Identification of denatured enzyme proteins in sodium dodecyl sulfate polyacrylamide gels

A simple modification of the immunological sandwich method of Muilerman et al. for the identification of denatured enzyme proteins in sodium dodecyl sulfate-polyacrylamide gels is described, enabling the method to be used in principle for any enzyme whose activity is not inhibited by binding to antibodies. An immunological sandwich consisting of denatured enzyme, antibodies, and native enzyme is formed on a nitrocellulose filter blot of the gel, the filter is divided into strips, and each strip is tested for enzyme activity. The presence of enzyme activity serves to identify the region in the gel containing denatured enzyme protein. Experiments with human lysosomal alpha-glucosidase as a model system are described. The method was applied to identify a protein of Mr 125,000 as the main component with UDPgalactose pyrophosphatase activity in a partially purified preparation of the enzyme from rat liver.     

Method for rapid removal of sodium dodecyl sulfate from polyacrylamide gels

A method is described for the isolation of hepatic microsomes by polyethylene glycol 6000 fractionation of the postmitochondrial fraction of liver homogenate. The procedure is simple and rapid requiring two centrifugation steps at 8000g for 10 min. The preparation has peptide patterns and levels of drug metabolic and other enzymatic activity similar to those of the microsomal fraction isolated by high-speed centrifugation and is referred to as polyethylene glycol 6000 microsomes. It is clarified with detergents and can serve as the starting material for the purification of microsomal proteins.     

Chromatographic recovery of polypeptides from copper-stained sodium dodecyl sulfate polyacrylamide gels.

A procedure of preparative electrophoresis is described in which proteins separated on sodium dodecyl sulfate gels, stained with copper and eluted by simple diffusion, are highly concentrated on a fluorocarbon packing and freed of small molecular weight substances, including sodium dodecyl sulfate and buffer components and gel-related substances. This method can be used for microscale preparations or it can be scaled up to recover milligram amounts of protein. The purified polypeptides, however denatured, are suitable for amino acid sequencing.     

Electrophoresis of proteins and DNA on horizontal sodium dodecyl sulfate polyacrylamide gels

An inexpensive Plexiglas apparatus which allows a simple and rapid preparation of horizontal polyacrylamide gels of different dimensions for different purposes, is described. Preparation of such gels is as easy and rapid as agarose gel preparation, and polymerized polyacrylamide gels are used to fractionate proteins or small DNA fragments using a common horizontal electrophoretic tank. This apparatus was used to electrophoretically fractionate proteins or DNA for immuno-blot analyses, particularirly in the study of the allergenic response to Parietaria judaica pollen in senescence, for Southern-blot hybridizations and in the study of DNA polymorphisms.     

Localization of cellular antigens in sodium dodecyl sulfate- polyacrylamide gels

A procedure is described for localizing antigen-antibody complexes in sodium dodecyl sulfate (SDS) polyacrylamide gels using 125I-labeled protein A from Staphylococcus aureus. We use the procedure to probe antigenic cross-reactivities between Strongylocentrotus and Chlamydomonas alpha- and beta-tubulins; we also domonstrate how the procedure can detect minor antibody species in an antiserum directed against a cell membrane.     

Thiol-dependent proteases of African trypanosomes. Analysis by electrophoresis in sodium dodecyl sulphate/polyacrylamide gels co-polymerized with fibrinogen

The proteases of several species of African trypanosomes were analysed by electrophoresis in sodium dodecyl sulphate/polyacrylamide gels containing fibrinogen or collagen. After electrophoresis the gels were incubated in the presence of enzyme activators and/or inhibitors and then stained with Coomassie brilliant blue. The areas where the proteolytic activity had degraded the fibrinogen did not stain and so formed clear bands against a blue background. The proteases were found to have pH optima between 5 and 6, and required dithiothreitol or 2-mercaptoethanol for full expression of their activity. They were inhibited by amino acid chloromethanes, iodoacetamide, p-chloromercuribenzoate and other inhibitors of the thiol-dependent proteases, as well as by the trypanocidal drugs berenil (4,4'-diamidinodiazoaminobenzene-diacetamidoacetate) and pentamidine [1,5-di-(4-amidinophenoxy)pentane-di-(2- hydroxyethanesulphonate)]. Trypanosoma evansi, Trypanosoma brucei brucei and Trypanosoma brucei gambiense each have a protease with a relative molecular mass, Mr, of 28 000. In addition they occasionally exhibit activity at higher Mr values (up to 105000). Trypanosoma congolense has a low-Mr protease (31 000) and may exhibit higher-Mr proteases (up to 150000). The protease profiles of Trypanosoma vivax differ from the other species, T. brucei or T. congolense, and are present in lesser amounts. The proteases of the cultured procyclic forms are present in much smaller amounts than those of the metacyclic or mammalian blood stream forms of these parasites. The catalytic properties and inhibition characteristics of these thiol-dependent enzymes suggest that they resemble the mammalian lysosomal cathepsins B and L.     

Electrophoretic behavior of protein dodecyl sulfate complexes in the presence of various amounts of sodium dodecyl sulfate.

This report describes the relationship between the amount of sodium dodecyl sulfate present in a sample solution and the electrophoretic mobility of the protein-dodecyl sulfate complexes. In order to determine the extent of any conformational changes in the proteins and to establish a correlation between any of these structural changes and the electrophoretic behavior, visible absorption spectra and circular dichroism spectra were obtained for heme proteins in the presence of the same amounts of surfactants as used in electrophoresis.From the results obtained, it is apparent that the amount of sodium dodecyl sulfate present in the sample solution must be taken into consideration when performing a separation. Optimum experimental conditions are chosen for attaining enhanced separation and a maximized linear range of molecular weights of proteins that can be accurately determined.     

Reverse staining of sodium dodecyl sulfate polyacrylamide gels by imidazole-zinc salts: sensitive detection of unmodified proteins.

We report here a modification to copper and zinc chloride staining methods. The introduction of a preincubation of the gels, prior to metal staining, with 0.2 M imidazole allows the formation of a homogeneous background for the subsequent precipitation of the metal chelate. The reported imidazole-zinc staining takes minutes, resulting in reproducible staining patterns with only slightly lower sensitivity than silver staining. The method allows efficient recovery of proteins from previously stained gels and is compatible with immunoidentification on Western blots and also with amino acid analysis and NH2-terminal sequence analysis of transferred proteins. A mechanism is proposed to explain the observed improvement in reproducibility and sensitivity of imidazole preincubation to zinc staining.     

The identification of rat intestinal membrane enzymes after electrophoresis on polyacrylamide gels containing sodium dodecyl sulphate.

Brush-border membranes were isolated from the rat small intestine and then treated with sodium dodecyl sulphate under non-reducing conditions at room temperature. Analysis of the solubilized components by polyacrylamide-gel electrophoresis identified three major glycoproteins that co-migrate with glucoamylase-maltase-sucrase, lactase and isomaltase-maltase-sucrase activities. High activities of alkaline phosphatase and trehalase were detectable, but they could not be attributed to distinct co-migrating protein bands. Analysis of mucosa from the distal small intestine by the same methods showed a pattern of bands different from that obtained with the proximal intestine, which appeared to correlate with the relative deficiency of some of the enzymes in the distal region.     

Aberrant migration of lipopolysaccharide in sodium dodecyl sulfate/polyacrylamide gel electrophoresis

Purified lipopolysaccharides of salmonellae strains were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Pre-electrophoresis of polyacrylamide gels had no apparent effect on one-dimensional silver-stained lipopolysaccharide profiles. However, without pre-electrophoresis, two-dimensional and three-dimensional patterns contained numerous bands with varied migration patterns compared to those in the one-dimension gels. The lipopolysaccharide was altered within the polyacrylamide gel during electrophoresis. Pre-electrophoresis of gels eliminated aberrant migration patterns.     

Electrophoretic behavior of micellar and monomeric sodium dodecyl sulfate in polyacrylamide gel electrophoresis with reference to those of SDS-protein complexes.

Electrophoretic behavior of sodium dodecyl sulfate (SDS) in polyacrylamide gels has been examined at various gel concentrations. Micellar SDS is subject to significant molecular sieving from the gels while monomeric SDS is virtually free from the effect. The two forms are in a rapid equilibrium with each other. The gel concentration, therefore, has a signifieant effect on the electrophoresis of SDS added in SDS-polyacrylamide gel electrophoresis. The simplified procedure of Stoklosa and Latz (1974, Biochem. Biophys. Res. Commun.58, 74–79), in which SDS is added only in sample solutions, has been criticized based on the results obtained.     

Electrophoretic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates: application to proenkephalin processing enzymes

A novel method is described for the zymographic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates such as [35S]methionine-labeled proenkephalin or 125I-labeled proinsulin. After electrophoresis the enzyme is reactivated and cleaves the radiolabeled in situ substrate into smaller peptides. These small peptides are able to diffuse out of the gel, leaving clear areas against a dark background when visualized by autoradiography. The technique can be used to detect as little as 200 fg of trypsin using only 50 ng (1.25 microCi) of [35S]proenkephalin. Soluble- and membrane-bound adrenal trypsin-like enzyme were isolated from bovine adrenal chromaffin granules. Both proteinases cleaved [35S]methionine-labeled proenkephalin but not 125I-labeled proinsulin. Moreover, both had a Mr of approximately 30,000. The potential of this technique for general use is discussed. An additional method using the synthetic fluorogenic substrate t-butoxycarbonyl Glu-Lys-Lys aminomethylcoumarin is also described.     

An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels

The use of 3,3′,5,5′-tetramethylbenzidine-H₂O₂ as a stain for the peroxidase activity of cytochrome P-450 (or cytochrome P-450 in sodium dodecyl sulfate polyacrylamide gels is described in this report. This reagent can be used to detect very low levels of heme-associated peroxidase activity. The blue-stained bands on polyacrylamide gels are distinet, and the color is stable. The stained gels can be photographed or scanned at 690 nm because the gel background remains clear. The stain is easily removed from the gels to permit subsequent protein staining. Staining first for peroxidase activity has no effect on the subsequent protein staining profile. The peroxidase activity of cytochrome P-450 (or cytochrome P-420) in immunoprecipitates in Ouchterlony double diffusion plates can also be detected using this reagent.     

Electrophoretic elution of macromolecules from polyacrylamide or agarose gels

A method for elution of micrograms of macromolecules from polyacrylamide and agarose gels is described. The recoveries were greater than 90% with three different macromolecules tested (28 to 360 kDa). An amount as small as 1 microgram of human serum albumin was eluted from polyacrylamide gel with 90% recovery. The eluted material is collected into a small chamber the size of which can be changed as required. Elution and concentration are achieved simultaneously and in one step under mild conditions. Sterile eluates can be obtained, if the apparatus is constructed under sterile conditions.     

Stability of aprA-subtilisin in sodium dodecyl sulfate

The effect of sodium dodecyl sulfate (SDS) on the structure and activity of aprA-subtilisin, a secreted bacterial serine protease which is 85% homologous to subtilisin BPN', was examined. The addition of SDS resulted in the slow conversion of the subtilisin from the intact protein to the completely unfolded form of the enzyme. No intermediates between these two populations were detected. This conversion was accompanied by decreased activity, disruption of tertiary structure, a change in the mobility of the protein when subjected to SDS-polyacrylamide gel electrophoresis, and an increase in the apparent Stokes radius of the protein. After 2 h in 1% SDS at 20 degrees C, 25% of the subtilisin was still intact and active. The amount of protein existing in the unfolded form was increased by increasing the length of time in SDS, by increasing the concentration of SDS, and by increasing the temperature of the subtilisin-SDS solution. Analysis of the dependence of the rate of unfolding on SDS concentration indicated that one SDS micelle can destroy two protein molecules. The activation energy for the SDS-induced denaturation of aprA-subtilisin was 20 kcal mol-1, indicating that unfolding of the protein could be the rate-limiting step.