glutathione 6.1%, 19.4% and 52.1% of desmopressin, respectively, were degraded. The masses of the conjugates after deconvolution measured by liquid chromatography and electrospray ionisation mass spectrometric detection were m/z 1069.67, m/z 1376.50, m/z 1683.40 and m/z 2138. These molecular masses, confirmed by Maldi-Tof-MS analysis, correspond with the masses of conjugates expected in theory. Under defined conditions, these results reveal that thiol-disulphide exchange reactions have a considerable impact on the alteration of peptide drugs and proteins.

S-Nitrosoglutathione/Glutathione Disulphide/Cu 2+-Dependent Stimulation of L-arginine Transport in Human Platelets

In this study, we have examined the effects of authentic nitric oxide (NO), NO + (NOBF 4), glutathione (GSH), glutathione disulphide (GSSG), and S-nitrosoglutathione (GSNO) in the presence and absence of Cu 2+, which thermally releases NO from S-nitrosothiols on the transport of L-arginine into the human platelet. The K M, apparent was unaffected by NO, NO +, GSH, and GSNO. However, Cu 2+ lowered K M, apparent by ∼2.85-fold. Cu 2+-dependent lowering of K M, apparent was also observed, albeit to a smaller extent when this ion was mixed with GSH (∼1.9-fold lower) and GSNO (∼2.0-fold). GSSG also lowered K M, apparent by ∼1.5-fold. The V max, apparent of L-arginine uptake was unaffected by NO, NO +, GSH, and Cu 2+. V max, apparent was stimulated by to the largest extent by GSNO (∼2.28-fold) and GSNO plus Cu 2+ (∼2.7-fold). GSSG and GSH plus Cu 2+ also increased V max, apparent by ∼1.9-fold. When these parameters are expressed in terms of transport efficiency (V max/K M) the largest effect of nearly 4.7-fold (over controls) was obtained by a combination of GSNO plus Cu 2+. These results suggest that platelet L-Arg transport is not affected either by NO or NO + but by a thiol-disulphide exchange reactions on the platelet L-Arg transporter, brought about by GSNO and GSSG. Based on these results, a GSNO/GSSG/Cu 2+ dependent regulatory mechanism for the uptake of L-arginine in human platelets has been proposed.

13C-n.m.r. of the cyanylated β-lactoglobulins: evidence that Cys-121 provides the thiol group of β-lactoglobulins A and B

The thiol groups of beta-lactoglobulins A and B have been cyanylated using [13C]KCN. The samples of [cyanato-13C]-cyanylated-beta-lactoglobulins A and B which we prepared had signals at 109.7 p.p.m. and 114.4 p.p.m. We conclude that the thiocyanate carbon having a chemical shift of 109.7 p.p.m. is in an apolar environment similar to a cyclohexane solvent, whereas the thiocyanate carbon having a chemical shift of 114.4 p.p.m. is in a polar environment similar to water. The signals with chemical shifts of 109.7 p.p.m. are assigned to the thiocyanate carbons of the native [cyanato-13C]cyanylated-beta-lactoglobulins A and B. We deduce that the signal at 114.4 p.p.m. is due to an irreversibly denatured/unfolded species produced by alkaline denaturation, which is caused by intramolecular thiol/disulphide exchange occurring during our cyanylation procedure. We propose that Cys-119 is cyanylated in the irreversibly denatured species and Cys-121 is cyanylated in the native [cyanato-13C]cyanylated-beta-lactoglobulins A and B. We suggest that the same intramolecular thiol-disulphide exchange reactions occurred when McKenzie and co-workers [McKenzie, Ralston and Shaw (1972) Biochemistry 11, 4539-4547] alkylated beta-lactoglobulins with iodoacetamide. Therefore the one mol of thiol/mol of monomer in the native beta-lactoglobulins is due to the thiol of Cys-121 and is not due to an equimolar mixture of Cys-119 and Cys-121 as they suggested.

Toxin-Targeted Design for Anticancer Therapy. I: Synthesis and Biological Evaluation of New Thioimidate Heterobifunctional Reagents

In an effort to obtain a more potent and specific immunotoxin for cancer therapy, we designed a series of heterobifunctional linkers characterized by a thioimidate group linked to a S-acetyl thiol (4, 5) or substituted aryldithio group (6-10). These ligands were synthesized by a Pinner-type process from the corresponding nitrile derivatives obtained by thiol-disulphide exchange reaction, reaction with substituted benzene-sulphenyl chloride, or other known procedures. To check the reagent of choice for immunoconjugate preparation, we studied thiol- disulphide exchange kinetics between the intermediate nitrile derivatives and cysteine. Among the tested aryldithio derivatives (6-10), we selected ethyl 3-(4-carboxamido-phenyldithio)propionthioimidate (CDPT, 9) for further studies. By analyzing the rate of incorporation of the linkers 4, 5, and 9 in a model immunoglobulin G protein, we found similar results with CDPT 9 and ethyl S-acetyl 3-mercaptopropionthioimidate ester hydrochloride (AMPT, 5) because both reagents showed a linear correlation between the number of introduced thiol groups and factors such as time and protein and reagent concentrations. Comparison of the two acetylthio-derivative ligands 4 and 5 showed that AMPT 5 was more stable toward deacetylation than ethyl S-acetyl 2-mercaptopropionthioimidate ester hydrochloride (AMAT, 4). By comparing the kinetic and biological parameters of seven new thioimidate linkers, we found that two of these (CDPT and AMPT) could be superior ligands for protein-protein conjugation. They offer advantages over the commercially available compounds, such as minimal perturbation of the protein structure, controlled reactivity, and good stability.

Kinetic and conformational factors involved in chemisorption and adsorption of proteins on mercaptopyridine-derivatized agarose

The kinetics of the thiol disulphide exchange reaction between thiol- and/or thioester-containing proteins were studied with the disulphide free in solution in the form of 2,2-dipyridyldisulphide and covalently attached to dextran, a water-soluble polymer in the form of N-ethyl-3-(2-pyridyldisulphanyl)propionamide dextran (Py-S 2-dextran) and 3-(2-pyridyldisulphanyl)-2-hydroxypropyl beaded agarose (Py-S 2-agarose), in order to investigate the effect of the polymer on the reaction. Analysis of the chemisorbed and adsorbed proteins revealed similarities in the initial adsorption process and we propose a model for adsorption—chemisorption to pyridine-derivatized beaded agarose gel.

Protein-disulphide isomerase and prolyl isomerase act differently and independently as catalysts of protein folding.

Two enzymes are now known that catalyse slow steps in protein folding. Peptidyl-prolyl cis-trans isomerase catalyses the cis-trans isomerization of Xaa-Pro peptide bonds in oligopeptides and during the refolding of several proteins. The other enzyme, protein-disulphide isomerase, accelerates the reactivation of reduced proteins, presumably by catalysis of thiol-disulphide exchange reactions. Recent evidence indicates that the beta-subunit of prolyl 4-hydroxylase, an enzyme involved in collagen biosynthesis, is identical with disulphide isomerase. On the basis of this important finding, it was suggested that disulphide isomerase accelerates protein folding, not by 'reshuffling' incorrect disulphide bonds, but in the same way as prolyl isomerase by catalysing proline isomerization which is known to be important for the folding of collagen and other proteins. Here we show that the catalytic activities of these two enzymes are different. Disulphide isomerase accelerates the reformation of native disulphide bonds during protein reoxidation. We find no evidence that this enzyme can catalyse the isomerization of proline peptide bonds, a reaction efficiently accelerated by prolyl isomerase. When both enzymes are present simultaneously during protein folding, they act independently of one another.

A competitive enzyme immunoassay of human β2-microglobulin based on separation by filtration

A competitive enzyme immunoassay has been developed for the determination of β2-microglobulin in undiluted human serum. In the assay a β2-microglobulin-β-galactosidase conjugate competes with β2-microglobulin from the sample for the binding to anti-β2-microglobulin antibodies bound to small size agarose particles (micro-Sepharose) via a double antibody.The conjugate is made by providing β2-microglobulin with ‘reactive disulphide structures’. This is achieved by the heterobifunctional reagent SPDP and the subsequent linkage of the formed derivative to β-galactosidase by a thioldisulphide exchange reaction. This procedure gives conjugates with high immunoreactivity and enzyme activity.The assay is fast and simple. The reagents are incubated together for 60 min in the wells of a Millititer plate and separation is performed by filtration. Bound enzyme-labelled β2-microglobulin is measured by incubation with substrate for 15 min. Both incubations are performed at room temperature without agitation. Due to the high capacity of the micro-Sepharose no sample predilution is needed. The use of the Millititer filtration system gives a rapid and efficient separation and the advantages of access to equipment for dispensing and reading adapted to the microtitre format.