On the heterogeneity of serum albumin: effect of sodium dodecyl sulphate on agarose chromatography of the protein

The chromatographic behaviour of serum albumin changes if it is dissolved in an aqueous solution of sodium dodecyl sulphate (SDS) and is left at room temperature or is heated. Instead of emerging as one peak from an agarose column it emerges as more than one peak and the protein in each when rechromatographed elutes in more than one peak. Electropherograms made in the presence of SDS of the protein in these peaks show multiple banding from proteins migrating with a wide range of molecular weights. The chromatographic and electrophoretic behaviour of serum albumin in the presence of SDS suggests that the protein is not a simple monomer and alternative explanations for this behaviour are discussed.     

Highly purified agarose as stacking gel in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.

A modified sodium dodecyl sulphage/polyacrylamide-gel-electrophoretic method is described that utilizes highly purified agarose as stacking gel. The same electrophoretic resolution of different marker proteins is found as when polyacrylamide is used as stacking gel, but the background staining seen when polyacrylamide is used as stacking gel is decreased.     

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.     

Determination of subunit molecular weights of canine renal (Na+,K+)-ATPase by low-angle laser light scattering coupled with high performance gel chromatography in the presence of sodium dodecyl sulfate

Subunit molecular weights of the (Na+,K+)-ATPase of canine renal outer medulla were estimated in the presence of sodium dodecyl sulfate (SDS) by the measuring system consisted of components connected in the following sequence: a TSK-gel G3000 SW column, a UV spectrophotometer, a low-angle laser light scattering photometer, and a differential refractometer. Polypeptide molecular weights of the alpha- and beta-subunits were determined to be 117,900 and 39,400, respectively. The measurement required the extinction coefficient at 280 nm of the sample polypeptide in addition to the outputs from the three detectors. The extinction coefficients at 280 nm of the alpha- and beta-subunits were determined to be 0.931 and 1.41 ml X mg-1 X cm-1, respectively by the quantitative amino acid analysis. The above procedure seems to be most appropriate to determine uniquely the composition of subunits molecular weights of an oligomeric membrane protein.     

Dissociation and denaturation behaviour of sesame alpha-globulin in sodium dodecyl sulphate solution.

The effect of anionic detergent, sodium dodecyl sulphate, on the major protein, alpha-globulin of sesame seed (Sesamum indicum L.) has been investigated by gel filtration, sedimentation velocity, viscosity, optical rotation, difference spectra and fluorescence measurements. The detergent causes dissociation of the protein first and then denaturation. In the detergent concentration range of .175-4.0 X 10(-"3) M four components are observed in the ultracentrifuge. The specific rotation of the protein increases with the detergent concentration above 2.5 x 10 (-3) M detergent suggesting conformational change; above 8 X 10(-"3) M detergent the value of -[alpha] does not change. The reduced viscosity etared however, increases above .25 X 10(-3) M detergent and does not attain a plateau value. The difference spectrum of the protein indicates that both tryptophan and tyrosine groups have been affected by the detergent. The fluorescence intensity decreases and the maxima shifts towards red in the detergent solution resulting in an "isoemissive point" at 355 nm. The double difference spectra in sucrose-detergent protein system show that below 5-0 X 10(-3) M detergent, the difference absorption and fluorescence spectrum result from the binding of the detergent near the chromophoric groups and are not due to conformational change. Binding studies by equilibrium dialysis indicate the presence of 50 binding sites in the protein and binding constant of 3-0 X 10(3).     

New method for analyzing the molecular weights of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Previously the method for determining protein molecular weights from SDS-PAGE depended on the accidental, only partial linearity of protein movement with the logarithm of its molecular weight. A new, mathematically rigorous method with supporting data is now described demonstrating that such movement is dependent upon the reciprocal of protein size. Experimental data, therefore, follow most closely a hyperbolic curve when plotted directly; it becomes linear and passes through the origin when movement is plotted vs the reciprocal of protein molecular weight. In the earlier method determination of the error of a measurement of molecular weight is very complex and never determined. In the method presented here such error is easily estimated and it is identical in both the hyperbolic and linear forms of data presentation. This method may eventually also allow other less-significant forces controlling movement such as protein charge to be analyzed and understood.     

Dynamics of molecular organization in agarose sulphate

Nongelling solutions of structurally regular chain segments of agarose sulphate show disorder–order and order–disorder transitions (as monitored by the temperature dependence of optical rotation) that are closely similar to the conformational changes that accompany the sol–gel and gel–sol transitions of the unsegmented polymer. The transition midpoint temperature (T_m) for formation of the ordered structure on cooling is ～25 K lower than T_m for melting. Salt-induced conformational ordering, monitored by polarimetric stopped-flow, occurs on a millisecond time scale, and follows the dynamics expected for the process 2 coil ? helix. The equilibrium constant for helix growth (s) was calculated as a function of temperature from the calorimetric enthalpy change for helix formation (ΔH_cal = ?3.0 ± 0.3 kJ per mole of disaccharide pairs in the ordered state), measured by differential scanning calorimetry. The temperature dependence of the nucleation rate constant (k_nuc), calculated from the observed second-order rate constant (k_obs) by the relationship k_obs = k_nuc(1 ? 1/s) gave the following activation parameters for nucleation of the ordered structure of agarose sulphate (1 mg mL^?1; 0.5M Me₄NCl or KCl): ΔH* = 112 ± 5 kJ mol^?1; ΔS* = 262 ± 20 J mol^?1 K^?1; ΔG*₂₉₈ = 34 ± 6 kJ mol^?1; (k_nuc)₂₉₈ = (7.5 ± 0.5) × 10⁶ dm³ mol^?1 s^?1. The endpoint of the fast relaxation process corresponds to the metastable optical rotation values observed on cooling from the fully disordered form. Subsequent slow relaxation to the true equilibrium values (i.e., coincident with those observed on heating from the fully ordered state) was monitored by conventional optical rotation measurements over several weeks and follows second-order kinetics, with rate constants of (2.25 ± 0.07) × 10^?4 and (3.10 ± 0.10) × 10^?4 dm³ mol^?1 s^?1 at 293.7 and 296.2 K, respectively. This relaxation is attributed to the sequential aggregation processes helix + helix → dimer, helix + dimer → trimer, etc., with depletion of isolated helix driving the much faster coil–helix equilibrium to completion. Light-scattering measurements above and below the temperature range of the conformational transitions indicate an average aggregate size of 2–3 helices.     

Effect of sodium dodecyl sulphate on the high molecular weight protein fraction of mustard (Brassica juncea) and rapeseed (Brassica campestris)

The effect of sodium dodecyl sulphate on mustard and rapeseed 12S protein has been monitored by the techniques of ultracentrifugation, viscosity, difference spectra and fluorescence spectrophotometry. At low concentration of sodium dodecyl sulphate (<3.47 mM) mustard protein undergoes aggregation and at higher concentrations it dissociates to 1.8 S protein, the dissociation being complete at 17.3 mM sodium dodecyl sulphate. The rapeseed protein, on the other hand, undergoes dissociation at all the concentrations of sodium dodecyl sulphate. The reduced viscosity values of mustard protein in the presence of the denaturant are higher than those of rapeseed protein. Similarly in difference spectra change in absorbance values of mustard protein are higher.’ The relative fluorescence intensity of the mustard protein increases with sodium dodecyl sulphate concentration, upto 0.87 mM and this is followed by fluorescence quneching at higher denaturant concentrations. However, with the rapeseed protein fluorescence quenching was observed at all concentrations of sodium dodecyl sulphate.     

Estimation of interaction between oriented immobilized green fluorescent protein and its antibody by high performance affinity chromatography and molecular docking

Although green fluorescence protein (GFP) and its antibody are widely used to track a protein or a cell in life sciences, the binding behavior between them remains unclear. In this work, diazo coupling method that synthesized a new stationary GFP was oriented immobilized on the surface of macro‐porous silica gel by a phase. The stationary phase was utilized to confirm the validation of injection amount‐dependent analysis in exploring protein–protein interaction that use GFP antibody as a probe. GFP antibody was proved to have one type of binding site on immobilized GFP. The number of binding site and association constant were calculated to be (6.41 ± 0.76) × 10^‐10 M and (1.39 ± 0.12) × 10⁹ M^‐1. Further analysis by molecular docking showed that the binding of GFP to its antibody is mainly driven by hydrogen bonds and salt bridges. These results indicated that injection amount‐dependent analysis is capable of exploring the protein–protein interactions with the advantages of ligand and time saving. It is a valuable methodology for the ligands, which are expensive or difficult to obtain. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

The binding of sodium dodecyl sulphate to bovine serum albumin at high binding ratios

The extent of binding of sodium dodecyl sulphate to bovine serum albumin at high binding ratios was investigated by gel filtration. The weight ratio of bound sodium dodecyl sulphate to bovine serum albumin increases with the NaCl concentration, and, except at low salt concentrations, with the concentration of sodium dodecyl sulphate. In the presence of 1.0g of sodium dodecyl sulphate/l, the binding ratio varied from 1.0 (at 0.04m-Na(+)) to 2.2 (at 0.44m-Na(+)). In the presence of 0.24m-Na(+), the binding ratio increased with sodium dodecyl sulphate concentration, from 0.9 (0.2g of sodium dodecyl sulphate/l) to 2.0 (5g of sodium dodecyl sulphate/l), at 26 degrees C, in a dilute sodium phosphate buffer. No significant dependence of the binding ratio upon temperature in the range 26-45 degrees C was observed. These results differ from those of Reynolds & Tanford (1970a) obtained by equilibrium dialysis.     

Separation of peptides dissolved in a sodium dodecyl sulfate solution by reversed-phase liquid chromatography: removal of sodium dodecyl sulfate from peptides using an ion-exchange precolumn

Separation of peptides by reversed-phase liquid chromatography is significantly affected by sodium dodecyl sulfate (SDS) in the sample solution. The strongly acidic group of SDS binds to the reversed-phase column where it serves as an ion exchanger and retards the elution of peptides. By using a DEAE precolumn connected in series to a reversed-phase column, the interference of SDS in the separation of peptides by reversed-phase chromatography can be significantly diminished. This simple method is applicable to the separation of peptide mixtures obtained by digestion of proteins extracted from SDS-polyacrylamide gels. Peptide production with some proteases in the presence of SDS was examined using the present method. Lysylendopeptidase was suitable for digestion in the presence of SDS, but V8 protease was not.     

Interaction of the myelin basic protein with the anionic detergent sodium dodecyl sulphate.

The interaction of the myelin basic protein and two peptides derived from it with the anionic detergent SDS (sodium dodecyl sulphate) was studied. At molar ratios of detergent/protein of up to approx. 20:1 the transient increase in turbidity (as measured by increases in A230) is proportional to the ratio. Between ratios of 30:1 and 100:1 the effect of the detergent is constant and maximal. At molar ratios exceeding 100:1 the transient increase in turbidity decreases with increasing amounts of detergent. With increasing ionic strength the rapid development of turbidity is inhibited, whereas the slow decay of turbidity is not affected. Neither of the peptide fragments produced by cleavage of the myelin basic protein at the single tryptophan residue, nor both when mixed, produce measurable turbidity when mixed with SDS. Under similar conditions poly-L-lysine of similar molecular size to the basic protein shows the increase in turbidity but not the decay. The interaction between the protein and SDS is interpreted in molecular terms, which involve the initial ionic interaction of the detergent with protein resulting in aggregation and turbidity in the solution. Within the aggregated complexes molecules rearrange to maximize hydrophobic interactions.