Sodium dodecyl sulfate enzymes

Similarly to quantitative determination of high surfactant concentrations, many alternative methods have been proposed for the quantitative determination of low surfactant concentrations. Tsuji et al. [270] developed a potentio-metric method for the microdetermination of anionic surfactants that was applied to the analysis of 5-100 ppm of sodium dodecyl sulfate and 1-10 ppm of sodium dodecyl ether (2.9 EO) sulfate. This method is based on the inhibitory effect of anionic surfactants on the enzyme system cholinesterase-butyryl-thiocholine iodide. A constant current is applied across two platinum plate electrodes immersed in a solution containing butyrylthiocholine and surfactant. Since cholinesterase produces enzymatic hydrolysis of the substrate, the decrease in the initial velocity of the hydrolysis caused by the surfactant corresponds to its concentration. Amounts up to 60 pg of alcohol sulfate can be spectrometrically determined with acridine orange by extraction of the ion pair with a mixture 3 1 (v/v) of benzene/methyl isobutyl ketone [271]. 

The number of different proteins in a membrane varies from less than a dozen in the sarcoplasmic reticulum to over 100 in the plasma membrane. Most membrane proteins can be separated from one another using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), a technique that has revolutionized their study. In the absence of SDS, few membrane proteins would remain soluble during electrophoresis. Proteins are the major functional molecules of membranes and consist of enzymes, pumps and channels, structural components, antigens (eg, for histocompatibility), and receptors for various molecules. Because every membrane possesses a different complement of proteins, there is no such thing as a typical membrane structure. The enzymatic properties of several different membranes are shown in Table 41-2. 

Clostridium sticklandii also expresses a proline reductase that can reduc-tively cleave proline to 8-aminovalerate (Seto and Stadtman 1976). PR was first purified by Seto and Stadtman (1976) by following the decomposition of proUne in the presence of dithiothreitol or NADH. They found PR to have a denatured mass of approximately 30kDa (sodium dodecyl sulfate-polyacrylomide gel electrophoresis SDS-PAGE) and a native size of approximately 300 kDa. The addition of selenite to the growth medium of C sticklandii did increase the specific activity of PR in extracts by threefold however, no selenium was detected in the purified enzyme. It should be noted that this purified enzyme had lost the ability to couple reduction of proline to NADH and thus probably was missing one or more components of the complete enzyme complex. 

This can be done by the Ouchterlony diffusion technique (see Fig. 2 and ref. 6). by enzyme-linked immunosorbent assay (see Chapter 15), or by Western blot, either using the purified protein or a more complex mixture of proteins containing the antigen of interest separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (see Chapter 20). 

Hager, D. A. and Burgess, R. R. (1980) Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem 109, 76-86. 

In the presence of sodium dodecyl sulfate (SDS), the apparent molecular weight was approximately 25,000, consistent with the presence of four subunits. The dissociation of the enzyme into half-molecules was also observed at alkaline pH (83). At pH 9.0 this dissociation was promoted by the addition of AMP in the cold and prevented by the addition of FDP. It was proposed that dissociation at alkaline pH was an extreme manifestation of a conformational change induced by AMP which was related to the allosteric effects observed with this effector. In this connection it is noteworthy that the enzyme desensitized to AMP inhibition by treatment with I2 or FDNB was also no longer susceptible to dissociation by AMP. 

Regardless of the chromatography method that is used, fractions containing subsets of the proteins in the sample are collected at the bottom of the column. Each of the fractions is assayed for enzyme activity. In addition, the complexity of the fraction is evaluated by separating the proteins in a small sample of the fraction using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This method separates proteins in mixtures based on their size. 

They are believed to enhance the transbuccal permeation by a mechanism that is similar to that of bile salts, namely, extraction of lipids, protein denaturation, inactivation of enzymes, and swelling of tissues [39], Sodium dodecyl sulfate is reported to have a significant absorption enhancing effect but may also produce damage to the mucosa [13]. The effect of sodium... 

Subtilisin BPN was prepared through a series of protein purification steps applied to the fermentation broth. These steps included ultrafiltration ethanol precipitation DEAE (diethyl-aminoethyl) Tris Acryl batch anionic exchange SP (sulfopropyl) Tris Acryl column cationic exchange and, concentration with an Amicon stirred cell. The enzyme purity was determined to be -951 via spectroscopic assays that measure the ratio of active enzyme to total protein. In addition, purity was verified via HPLC and SDS-page (sodium dodecyl sulfate polyacrylamide gel electrophoresis). 

Creatine kinase was purified from rabbit muscle by the method of Kuby et al, (4). Rabbit muscle pyruvate kinase was purchased from Boehringer. Porcine muscle adenylate kinase was purchased from Sigma, and was further purified by gel filtration on Sephadex G-50. The enzymes were homogeneous as judged by their specific activities and by their migration as single components in sodium dodecyl sulfate gel electrophoresis. Proton NMR spectra at 250 MHz of 0.5-2.0 mM enzyme sites in 0 solution were obtained with a Bruker WM 250 MHz pulse FT spectrometer at 25°. At least 256 transients were accumulated over 8192 data points using 16 bit A/D conversion. Relaxation rates and histidine pK values were determined by standard NMR methods (5, 6),... 

Figure 10. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of products of limited proteolysis of the debranching enzyme with trypsin. The molecular weights shown of the various bands were determined by the methodology described previously (26). The ratio of debrancher to trypsin was 100 to 1. The incubation was conducted for 60 minutes at 25°. The gel stain was Coomassie Brilliant Blue and the absorbance was measured at 600 nm using a Gilford gel scanner with a 0-1 O.D. chart scale (36).

The sample (GOx-PPNVP/PEUU) was removed from the GOx solution and washed exhaustively to remove adsorbed enzyme by continuous stirring in 900 mL of sodium dodecyl sulfate (SDS, Sigma, 2% v/v in H2O), Triton X-100 (octyl phenoxy polyethoxyethanol, Sigma, 2% v/v in H2O), and 20 mM sodium phosphate (pH 7.0) for 24 h each at 5°C and stored in 20 mM sodium phosphate (pH 7.0) at 4°C. It has been shown that this wash and storage procedure is effective in removing adsorbed GOx and does not denature the immobilized GOx (11). 

There is evidence that protein structures are also responsible for cell cohesion in nonpalmo-plantar stratum corneum. When punch biopsies of normal human gluteal skin were incubated in a buffer containing a mixture of the zwitterionic surfactant /V,/V,-dimethyldodecylamine and the anionic surfactant sodium dodecyl sulfate,11 there was dissociation of cells in the stratum corneum but not in the rest of the epidermis. The cell dissociation took place only in the presence of EDTA and was inhibited by the serine protease inhibitor aprotinin.12 Suzuki et al.13,14 presented evidence that spontaneous cell dissociation in nonpalmo-plantar stratum corneum could be inhibited by a combination of inhibitors of trypsin-like and chymotrypsin-like enzymes. Thus, nonpalmo-plantar stratum corneum contains endogenous proteases that mediate cell dissociation. 

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