The binding of sodium dodecyl sulphate to various proteins

1. The binding of sodium dodecyl sulphate to proteins by equilibrium dialysis was investigated. 2. Most of the proteins studied bound 90-100% of their weight of sodium dodecyl sulphate. 3. The glycoproteins studied bound 70-100% of their weight of sodium dodecyl sulphate, calculated in terms of the polypeptide moiety of the molecule. 4. Proteins not containing S.S groups bound about 140% of their weight of sodium dodecyl sulphate. 5. Reduction of four proteins containing S.S groups caused a rise in sodium dodecyl sulphate binding to 140% of the weight of protein. 6. The apparent micellar molecular weights of the protein-sodium dodecyl sulphate complexes were measured by the dye-solubilization method; they were all found to have approximately the same micellar molecular weight (34000-41000) irrespective of the molecular weight of the protein to which they were attached.     

Pressure stability of proteins at their isoelectric points

Yeast alcohol dehydrogenase is slowly denatured at moderate hydrostatic pressure (t(1/2) approximately equals 30 min at 2 kbar and pH 7). The extent of denaturation is pH dependent with maximal stability near the isoelectric point of the protein, pH = 5.4. While not a surprising finding, it appears that this phenomenon has not been documented before (or at least not identified) despite many investigations into the pressure stability of proteins. Consideration of changes in the net charge of proteins far from their isoelectric points may explain other pressure effects as well.     

Two-dimensional separation of chloroplast membrane proteins by isoelectri focusing and electrophoresis in sodium dodecyl sulphate

Proteins of chloroplast subfragments enriched in Photosystem I and Photosystem II electron flow activity have been analyzed by two-dimensional polyacrylamide gel electrophoresis. In the first dimension, polyacrylamide gel isoelectric focusing (pH 5–7) was used in the presence of Triton X-100, followed at right angle by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Characteristic fingerprints were obtained for the Photosystem I and II fractions and a correlation between the major proteins separated by isoelectric focusing and the major polypeptides separated by undimensional SDS electrophoresis was established. Two dominant spots of 68 000 and 60 000 daltons appeared in the two-dimensional patterns of Photosystem I fractions $\text{p}\text{I}$ values about 5.6; two spots with molecular weights of 33 000 and 23 000 were characteristics for Photosystem II fractions $\text{p}\text{I}$ values about 5.3 and 6.3). Photosystem I fractions were furthermore characteristics by a series of spots in the 44 000–33 000 range $\text{p}\text{I}$ values from about 5.9 to 6.8). The two-dimensional system revealed that (a) several SDS-polypeptides have multiple forms differing in charge only, (b) some proteins separated by isoelectric focusing are resolved in the second dimensional into polypeptides of different size. The two-dimensional method combining Triton X-100 isoelectric focusing' and SDS electrophoresis provides a higher degree of resolution than either of the unidimensional methods thus allowing a detailed analysis of chloroplast membrane proteins.     

Mobility of sodium dodecyl sulphate - protein complexes.

Reduced and unreduced lysozyme aggregates formed by formaldehyde cross-linking comprise a set of model compounds for studying the effects of protein conformation on the electrophoretic mobilities of sodium dodecyl sulphate-protein complexes. The reduced aggregates were indistinguisable from normal proteins, but the unreduced aggregates migrated anomalously fast by about 14%. Contrary to expectations, plots of logarithm Rf versus Kr (retardation coefficient) failed to reveal an unusual conformation for the unreduced aggregates. Thus the anomalous mobility caused by several intramolecular disulphide bonds escaped detection by the above two diagnostic plots. Also included in this paper is a discussion of the implications of these results with regard to current models for sodium dodecyl sulphate-protein complexes.     

Sodium dodecyl sulphate electrophoresis of urinary proteins

The analysis of urinary proteins and their identification are discussed, particularly in regard to the technique of sodium dodecyl sulphate electrophoresis in polyacrylamide gradient gels. Urine collection, storage and preparation are evaluated, especially in regard to problems connected with concentration and dialysis of such samples. The instrumental approach to sodium dodecyl sulphate polyacrylamide gel electrophoresis represented by the Phast System appears to be particularly valuable in routine clinical analysis of urine specimens, since no sample pretreatment is required. The following types of proteinurias are evaluated: (a) orthostatic proteinurias; (b) post-renal proteinurias; (c) Bence—Jones proteinuria; (d) lower and upper urinary tract infection (cystitis and pyelonephritis) and (e) diabetes mellitus proteinurias.     

Cyanogen bromide cleavage of proteins in sodium dodecyl sulphate/polyacrylamide gels. Diagonal peptide mapping of proteins from epidermis.

A two-dimensional electrophoretic procedure employing CNBr has been devised for the analysis of proteins in sodium dodecyl sulphate/polyacrylamide gels. The technique allows the detection of an unusual class of epidermal proteins that lack methionine. The proteins have been identified by this method in newborn mouse, rat, and rabbit, because they are stable in the presence of CNBr and consequently lie on a diagonal. Adult human epidermis also contains CNBr-stable proteins, but in lesser amounts than in the newborn rabbit or newborn rodents. The methionine-containing proteins (i.e., the keratins) are degraded by CNBr into a series of unique and characteristics peptides which lie below the diagonal. Inter- and intra-species similarities and differences exist between the individual keratins, depending on the number and distribution of their methionine residues. The peptide-map patterns for the rodent and lagomorph proteins are more similar to each other than to that for the human proteins. The maps for rat and rabbit skin proteins are the most similar. We conclude that the epidermal keratins are a closely related, yet individually distinct, group of proteins that are found in conjunction with a class of proteins that lack methionine. The latter proteins are related to the histidine-rich basic protein, an epidermal structural protein that aggregates with keratin filaments.     

Interaction of sodium dodecyl sulphate with elastin polypeptides

The interaction that occurs between polypeptidic fragments of elastin and sodium dodecyl sulphate (SDS) as a model of natural amphiphilic substances has been studied by means of thermal concerration of the elastin fragments in the presence of detergents, by solubilization of a lipophilic dye, and by means of gel permeation chromatography. It was found that elastin polypeptides interact with SDS giving mixed micelles. This finding seems to be especially relevant in the tissues, revealing enhanced degradation of elastin and accumulation of lipophilic substances (e.g. in atheromatous plaques). In such tissues, elastin polypeptides formed could interfere with the formation of the normal elastic fibre by means of their interaction with amphiphilic substances.     

Utilization of sodium dodecyl sulphate by denitrifying bacteria under anaerobic conditions

Abstract Analysis of the exopolysaccharides from Rhizobium japonicum USDA191 showed substantial amounts of mannose (22.5%) and uronic acids (13.4%). These sugars are normally absent in the fast growers like Rhizobium meliloti . In addition USDA191 contained pyruvate (5.1%), which is normally absent in slow-growing strains of Rhizobium japonicum . The heterogeneity in the exopolysaccharide composition of strain 191 indicates a closer similarity with the slow-growing Rhizobium japonicum . SDS-urea polyacrylamide gel analysis of the lipopolysaccharides also reflects this heterogeneity.     

Serial analysis of zein by isoelectric focusing and sodium dodecyl sulfate gel electrophoresis

Zein, the major storage protein of maize (Zea mays L.) endosperm, was extracted from a number of inbreds with alcohol plus a reducing agent. Isoelectric focusing (IEF) separated total zeins into 41 components, while sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separated total zeins into about 15 components. Each procedure gave characteristic patterns of zein bands for a number of maize inbreds. IEF and SDS-PAGE were used serially so that each band separated by IEF could be assayed as an individual SDS-PAGE sample. Some IEF bands revealed only a single band after SDS-PAGE, while others revealed two or more bands. A nomenclature system is presented which integrates the two separation systems with information about chromosome locations of zein genes, maize mutations which affect zein synthesis, and inbred sources for different zeins. SDS-PAGE of zein gives apparent molecular masses which vary widely according to the standards used and the properties of the gels, therefore an artificial nomenclature for identifying zein bands after SDS-PAGE is presented. The new nomenclature provides a flexible system which is useful and can be conveniently used in different laboratories.     

Removal of sodium dodecyl sulfate from proteins.

A convenient and relatively simple electrodialysis method for the removal of sodium dodecyl sulfate (SDS) from proteins is described. Six samples can be processed simultaneously. The kinetics of removal of SDS from proteins by equilibrium dialysis and electrodialysis have been studied.     

Two-dimensional isoelectric focussing/sodium dodecyl sulphate polyacrylamide gel electrophoretic mapping and some molecular characteristics of the proteins of the adult guinea-pig small intestinal microvillus membrane

The adult guinea-pig small intestinal microvillus membrane was purified approximately 25-fold by both cation-precipitation and differential centrifugation methods. Comparison by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed no substantial differences in polypeptide composition between the two preparations. One-dimensional SDS-PAGE and two-dimensional isoelectric focussing (IEF)/SDS-PAGE, together with Coomassie-blue, silver and lectin-staining, showed three major high molecular weight polypeptides, Mr 108 000, 116 000 and 127 000, as well as a 47 kDa protein (actin), as major constituents of the membrane. The proteins of Mr 108 000 and 116 000 were strongly concanavalin A reactive. A detailed two-dimensional IEF/SDS-PAGE map of the membrane was constructed. Sodium carbonate treatment showed the two concanavalin A-reactive glycoproteins, Mr 108 000 and 116 000, comprising the sucrase-isomaltase complex, to be loosely-associated 'extrinsic' microvillus membrane proteins. Two proteins, Mr 127 000 and 135 000, were tightly-associated 'intrinsic' microvillus proteins. Despite regional differences in specific activity of some small intestinal microvillar enzymes, most noticeably enterokinase (EC 3.4.21.9) and dipeptidyl peptidase IV (EC 3.4.14.x), no substantial regional differences were seen in microvillus membrane polypeptide composition. In contrast, a substantial increase in the major high molecular weight proteins of Mr 108 000 and 116 000 accompanied a 10-fold rise in sucrase-isomaltase activity, and loss of a major protein of Mr 131 000 accompanied the complete loss of lactase activity from the membrane during postnatal development.     

Collagen fibril formation in the presence of sodium dodecyl sulphate.

Sodium dodecyl sulphate (SDS) was used to weaken both the electrostatic and the hydrophobic interactions during collagen fibrillogenesis in vitro. The rate and extent of fibril formation as well as fibril morphology were affected by SDS concentration. Both the formation of large fibrils at 0.3 mM-SDS and the complete cessation of fibril formation at 0.5 mM-SDS were considered to be the result of SDS-induced conformational changes in the non-helical telopeptides. A possible mechanism of SDS interaction with the N-terminal and the distal region of the C-terminal telopeptides is offered.     

Solubilization of isolated central-nervous-system myelin preparations by the amniotic detergent sodium dodecyl sulphate.

The mechanism for the solubilization of isolated central-nervous-system myelin by sodium dodecyl sulphate was studied in detail. The release of protein and phospholipid to the 100000 g x 1 h supernatant fraction is dependent on the total amount of detergent relative to the amount of membrane present and on the ionic strength of the solubilization system. Gel-filtration analysis of supernatant fractions indicate that at suboptimal concentrations of detergent these contain lipid-protein complexes. The complete dissociation of the individual protein components from lipid is dependent on the total amount of sodium dodecyl sulphate present in the system. The results indicate that for the analysis of membrane components in sodium dodecyl sulphate it is essential that sufficient detergent is present.     

Anomalous migration of leghaemoglobin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.

The estimate of the molecular weight of leghaemoglobin by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis is about 20% too low. This is due to an anomalously high limiting relative mobility. Leghaemoglobin binds 1.4 g of sodium dodecyl sulphate/g of protein with a concomitant decrease in the helical content from 71-72% to 49-51%.