Free Sulphydryl Groups Research Articles

α-Tocopherol/lipid ratio in blood is decreased in patients with Leber's hereditary optic neuropathy and asymptomatic carriers of the 11778 mtDNA mutation

OBJECTIVESLeber's hereditary optic neuropathy (LHON) is a maternally inherited disease characterised by acute or subacute bilateral visual loss in young patients. The primary aetiological event is a mutation in the...

Structural Models of Limit Dextrinase Inhibitors from Barley

Two limit dextrinase inhibitors (designated low and high pI inhibitors) isolated from barley had a high sequence homology with a protein sequence coded by a mRNA previously found in barley and thought to code for alpha -amylase-trypsin inhibitor. The inhibitors did not inhibit trypsin or alpha -amylases from malt, T. molitor (yellow mealworm) or pig pancreas under the experimental conditions where they inhibited limit dextrinase. Each inhibitor was heterogeneous and appeared to have «ragged» carboxyl ends. A molecular model of the inhibitor proteins was developed which showed that of the nine cysteine residues in the molecules, eight were likely to be involved in intramolecular disulphide bonds, leaving one free sulphydryl group. The low pI inhibitor had a major component of molecular mass 12 928 that contained a glutathione residue, probably bound to the free sulphydryl group. The high pI inhibitor had two major components with molecular masses of 12 686 and 12 744 and these may contain cysteine residues bound to the free sulphydryl group. Further analysis of the model showed structural differences between the limit dextrinase inhibitors and alpha -amylase-trypsin inhibitors of the same family.

Apparent chemical composition of nine commercial or semi‐commercial whey protein concentrates, isolates and fractions

Summary Analytical results are given for whey powders prepared on a commercial or semi‐commercial scale by three companies. Altogether, five preparations enriched in β‐lactoglobulin, four whey protein isolates and a fraction enriched in α‐lactalbumin were analyzed for protein composition, including %β‐lactoglobulin, α‐lactalbumin, bovine serum albumin, casein (glyco) macropeptide and the main triglycerides. Protein composition was determined by high pressure gel permeation and reversed phase liquid chromatography and by capillary zone electrophoresis. The extent of modification of the native β‐lactoglobulin structure was also measured through the degree of lactosylation and the fraction of accessible free sulphydryl groups. One significant finding was that the calculated recovery of protein following quantitation of the chromatogram or electropherogram was seldom above 90% and occasionally below 60% of that loaded onto the column or capillary, raising doubts as to the reliability of the analytical results. Extrapolation by linear regression to 100% recovery allowed estimates to be made of the true β‐lactoglobulin composition of the samples. The nine samples could be placed into three distinct groups with estimated true β‐lactoglobulin weight % of 70.9 ± 1.1, 62.0 ± 3.4 and 39.5 ± 4.9. Physico‐chemical properties of the group of samples are reported elsewhere (Holt et al., 1999).

Isolation and characterisation of four trypsin-chymotrypsin inhibitors from lentil seeds

Twenty-three proteinase inhibitors were isolated from Syrian local small lentils (Lens culinaris) by ammonium sulphate fractionation of the acidic extract followed by affinity chromatography on anhydrotrypsin-Sepharose. They all inhibited human and bovine trypsin and chymotrypsin. Three inhibitors (LCI-1.7, -3.3 and -4.6) were separated and purified to homogeneity by anion exchange chromatography and preparative isoelectric focusing (IEF) with immobilised pH gradients; a fourth (LCI-2.2) required additional reversed-phase high-pressure liquid chromatography. The four inhibitors were similar in their amino acid composition, with high cystine and aspartic acid/asparagine content, and lack of free sulphydryl groups, methionine and tryptophan. The calculated minimum number of amino acid residues per molecule, the calculated molecular masses confirmed by get liquid chromatography, gel-permeation high-pressure liquid chromatography and sodium-dodecylsulphate polyacrylamide get electrophoresis, and the isoelectric points determined by IEF (immobilised pH gradients and carrier ampholytes) were 84, 77, 68 and 60 residues per molecule, 9200, 8500, 7200 and 6750, and 5.26, 5.88, 6.80 and 7.80 for LCI-1.7, -2.2, -3.3 and -4.6, respectively. All four inhibitors inhibited human trypsin less than bovine trypsin, and human chymotrypsin more than the bovine enzyme. All these properties are in accordance with the classification of the four lentil inhibitors as members of the Bowman-Birk proteinase inhibitor family.

Protein stability function relations: β-lactoglobulin-A sulphydryl group reactivity and its relationship to protein unfolding stability

The effect of protein stability on the reactivity of the free sulphydryl (SH) group in β-lactoglobulin-A ( β-LgA) provides a model for the study of protein stability-function relations (PSFR). The free energy change for protein unfolding (Δ G O) and SH group exposure (Δ G SH) were determined from (i) the urea unfolding curve for β-LgA and (ii) the kinetics of β-LgA SH/disulphide exchange with 2-pyridine disulphide (2-PDS) in 0–8 M urea (pH 3). Protein unfolding profiles determined from extrinsic fluorescence and SH-group reactivity measurements were not coincident. β-LgA formed a stable intermediate ( X) state in the presence of 4 M urea with Δ G O=20 (±0.03) kJ/mol. From the low rate of SH/disulphide exchange in 4 M urea, the SH-group within β-LgA was efficiently masked within the X-state. SH reactivity increased after β-LgA was unfolded in 6–8 M urea with, Δ G SH=43(±6.4) kJ/mol. Such results are discussed in terms of possible interrelationships between protein unfolding stability and SH reactivity in β-LgA.

Primary structure of 2S albumin from seeds of Lupinus cosentinii

The 2S albumin from seeds of Lupinus cosentinii Guss. was purified, and the complete amino acid sequences of the dominating small and large subunit were determined by automated Edman degradation of the reduced and S-pyridylethylated polypeptides and of their enzymatic fragments. The small subunit of the 2S albumin consists of 35 amino acid residues resulting in a molecular mass (M r) of 4233. The large subunit contains 73 amino acid residues (M r = 8627). The two polypeptide chains are linked by two interchain disulphide bonds. In addition, the large polypeptide contains two intrachain disulphide bridges and one free sulphydryl group. A high degree of homology (88–89%) exists between the primary structure of the 2S albumin from L. cosentinii and those from other Lupinus species. The positions of the cysteines and of some other amino acids are conserved not only in most of the Dicotyledoneae 2S albumins sequenced so far but also in other storage proteins.

Evaluation of the mutagenicity of acetochlor to male rat germ cells

Male rat dominant lethal (DL) assays conducted on the herbicide acetochlor are described. Single dose studies conducted at the maximum tolerated dose (MTD, ≤1000 mg/kg) produced no effects on any of the DL assay parameters at any of the ten weekly sampling periods. It is concluded that acetochlor is non-mutagenic to rat germ cells. Due to initial limited knowledge of the MTD of acetochlor it was also evaluated in the DL assay at a dose level of 2000 mg/kg. At this high dose level severe bodyweight loss and some deaths occurred among the treated animals. In addition, reduced implantations and reduced pregnancy rates were observed at the third sampling period (18–25 days post dosing) in the absence of an increase in early post-implantation deaths. These results indicated that the use of supra-MTD doses of acetochlor had reduced the fertility of the treated males leading to the production of a pseudo-DL assay response, as alerted to and defined by Ehling. Although several such pseudo-DL assay responses have been described, none have been explained mechanistically. It was therefore decided to pursue the effects seen in the DL assay when using supra-MTD doses of acetochlor. Ova analysis of female rats mated with male rats exposed to 2000 mg/kg acetochlor revealed unfertilized ova at the critical third sampling time. Normal fertilization of ova was observed at the first and fifth sampling period and, for a dose of 200 mg/kg acetochlor, at the third sampling period. The magnitude and temporal nature of these effects confirmed the induction of a pseudo-DL assay response, and studies were then undertaken to probe its genesis. Rats treated with 2000 mg/kg acetochlor had normal testicular and epididymal pathology and normal sperm numbers and sperm motility at the critical third sampling period. Despite a small reduction in testicular and epididymal glutathione levels 12 h after exposure to 2000 mg/kg acetochlor, testicular LDH and LDH-X enzyme levels were unaffected. Further, no reduction in the level of free sulphydryl groups (-SH) were observed in epididymal caput sperm heads isolated 0.5, 7 or 14 days after treatment of male rats with 2000 mg/kg acetochlor. The only sperm parameter affected by treatment with 2000 mg/kg acetochlor was an increase in epididymal cauda sperm with head abnormalities. The non-specific nature of this effect was considered inadequate to explain fully the high dose fertility effects seen in the DL assays, which therefore remain unexplained. The present data establish that acetochlor is non-mutagenic to rat germ cells. They also confirm the importance of segregating mutagenic and fertility effects in the DL assay, and emphasize the need for appropriate dose-setting studies prior to the conduct of rodent genetic toxicity assays.

Chromatographic elution profiles, electrophoretic properties and free amino and sulphydryl group contents of commercial sodium caseinates

Abstract The proteins in commercial sodium caseinates and laboratory-prepared, unheated sodium caseinate were studied using anion and cation exchange FPLC, gel permeation FPLC and alkaline urea-PAGE, and free amino and sulphydryl groups were analysed. Anion and cation exchange FPLC profiles showed that the charged residues of constituent proteins in the caseinates were modified to different extents. Commercial caseinates showed an extra peak (pre-αs1-casein) on cation exchange FPLC, which eluted at a lower salt concentration than that required to elute αs1-casein; there was little pre-αs1-casein in the laboratory-prepared caseinate. Gel permeation FPLC showed that the caseinates contained different levels of high molecular weight proteins which were present at very low levels in the laboratory-prepared caseinate. Alkaline urea-PAGE gave good resolution of all proteins in the laboratory-prepared caseinate while in the commercial caseinate samples, protein bands were smeared and αs2-casein was less pronounced. The laboratory-prepared caseinate had a higher content of free amino and sulphydryl groups than the commercial caseinates.

Cloning, expression, purification, and characterization of the major core protein (p26) from equine infectious anemia virus

The gene coding for the major core protein (p26) of the lentivirus equine infectious anemia virus (EIAV) was cloned from EIAV infected serum, expressed in E. coli, and the resultant protein purified to electrophoretic homogeneity. The protein was expressed in a soluble form and was purified by conventional protein separation methods. When analyzed by SDS-PAGE, under both reducing and non-reducing conditions, the purified protein migrated as a 26 kDa monomer. Recombinant p26 (rp26), therefore, does not contain any intermolecular disulfide bond. Gel filtration chromatography also indicated that the protein occurs as a monomer in solution. Labeling of free sulphydryl groups with [1- 14C]iodoacetamide suggests that none of the three cysteine residues of rp26 is involved in intramolecular disulfide bonds. The circular dichroism spectrum of rp26 was consistent with the following assignment of secondary structure elements: 51% α-helix, 15% β-turn, and 34% aperiodic. Fluorescence spectroscopy revealed that the three tryptophan residues in rp26 occupy two different environments. These data support the conclusion that the recombinant protein is folded into an ordered and probably native conformation. Immunoblotting and enzyme immunoassay with EIAV infected sera demonstrated that recombinant p26 protein may be useful for diagnostic purposes.

Dopamine and iron induce apoptosis in PC12 cells.

Recent studies have shown that Fe2+ increases the oxidation of monoamines such as serotonin, dopamine and related toxins and that the formed oxidation products can undergo co-valent binding to free sulphydryl groups of proteins such as actin and "serotonin binding proteins" which are present in soluble brain extracts. Here we have tested the ability of ferrous iron to induce [3H]dopamine association to cytoplasmic proteins and we have established that a similar oxidation mechanism evidenced in vitro studies could be applied in cell culture. When PC12 cells were incubated with ferrous iron (ferrocene), the binding of [3H]dopamine to proteins was found to be two fold increased with respect to control. The iron is likely to accelerate the oxidation of dopamine to produce quinones which covalently bind to proteins and induce high-molecular protein aggregates. We evidenced that dopamine/iron combination induced cell death in undifferentiated PC12 cells via an active cellular process evaluated in terms of morphological and biochemical changes indicative of apoptosis. We also demonstrated induction of lipid peroxidation when dopamine and ferrocene were present in high concentrations. Moreover, ascorbic acid diminished apoptosis but not the lipid peroxidation process. It might indicate that ferrocene and dopamine could produce oxidative stress of a different nature. These results show that the actions of dopamine and iron are essential in the induction of apoptosis and lipid peroxidation. However, there is no necessary casual link between lipid peroxidation and apoptosis. Our data also suggest that iron is capable of increasing the cytotoxicity of dopamine merely by increasing its rate of oxidation and without intervention of the monoamine oxidase B enzyme and, hence, both phenomenons may occur independently from each other in rat pheochromocytoma PC12. These observations may have relevance to the understanding of the mechanism by which dopaminergic neurones are destroyed in some neurodegenerative disorders.

Phosphorylation of complement component C3 and C3 fragments by a human platelet protein kinase. Inhibition of factor I-mediated cleavage of C3b.

Phosphorylation of C3 in vitro has been shown previously to lead to significantly altered function of the protein. Platelets are known to contain and release considerable amounts of protein kinases and ATP, which are prerequisites for protein phosphorylation. The aim of the present study was to investigate whether C3 is phosphorylated extracellularly by human platelets. Platelet-rich plasma was stimulated by human aggregated gamma-globulin or ADP. The remaining cells were removed by centrifugation, and the plasma was incubated with [gamma-32P]ATP. After precipitation with Sepharose-bound Abs to C3c followed by SDS-PAGE, it was shown that C3 was phosphorylated in the alpha-chain by a protein kinase dependent on Mn2+, Ca2+, or Mg2+ ions. The supernatant from washed, activated platelets was incubated with purified C3 or soluble or activated thiol Sepharose-bound C3b, together with [gamma-32P]ATP. Phosphorylation was seen in the alpha-chain of C3, and to the same extent in the alpha'-chain of both C3b preparations. The analysis of acid hydrolysate demonstrated that C3 contained 32P-labeled Thr and 32P-labeled Ser. After extensive proteolysis with trypsin, the major phosphorylation site was located to a peptide of 3 to 4 kDa that was bound to the activated thiol Sepharose via the free sulphydryl group in the C3d fragment. Incubation of phosphorylated C3b with factors I and H showed that phosphorylation inhibited the cleavage of the alpha'-chain of C3b. The results in this study suggest that phosphorylation is a regulator of C3 during platelet activation induced, for example, by immune complexes.

A genetic variant of albumin (albumin Asola; Tyr140-->Cys) with no free -SH group but with an additional disulfide bridge.

A slow migrating variant of human serum albumin, present in lower amount than the normal protein, has been detected by routine clinical electrophoresis at pH 8.6 in two members of a family living in Asola (Lombardia, Italy). Ion-exchange chromatography of serum samples failed to separate the normal protein from the variant. Analysis of the albumin peak by SDS/PAGE revealed that the variant had a lower apparent molecular mass than its normal counterpart. However, the abnormal band was not detectable when the separation was performed under reducing conditions or when both albumins were carboxymethylated. Isoelectric-focusing analysis of CNBr fragments localized the mutation to fragment CNBr 3 (residues 124-298). This fragment was isolated on a preparative scale and subjected to tryptic digestion. Sequence determination of the abnormal tryptic peptide revealed that the variant arises from a Tyr140--> Cys substitution. This result was confirmed by DNA sequence analysis, which showed a single transition of TAT-->TGT at nucleotide position 5074. Despite the presence of an additional cysteine residue, several lines of evidence indicated that albumin Asola has no free -SH group; therefore, we propose the formation of a new S-S bond between Cys140 and Cys34, the only free sulphydryl group present in the normal protein. The relatively low level of the variant in serum and its abnormal mobility on cellulose acetate electrophoresis and SDS/PAGE are probably caused by a gross conformational change of the molecule induced by the new S-S bridge.

A Genetic Variant of Albumin (Albumin Asola; Tyrl40Cys) with no Free-SH Group But with an Additional Disulfide Bridge

A slow migrating variant of human serum albumin, present in lower amount than the normal protein, has been detected by routine clinical electrophoresis at pH 8.6 in two members of a family living in Asola (Lombardia, Italy). Ion-exchange chromatography of serum samples failed to separate the normal protein from the variant. Analysis of the albumin peak by SDS/PAGE revealed that the variant had a lower apparent molecular mass than its normal counterpart. However, the abnormal band was not detectable when the separation was performed under reducing conditions or when both albumins were carboxymeth-ylated. Isoelectric-focusing analysis of CNBr fragments localized the mutation to fragment CNBr 3 (residues 124–298). This fragment was isolated on a preparative scale and subjected to tryptic digestion. Sequence determination of the abnormal tryptic peptide revealed that the variant arises from a Tyr140Cys substitution. This result was confirmed by DNA sequence analysis, which showed a single transition of TATTGT at nucleotide position 5074. Despite the presence of an additional cysteine residue, several lines of evidence indicated that albumin Asola has no free-SH group; therefore, we propose the formation of a new S-S bond between Cys140 and Cys34, the only free sulphydryl group present in the normal protein. The relatively low level of the variant in serum and its abnormal mobility on cellulose acetate electrophoresis and SDS/PAGE are probably caused by a gross conformational change of the molecule induced by the new S-S bridge.

A comparative study of the interaction of Myocrisin with albumin and γ-globulin

The reactions of Myocrisin with albumin and γ-globulin have been investigated using atomic absorption spectrometry and gel filtration. Albumin rapidly binds gold up to the levels predicted from the concentration of free sulphydryl groups present in the protein. However, in the presence of glutathione, gold incorporation is increased, suggesting that in vivo, free thiols (glutathione, thiomalate) may facilitate gold uptake by the protein. In comparison, γ-globulin is found to be capable of binding up to one atom of gold per molecule of protein in a slow reaction which requires high Myocrisin concentrations.

Analysis of free reduced and free oxidised glutathione in wheat flour

A rapid and sensitive high-performance liquid chromatography (HPLC) method is described for the determination of reduced and oxidised glutathione (GSH and GSSG, respectively) in wheat flour. The procedure, in which the various steps were optimised, involves extraction with 5% (w/v) perchloric acid (PCA) so as to prevent sulphydryl-disulphide (SH/SS) interchange reactions and to separate PCA-extractable peptides from proteins, the latter being insoluble in PCA. After S-carboxymethylation of free sulphydryl groups by alkylation with iodoacetic acid (IAA) and dinitrophenylation of free amino groups with 1-fluoro-2,4-dinitrobenzene (FDNB), the derivatives are analysed by HPLC on an amino-bonded phase silica column. Recoveries of GSH and GSSG standards added during extraction averaged 98% and 86%, respectively, and detector responses were linear up to 100 nmol GSH/g flour and 50 nmol GSSG/g flour. GSH levels in ‘straight-run’ Brabender Quadrumat Junior-milled white flours from single samples of grain of 11 U.K.-grown wheat cultivars ranged from 18 to 89 nmol/g flour and GSSG levels from 12 to 22 nmol/g flour. The ratio of GSH/GSSG varied between 0·9 and 5·9. No clear relationships were noted for this limited sample set between either the flour GSH or GSSG contents or the GSH/GSSG ratios and the bread making classifications of these cultivars. Storage of white flour samples at 4 °C for 90 days resulted in substantially reduced values for both GSH and GSSG necessitating standardisation of the time between milling and subsequent analysis.

Non‐adrenergic, non‐cholinergic relaxation of the bovine retractor penis muscle: role of S‐nitrosothiols

1. This study examined the possibility that an S-nitrosothiol, rather than nitric oxide, functions as the non-adrenergic, non-cholinergic (NANC) inhibitory neurotransmitter in the bovine retractor penis (BRP) muscle. 2. Treatment of BRP muscle with either of two sulphydryl inactivating agents, diamide (1 mM) and N-ethylmaleimide (0.3 mM), inhibited NANC relaxation and this was prevented by pretreating tissues with L-cysteine (3 mM), L-glutathione (3 mM) or dithiothreitol (3 mM). Inhibition was not specific, however, since the inactivating agents also inhibited the relaxant actions of authentic nitric oxide (0.3 microM), glyceryl trinitrate (0.001-1 microM) and isoprenaline (0.01-1 microM). 3. Reacting nitric oxide with L-cysteine in nominally oxygen-free solution at pH 3, followed by purging to remove free nitric oxide and neutralisation, produced greater and more prolonged relaxant activity when assayed on rabbit aortic rings than could be attributed to nitric oxide alone. H.p.l.c. analysis of the mixture identified a new peak distinct from either L-cysteine or nitric oxide which was responsible for the relaxant activity. The spectral absorption of this new compound had two bands with peaks at 218 and 335 nm. 4. Using a series of structural analogues of L-cysteine (all at 15 mM) it was found that removal of the carboxyl group (L-cysteamine), replacement of the carboxyl with an ester function (L-cysteine methyl ester) or substitution at the amino group (N-acetyl-L-cysteine) had no effect on the ability to generate relaxant activity upon reaction with nitric oxide (0.1 mM). In contrast, substitution at the sulphydryl group (S-methyl-L-cysteine, L-cysteinesulfinic acid and L-cysteic acid), or formation of disulphides(L-cystine and L-cystamine) led to a complete loss of ability to generate relaxant activity. L-Glutathione was also able to react with nitric oxide to produce relaxant activity, and this too was blocked upon substitution of the free sulphydryl group (S-methyl-L-glutathione). A free sulphydryl group was therefore required to generate relaxant activity following reaction with nitric oxide.5. Reacting L-cysteine (10 mM) with nitric oxide (~ 3 mM) under more stringent oxygen-free conditions followed by purging to remove free nitric oxide resulted in the generation of low relaxant activity and small absorption peaks at 218 and 335 nm and these were unaffected upon exposure to the air. In contrast, admitting air to the reaction chamber before purging enhanced both relaxant activity and the absorption peaks at 218 and 335 nm by some 40 fold and the solution turned pink due to the appearance of another absorption peak at 543 nm. This enhanced relaxant activity was not due to nitrogen dioxide being the reactive species, since at 0.1 mM this gas failed to react with L-cysteine to generate relaxant activity, and at 1 mM generated less activity than the equivalent concentration of nitricoxide.6. The relaxant activity generated by reacting nitric oxide with L-cysteine or L-glutathione was abolished following treatment with haemoglobin (3 MicroM), methylene blue (10 MicroM) or Nmethylhydroxylamine(100 MicroM), but was unaffected by N0-nitro-L-arginine (30 MicroM). Furthermore, two agents that generate superoxide anion, pyrogallol (0.1 mM) and hydroquinone (0.1 mM), also inhibited this relaxant activity as well as that induced by authentic nitric oxide (0.3 MicroM) but as previously reported, had no effect on relaxation induced by NANC nerve stimulation. Superoxide dismutase(100 u ml1) reversed the actions of pyrogallol and hydroquinone but had no effect on NANC relaxation.7. In conclusion, the reaction of nitric oxide with L-cysteine or L-glutathione generates relaxant activity which exceeds that of nitric oxide alone and probably results from formation of S-nitrosocysteine and S-nitrosoglutathione, respectively. The effects of pyrogallol and hydroquinone suggest that the NANCneurotransmitter is a superoxide anion-resistant, nitric oxide-releasing molecule and that neither S-nitrocysteine nor S-nitrosoglutathione is a suitable candidate for this.

Effects on Gluten of Heating at Different Moisture Contents. II. Changes in Physico-Chemical Properties and Secondary Structure

When gluten is heated at 80°C for 30 min, dramatic changes are observed in its physico-chemical properties if moisture content exceeds 20%. To understand these changes further we characterised the extractability, sulphydryl/disulphide content, conformation and hydrophobicity of the proteins extensively. Gel-filtration chromatography (GFC) on Sepharose C1-6B column of a 1·5% (w/v) sodium dodecyl sulphate (SDS) extract of heated gluten resulted in a void volume peak consisting of glutenin aggregates with a relative molecular mass (Mr) greater than 1 × 106. This peak was separated further on a Biogel A-150m column into three fractions. The amount of the glutenin fraction with the greatest Mr (Mr > 1 × 108) extracted from fully-denatured gluten was only 5% of that from the unheated sample. Meanwhile, the amounts of free sulphydryl groups were decreased, indicating an increase in disulphide bonds. Labelling studies with a fluorescent label specific for sulphydryl groups in large subunits (Mr > 4 × 104) were decreased, but not in the subunits around Mr 2 × 104. Heating gluten at a moisture content above 20% also affected the conformation of the glutenin, but not that of the gliadin, albumins and globulins isolated by GFC, as indicated by circular dichroism measurements of proteins extracted in 1·5% (w/v) SDS. The association constant and the number of binding sites of the hydrophobic ligand 8-anilino-naphthalene sulphonic acid decreased when gluten with a moisture content of 13% or higher was heated. The decrease in hydrophobicity may be explained by hypothesising that, upon heating, hydrophobic groups are exposed to such an extent that the protein aggregates. After aggregation the hydrophobicity is decreased. The results indicate that, after heating gluten at different moisture contents, the first major change to be observed at moisture contents below 20% is a decrease in extractability of large glutenin aggregates and in hydrophobicity of the acetic acid unextractable protein. At higher moisture contents, also, disulphide bonding and irreversible conformational changes can be observed.

The polymerization of fibrinogen Dusart (Aα 554 Arg→Cys) after removal of carboxy terminal regions of the Aα-chains

The six polypeptide chains of normal fibrinogen are covalently linked by interchain disulphide bonds, and there are no free sulphydryl groups. Fibrinogen Dusart is a congenital fibrinogen variant in which A alpha 554 Arg is replaced by Cys; albumin is disulphide linked to these fibrinogen molecules, possibly at A alpha 554 Cys. Functionally, Dusart fibrinogen displays markedly abnormal fibrin polymerization, characterized by delayed lateral fibril association and matrix fibre bundles that are thinner than normal fibrin bundles. These observations are consistent with experiments suggesting that the carboxy terminal region of the A alpha-chain contains a polymerization domain that participates in lateral fibril associations. In order to investigate the location and the effect of albumin binding to Dusart fibrinogen, we examined the fibrinogen by electron microscopy, and compared the polymerization and ultrastructure of fibrin prepared from normal fibrinogen containing intact A alpha-chains (fraction I-2) or plasmin degraded fibrinogen molecules lacking carboxy terminal regions of A alpha-chains (fraction I-9D), with fibrin prepared from Dusart fraction I-2 and I-9D. Most bound albumin was released from Dusart fibrinogen by plasmin degradation involving the A alpha-chains. Nevertheless, we were able to visualize albumin molecules remaining covalently bound to Dusart I-9D as well as to Dusart I-2 fibrinogen, as distinct globular domains situated near the fibrinogen D domain. The presence of albumin in these fractions was confirmed by Western blotting using anti-albumin. Dusart fibrin polymerized much more slowly than normal I-2, as previously reported, whereas polymerization of Dusart I-9D fibrin was faster than Dusart I-2 and nearly the same as normal I-9D fibrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of other whey proteins on the heat-induced aggregation of α-lactalbumin

Abstract The thermally induced aggregation of α-lactalbumin in solution and in the presence of serum protein-free casein micelles has been studied using gel permeation chromatography. No aggregation of α-lactalbumin was detected after heating at 90°C for 24 min. However, addition of β-lactoglobulin or serum albumin to the serum protein-free casein micelles + α-lactalbumin system caused aggregation of the α-lactalbumin, the rate and extent of this aggregation being dependent upon the concentration of free sulphydryl groups present in the other whey protein. It is therefore the sulphydryl group which is important and which appears to function by inducing cleavage of intramolecular disulphide bonds in the α-lactalbumin. This leads to the formation of intermolecular disulphide bridges and hence to aggregation of the α-lactalbumin.

Measurement of total plasma and cerebrospinal fluid homocysteine by fluorescence following high-performance liquid chromatography and precolumn derivatization with o-phthaldialdehyde

Precolumn derivatization of amino acids with o-phthaldialdehyde followed by high-performance liquid chromatographic separation and fluorescence detection is used in many clinical and experimental laboratories for the measurement of primary amino acids. This technique was adapted for the measurement of homocysteine in plasma and cerebrospinal fluid (CSF) following alkylation of the free sulphydryl group with iodoacetate. The minimum detection limits are less than 1 μ M in plasma and 80 n M in CSF. Within-day and between-day coefficients of variation for plasma and for CSF are less than 10%. Values for normal plasma homocysteine range from 6.04 to 16.2 μ M and for CSF from 0.28 to 0.66 μ M.