Formation Of Intermolecular Disulphide Bonds Research Articles

Study of structural changes of gluten proteins during bread dough mixing by Raman spectroscopy

The aim of the present study was to evaluate Raman spectroscopy in determining changes that occur in the structure of gluten proteins induced during bread dough mixing. Raman spectra were measured directly within the dough. Three particular phases of mixing were studied: under-mixing, optimum mixing and over-mixing. A thiol blocking reagent, Tris(2-carboxyethyl)phosphine (TCEP) was then used to reduce disulphide bonds within proteins to confirm the important role of disulphide bridges in gluten network formation. For the control dough, the most important changes occurred during the optimum mixing phase when an increase in intermolecular disulphide bonds, anti-parallel β-sheet and α-helix structures was observed, combined with the hydrophobic burial of tryptophan and tyrosine residues. The addition of TCEP appeared to effectively reduce the formation of intermolecular disulphide bonds, anti-parallel β-sheet and α-helix structures and lead to a more disordered secondary protein structure.

Super-Resolution Fluorescence Microscopy Reveals Clustering Behaviour of Chlamydia pneumoniae's Major Outer Membrane Protein.

Simple SummaryChlamydia is an infamous sexually transmitted bacterium that also has a less well-known role in human respiratory infections, which has evolved a unique cell structure to enable its survival within the body. Covering the surface of this infectious cell is a strong mesh-like network made up of many different proteins which protects the cell against damage. This research focussed on the most abundant protein within this mesh, the Major Outer Membrane Protein (MOMP), and introduced a series of mutations designed to prevent the mesh from forming completely. The effect of the mutations was visualised by adding a bright fluorescent dye to each MOMP, which was then examined with a high-resolution fluorescence microscope capable of showing us each individual cell and the MOMPs at their surface. With statistical analysis, we observed that certain mutations disrupted the connections between MOMPs, giving us greater insight into how Chlamydia forms these interactions. Chlamydia is an extremely prevalent disease amongst the global population, and whilst treatable, there is currently no available vaccine. By researching Chlamydia’s biology and its method of evading our immune system, we can not only further our understanding of this complex bacterium, but also develop novel therapeutics for its treatment and prevention.Chlamydia pneumoniae is a Gram-negative bacterium responsible for a number of human respiratory diseases and linked to some chronic inflammatory diseases. The major outer membrane protein (MOMP) of Chlamydia is a conserved immunologically dominant protein located in the outer membrane, which, together with its surface exposure and abundance, has led to MOMP being the main focus for vaccine and antimicrobial studies in recent decades. MOMP has a major role in the chlamydial outer membrane complex through the formation of intermolecular disulphide bonds, although the exact interactions formed are currently unknown. Here, it is proposed that due to the large number of cysteines available for disulphide bonding, interactions occur between cysteine-rich pockets as opposed to individual residues. Such pockets were identified using a MOMP homology model with a supporting low-resolution (~4 Å) crystal structure. The localisation of MOMP in the E. coli membrane was assessed using direct stochastic optical reconstruction microscopy (dSTORM), which showed a decrease in membrane clustering with cysteine-rich regions containing two mutations. These results indicate that disulphide bond formation was not disrupted by single mutants located in the cysteine-dense regions and was instead compensated by neighbouring cysteines within the pocket in support of this cysteine-rich pocket hypothesis.

Controlled release matrix tablet formulation using synthesized N-acyl Thiolated Chitosan derivatives

Chitosan derivative was successively synthesized by initial preparation of acyl thiourea reagent using ammonium thiocyanate and making it to react with at primary amino groups of chitosan and then reduced to yield Thiolated Chitosan. Due to the formation of disulphide bonds with mFucus glycoproteins, mucoadhesiveness is augmented. The thiol groups were then quantified using Ellman’s reagent. The derivatives inherit good swelling property in neutral and alkaline media. The different derivatives containing thiol groups were formulated into tablets using reference drug for evaluation. The Thiolated Chitosan display in situ gelling features due to the pH-dependent (alkaline pH) formation of inter-molecular disulphide bonds which makes the application of Thiolated Chitosan on intestinal mucosa and can guarantee prolonged controlled release of embedded therapeutic ingredients.

Lipase Immobilized on Magnetic Nanoparticles: A New Tool for Synthesis of Disulphide Compounds

The study describes chemo-enzymatic synthesis of organic disulphide compounds. The reaction was initiated by hydrolysis of thiol acetates using hydrolytic enzyme lipase (PPL) immobilized on to magnetic nanoparticles and subsequent formation of organic disulphide compounds. Lipase was immobilized on to the magnetic nanoparticles by co-precipitation method via epichlorohydrin chitosan cross-linking, under mild and eco-friendly conditions. The immobilized lipase enzyme exhibited broad range of substrate specificity in synthesizing disulphide compounds, which involves both intra and inter-molecular disulphide bond formation under anaerobic conditions. The disulphide compounds synthesized also show a promising antimicrobial activity.

Identification of a Group of Novel γ-Gliadin Genes

γ-Gliadins are an important component of wheat seed storage proteins. Four novel γ-gliadin genes (Gli-ng1 to Gli-ng4) were cloned from wheat (Triticum aestivum) and Aegilops species. The novel γ-gliadins were much smaller in molecular size when compared to the typical γ-gliadins, which was caused by deletion of the non-repetitive domain, glutamine-rich region, 3' part of the repetitive domain, and 5' part of the C-terminal, possibly due to illegitimate recombination between the repetitive domain and the C-terminal. As a result, Gli-ng1 and Gli-ng4 only contained two and three cysteine residues, respectively. Gli-ng1, as the representative of novel γ-gliadin genes, has been sub-cloned into an Escherichia coli expression system. SDS-PAGE indicated that the both cysteine residues of Gli-ng1 could participate in the formation of intermolecular disulphide bonds in vitro. Successful cloning of Gli-ng1 from seed cDNA of T. aestivum cv. Chinese Spring suggested that these novel γ-gliadin genes were normally transcribed during the development of seeds. Phylogenic analysis indicated that the four novel γ-gliadin genes had a closer relationship with those from the B (S) genome of wheat.

Use of FCS to Study Protein Denaturation and Aggregation

Fluorescence Correlation Spectroscopy (FCS) is a powerful technique for studying the diffusive dynamics of fluorophore-tagged biomolecules and thereby drawing inferences about their conformational states and transitions. We have employed FCS to investigate the denaturation and aggregation pathways of bovine immunoglobulin (IgG), a multi-subunit all beta-sheet Y-shaped protein prone to aggregation. Each upper arm of the Y consists of a light and a heavy chain connected by cysteine-shy;cysteine disulphide bonds, and the two heavy chains are joined at the bend by more disulphides. While majority of proteins show continuous increase of hydrodynamic radius (Rg) when presented with increasing denaturant concentration, IgG exhibits strikingly different behavior under similar conditions. Its Rg initially decreases with increasing concentration of GdnHCl (from 0 to 3 M) and then goes through two pronounced maxima at 4 M and 7 M [GdnHCl]. We propose that the initial contraction is related to sheet-to-helix transition at the flexible bends of the Y-arms of the protein. The first maximum of Rg is attributed to electrostatic repulsions and their screening by Gdn+ and Cl- ions; the second is shown to arise from disulphide-mediated effects.Previous studies have proposed that IgG aggregation is initiated by intermolecular disulphide bond formation. We studied the aggregation kinetics of IgG by monitoring the time evolution of characteristic diffusion time (τD) under two different conditions: with the cysteine residues free to interact or blocked. The progress curves (τD vs.elapsed time) were sigmoidal in both cases, but the delay time was almost an order of magnitude longer, and the apparent polymerization rate half as fast, for the case where the cysteines were blocked than when they were free. This result indicates that intermolecular disulphide bond formation is a key factor driving the aggregation process of IgG.

Identification of novel α-gliadin genes

Ten novel α-gliadin genes (Gli-ta, Gli-turg1, Gli-turg2, Gli-turg3, Gli-turg4, Gli-turg5, Gli-turg6, Gli-cs1, Gli-cs2, and Gli-cs3) with unique characteristics were isolated from wheat (Triticum aestivumL.), among which Gli-cs1, Gli-cs2, Gli-cs3, and Gli-turg6 were pseudogenes. Gli-cs3 and nine other sequences were much larger and smaller, respectively, than the typical α-gliadins. This variation was caused by insertion or deletion of the unique domain I and a polyglutamine region, possibly the result of illegitimate recombination. Consequently, Gli-cs3 contained 10 cysteine residues, whereas there were 2 cysteine residues only in the other nine sequences. Gli-ta/Gli-ta-like α-gliadin genes are normally expressed during the development of seeds. SDS-PAGE analysis showed that in-vitro-expressed Gli-ta could form intermolecular disulphide bonds and could be chain extenders. A protein band similar in size to Gli-ta has been observed in seed extracts, and mass spectrometry results confirm that the band contains small molecular mass α-gliadins, which is a characteristic of the novel α-gliadins. Mass spectrometry results also indicated that the two cysteine residues of Gli-ta/Gli-ta-like proteins participated in the formation of intermolecular disulphide bonds in vivo.

The influence of bovine serum albumin on β-lactoglobulin denaturation, aggregation and gelation

The effect of bovine serum albumin (BSA) on the heat-induced denaturation, aggregation and subsequent acid-induced gelation of β-lactoglobulin ( β-lg) was investigated in this work. Changes in the denaturation kinetics of β-lg during heating at 78 °C were determined by monitoring the disappearance of the native protein by reverse-phase chromatography. Replacing β-lg with increasing amounts of BSA, while keeping the total protein concentration constant at 5% (w/w), significantly increased the denaturation rate of β-lg from 2.57±0.30×10 −3(g L −1) (1− n) s −1 to 5.07±0.72×10 −3(g L −1) (1− n) s −1 ( β-lg: BSA ratio of 3:1 w/w). The reaction order for β-lg was 1.40±0.09. Partial replacement of β-lg with BSA ( β-lg: BSA ratio of 3:1 w/w) significantly increased the reaction order to 1.67±0.13. Heat-induced aggregates between β-lg and BSA were studied by dynamic light scattering, two-dimensional electrophoresis and size exclusion chromatography. The partial replacement of β-lg with BSA significantly changed the gelling properties of the acid-induced gels. A rapid rate of acidification resulted in a significant decrease, while a slow acidification rate resulted in a significant increase in gel strength. Size exclusion chromatography demonstrated that intermolecular disulphide bond formation occurred during both heat-induced denaturation/aggregation and subsequent acid-induced gelation. Results clearly indicate that BSA contributed to the formation of these disulphide bonds.

Characterization of Low Molecular Weight Glutenin Subunit Gene Representing Glu-B3 Locus of Indian Wheat Variety NP4

Low molecular weight (LMW) glutenin subunits represent major part (30%) of storage proteins in wheat endosperm and determine the quality of dough. Despite their importance few LMW glutenin genes have been characterized so far and none from Indian wheat variety. In the present investigation PCR technique was employed to characterize LMW-GS gene representing Glu-B3 locus from Indian bread wheat cultivar NP4. The deduced protein sequence coded by Glu-B3 locus of LMW-GS gene from NP4 showed the presence of regular structure of the repetitive domain with varying numbers of glutamine (Q) residues and the presence of 1st cysteine residue within the repetitive domain at 40th position in mature polypeptide. Such structure might increase and stabilize the gluten polymer through intermolecular interactions of the large numbers of glutamine side chains and cysteine residues for intermolecular disulphide bond formation leading to stronger dough quality of NP4. Moreover, Glu-B3 specific primers could also be used for identifying 1BL/1RS translocation in addition to amplifying LMW glutenin genes. There was no amplification in 1B/1R translocation lines as short arm of wheat was replaced by short arm of rye chromosome in these lines. Such information can be useful in wheat improvement for dough properties for better chapati and bread quality.

Substitution of Serine for Non-disulphide-bond-forming Cysteine in Grass Carp (Ctenopharygodon Idellus) Growth Hormone Improves In Vitro Oxidative Renaturation

Native grass carp (Ctenopharygodon idellus) growth hormone, has 5 cysteine amino acid residues, forms two disulphide bridges in its mature form. Recombinant grass carp growth hormone, when over-expressed in E. coli, forms inclusion bodies. In vitro oxidative renaturation of guanidine-hydrochloride dissolved recombinant grass carp growth hormone was achieved by sequential dilution and stepwise dialysis at pH 8.5. The redox potential of the refolding cocktail was maintained by glutathione disulphide/glutathione couple. The oxidative refolded protein is heterogeneous, and contains multimers, oligomers and monomers. The presence of non-disulphide-bond-forming cysteine in recombinant grass carp growth hormone enhances intermolecular disulphide bond formation and also nonnative intramolecular disulphide bond formation during protein folding. The non-disulphide-bond-forming cysteine was converted to serine by PCR-mediated site-directed mutagenesis. The resulting 4-cysteine grass carp growth hormone has improved in vitro oxidative refolding properties when studied by gel filtration and reverse phase chromatography. The refolded 4-cysteine form has less hydrophobic aggregate and has only one monomeric isoform. Both refolded 4-cysteine and 5-cysteine forms are active in radioreceptor binding assay.

Oxidative stress inhibits MEKK1 by site-specific glutathionylation in the ATP-binding domain.

Many intracellular signalling events are accompanied by generation of reactive oxygen species in cells. Oxidation of protein thiol groups is an emerging theme in signal-transduction research. We have found that MEKK1 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase kinase 1], an upstream activator of the SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase) pathway, is directly inhibited by cysteine alkylation using NEM (N-ethylmaleimide). The related kinase, ASK1 (apoptosis signal-regulating kinase 1), was not inhibited, but was instead activated by NEM. Inhibition of MEKK1 requires a single unique cysteine residue (Cys1238) in the ATP-binding domain of MEKK1. Oxidative stress induced by menadione (2-methyl-1,4-naphthoquinone) also inhibited MEKK1, but activated ASK1, in cells. MEKK1 inhibition by menadione also required Cys1238. Oxidant-inhibited MEKK1 was re-activated by dithiothreitol and glutathione, supporting reversible cysteine oxidation as a mechanism. Using various chemical probes, we excluded modification by S-nitrosylation or oxidation of cysteine to sulphenic acid. Oxidant-inhibited MEKK1 migrated normally on non-reducing gels, excluding the possibility of intra- or inter-molecular disulphide bond formation. MEKK1 was inhibited by glutathionylation in vitro, and MEKK1 isolated from menadione-treated cells was shown by MS to be modified by glutathione on Cys1238. Our results support a model whereby the redox environment within the cell selectively regulates stress signalling through MEKK1 versus ASK1, and may thereby participate in the induction of apoptosis by oxidative stress.

The sites for fatty acylation, phosphorylation and intermolecular disulphide bond formation of influenza C virus CM2 protein.

The sites for fatty acylation, disulphide bond formation and phosphorylation of influenza C virus CM2 were investigated by site-specific mutagenesis. Cysteine 65 in the cytoplasmic tail was identified as the site for palmitoylation. Removal of one or more of three cysteine residues in the ectodomain showed that all of cysteines 1, 6 and 20 can participate in the formation of disulphide-linked dimers and/or tetramers, although cysteine 20 may play the most important role in tetramer formation. Furthermore, it was found that serine 78, located within the recognition motifs for mammary gland casein kinase and casein kinase I, is the predominant site for phosphorylation, although serine 103 is phosphorylated to a minor extent by proline-dependent protein kinase. The effects of acylation and phosphorylation on the formation of disulphide-linked oligomers were also studied. The results showed that, while palmitoylation has no role in oligomer formation, phosphorylation accelerates tetramer formation without influencing dimer formation. CM2 mutants defective in acylation, phosphorylation or disulphide bond formation were all transported to the cell surface, suggesting that none of these modifications is required for proper oligomerization. When proteins solubilized in detergent were analysed on sucrose gradients, however, the mutant lacking cysteines 1, 6 and 20 sedimented as monomers, raising the possibility that disulphide bond formation, although not essential for proper oligomerization, may stabilize the CM2 multimer. This was supported by the results of chemical cross-linking analysis, which showed that the triple-cysteine mutant can form multimers.

Identification of the cysteine residues implicated in the formation of alpha 2 and alpha/beta dimers of rat meprin.

Meprin (endopeptidase-24.18; EC 3.4.24.18) is a multisubunit zinc-metallopeptidase found in the brush-border membranes of rodent kidney and human intestine. The alpha and beta subunits of meprin are disulphide-linked to form either soluble alpha 2 homodimers or membrane-associated alpha/beta heterodimers. The aim of the present study was to identify the cysteine residue(s) implicated in the formation of alpha 2 and alpha/beta dimers and to investigate the effects of dimerization on intracellular transport and processing of the alpha subunit. Three cysteine residue candidates for the formation of disulphide bonds in the alpha subunit were selected by hydrophobic cluster analysis. These residues, located at positions 309, 560 and 562, were mutated to serine residues. When the resulting alpha subunit mutants were expressed alone in COS-1 cells, the alpha C560S and alpha C562S mutants were found to be secreted as alpha 2 homodimers whereas the alpha C309S mutant was found as monomers in the culture medium. In double-transfection experiments with the wild-type beta subunit, the alpha C560S and alpha C562S mutants behaved exactly as the wild-type alpha subunit and formed membrane-bound alpha/beta heterodimers. In contrast, the alpha C309S mutant was not retained at the cell surface but rather secreted as monomers in the culture medium, as was found in the simple transfection experiment. These results show that, despite the normal expression level and folding of the protein in a transport-competent from, the alpha C309S mutant is unable to form alpha 2 homodimers or alpha/beta heterodimers. This suggests that Cys309 is the unique residue of the alpha subunit implicated in the alpha 2 and alpha/beta dimerizations. Hydrophobic cluster analysis of the alpha and beta subunit sequences predicts that Cys309 is similar to Cys306 of the beta subunit. We mutated the latter residue to a serine and expressed the beta C306S mutant and the wild-type alpha subunit in the same COS-1 cells. No beta 2 or alpha/beta dimers were observed on immunoblotting, showing that Cys306 of the beta subunit is required for the formation of intermolecular disulphide bonds both in beta 2 homodimers and in alpha/beta heterodimers. Taken together, these results suggest that the alpha/beta heterodimeric form of meprin is held together by a single disulphide bond linking Cys309 in the alpha subunit to Cys306 in the beta subunit.

Redox properties of wild-type, Cys69Ala, and Cys69Ser Azotobacter vinelandii flavodoxin II as measured by cyclic voltammetry and EPR spectroscopy,.

This study deals with the detailed electrochemistry and complete EPR-monitored titrations of flavodoxin II of Azotobacter vinelandii (ATCC 478). Since wild-type flavodoxin dimerises via intermolecular disulphide bond formation between Cys69 residues, Cys69 has been replaced by both an alanine and a serine residue. Redox properties of the C69A and C69S flavodoxin mutants were compared to those of wild-type flavodoxin. In the presence of the promotor neomycin, C69A and C69S flavodoxin showed a reversible response of the semiquinone/hydroquinone couple at the glassy carbon electrode. However, the addition of dithiothreitol proved to be necessary for the stabilisation of the wild-type flavodoxin response. EPR-monitored redox titrations of wild-type and C69A flavodoxin at high and low pH confirmed the redox potentials measured using cyclic voltammetry. The pH dependence of the semiquinone/hydroquinone redox potentials cannot be described using a model assuming one redox-linked pK. Instead, the presence of at least two redox-linked protonation sites is suggested: pKred.1 = 5.39 +/- 0.08, pKox = 7.29 +/- 0.14, and pKred.2 = 7.84 +/- 0.14 with Em.7 = -459 +/- 4 mV, and a constant redox potential at high pH of -485 +/- 4 mV. The dependence of the semiquinone/hydroquinone redox potential on temperature is -0.5 +/- 0.1 mV . K(-1), yielding delta H degrees = 28.6 +/- 1.5 kJ . mol(1) and delta S degrees = -50.0 +/- 6.2 J . mol(-1) . K(-1). No significant differences in redox properties of wild-type, C69A, and C69S flavodoxin were observed. The electrochemical data suggest that replacement of Cys69 in the vicinity of the FMN by either an alanine or a serine residue does not alter the dielectric properties and structure of A. vinelandii flavodoxin II.

Biochemical properties of recombinant human beta-glucuronidase synthesized in baby hamster kidney cells.

The cDNA sequence encoding human beta-glucuronidase [Oshima, Kyle, Miller, Hoffmann, Powell, Grubb, Sly, Troplak, Guise and Gravel (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 685-689] was expressed in baby hamster kidney (BHK) cells. After purification from the culture supernatant in one step by use of immunoaffinity chromatography, the biochemical properties of the enzyme were examined. With a pH optimum of 4.0, a Km of 1.3 mM and thermal stability up to 68 degrees C, this protein has characteristics very similar to those described for beta-glucuronidase from human placenta [Brot, Bell and Sly (1978) Biochemistry 17, 385-391. However, the recombinant product has several structural properties not previously reported for beta-glucuronidase isolated from natural sources. First, recombinant beta-glucuronidase is synthesized as a tetramer consisting of two disulphide-linked dimers. As can be inferred from the cDNA sequence, the enzyme possesses five cysteine residues after cleavage of the signal peptide. By introducing a C-terminal truncation, we eliminated the last cysteine at position 644. In the mutant, covalent linkage between two monomers is no longer observed, indicating that Cys-644 is involved in intermolecular disulphide-bond formation. The functional role of the disulphide bond remains elusive, as it was shown that (i) intracellular transport of the mutant is not impaired and (ii) it is still able to form an enzymically active tetramer. A second feature that has not previously been observed for beta-glucuronidase from any origin is the existence of two enzymically active species for recombinant beta-glucuronidase, when examined by gel filtration on a TSK 3000 column. With apparent molecular masses of 380 kDa and 190 kDa we propose that they represent tetramers and dimers respectively. Partial N-terminal sequencing and electrophoresis under denaturing conditions revealed that the dimers consist of subunits that have been proteolytically processed at their C-terminus losing 3-4 kDa in peptide mass. Controlled proteolysis demonstrates that the enzyme's overall protein backbone as well as its activity are resistant to a number of proteases. Only the C-terminal portion is susceptible to protease action, and the disulphide-linked form is readily converted into non-disulphide-bonded subunits. Pulse-chase analysis shows that human beta-glucuronidase remaining intracellular in BHK cells after synthesis undergoes a similar proteolytic processing event, i.e. a reduction in mass of 3-4 kDa.(ABSTRACT TRUNCATED AT 400 WORDS)

The predicted amino acid sequence of the spheroidin protein from Amsacta moorei entomopoxvirus: lack of homology between major occlusion body proteins of different poxviruses.

Entomopoxviruses replicate in the cytoplasm of insect cells and characteristically produce occlusion bodies which serve to protect the virion from the environment; the major component of these bodies is a protein called spheroidin. We have previously identified and sequenced the gene encoding the major occlusion body protein of eastern spruce budworm (Choristoneura biennis) entomopoxvirus (CbEPV) and found it to encode a 47K polypeptide which aggregates due to the formation of intermolecular disulphide bonds. In this publication we demonstrate that the insect poxvirus of Amsacta moorei produces spheroidin with a unit Mr of 114.8K. The gene for this protein was cloned and sequenced, and the predicted polypeptide was demonstrated to contain 38 cysteine residues, a leucine zipper for possible protein-protein interactions and 14 potential Asn-linked glycosylation sites. Other than possessing a large number of sulphydryl groups, this protein showed no homology to its analogue found in cells infected with CbEPV. Antibodies directed against occlusion body proteins of the two viruses also failed to cross-react significantly on Western blots. In addition, nucleic acid probes prepared from the two different genes did not cross-hybridize on Southern blots of genomic DNA prepared from the viruses. Finally, the occlusion body proteins from the two insect viruses were compared with the A-type inclusion body protein of cowpox virus. Again, little homology between these proteins was evident, with the exception of a generally high cysteine content and a similarity between their late gene promoters. We conclude that the major occlusion body proteins of different poxviruses possess diverse primary structures, but all are capable of yielding large aggregates through the formation of disulphide bonds.

Stability of human interferon-ß1: oligomeric human interferon-ß1 is inactive but is reactivated by monomerization

Human interferon-beta 1 is extremely stable is a low ionic strength solution of pH 2 such as 10 mM HCl at 37 degrees C. However, the presence of 0.15 M NaCl led to a remarkable loss of antiviral activity. The molecular-sieve high-performance liquid chromatography revealed that, whereas completely active human interferon-beta 1 eluted as a 25 kDa species (monomeric form), the inactivated preparation eluted primarily as a 90 kDa species (oligomeric form). The specific activity (units per mg protein) of the oligomeric form was approx. 10% of that of the monomeric form. This observation shows that oligomeric human interferon-beta 1 is apparently in an inactive form. When the oligomeric eluate was resolved by polyacrylamide gel containing sodium dodecyl sulphate (SDS), it appeared to be monomeric under non-reducing conditions. Monomerization of the oligomeric human interferon-beta 1 by treatment with 1% SDS, fully regenerated its antiviral activity. These results suggest that the inactivation of the human interferon-beta 1 preparation was caused by its oligomerization via hydrophobic interactions without the formation of intermolecular disulphide bonds. These oligomers can be dissociated by SDS to restore biological activity.

Haemoglobin properties and polymerization in the marine teleost Lophius americanus (Goosefish)

1. 1. The red cells of the marine teleost Lophius americanus (goosefish) contain a haemoglobin which is easily auto-oxidized at neutral pH, has a large number of sulphydryl groups (16 per tetramer) and polymerizes in vitro by intermolecular disulphide bond formation. 2. 2. The protein is considerably less resistant to urea denaturation than ocean pout or human. The urea denaturation mid-point for goosefish Hb of 4.3 M compares with 5.8–6.0 for ocean pout and human. 3. 3. Structurally, goosefish haemoglobin is a heterotetramer with two dissimilar sub-units and appears to have the form α 2 β 2.

The nature of disulphide bonds in rat lens proteins.

The nature of disulphide bonds in 3, 7 and 24 month old rat lens proteins has been determined in order to examine the possibility that there is a direct correlation between intermolecular disulphide bond formation and hardening of the lens during ageing. Significant levels of S-S were found in all of the crystallins, as well as in the insoluble proteins. However, no substantial changes in the molecular weight distribution of the proteins, or their constituent polypeptides, were observed after the reduction of these bonds. This indicates that almost all of the S-S bonds are intramolecular. It would appear that the hardening of the nucleus is not a result of intermolecular disulphide crosslinking during ageing. Instead, our observations suggest that intramolecular disulphide bonds may make the gamma-crystallin molecule more compact, thereby promoting closer packing in the nucleus where the concentration of this protein is highest.

The isolation of nuclear envelopes. Effects of thiol-group oxidation and of calcium ions

The effects of (a) oxidative cross-linking of protein thiol groups and (b) the presence or absence of Ca2+ ions on rat liver nuclear-envelope isolation were studied. Two envelope-isolation procedures were compared: a well characterized low-ionic-strength method and a recently developed high-ionic-strength method. The latter method seems preferable to the former in respect of lower intranuclear contamination of the envelopes, suppression of endogenous serine proteinase, and maintenance of high specific activities of envelope-associated enzymes. In both procedures, however, the presence of Ca2+ gave rise to a rapid, apparently irreversible, contamination of the envelopes by intranuclear material. This effect was half-maximal at 20 microM-Ca2+. In addition, the envelopes became contaminated with intranuclear material by a Ca2+-independent mechanism, apparently resulting from N-ethylmaleimide-sensitive intermolecular disulphide-bond formation. This oxidative process seemed to have two major kinetic components (half-life, t1/2, approx. 2 min and 10 min). In view of these findings, it is recommended that (i) for most purposes, nuclear envelopes be isolated by the newly developed high-ionic-strength procedure, (ii) irrespective of the method used, Ca2+-chelators be included in all the buffers, (iii) thiol-group oxidation be prevented or reversed during the procedure.