Sulfhydryl groups , determination

Sulfhydryl Groups, Determination in Biological Substances (Chinard... 

After It has been determined which chain In the variant Is aberrant, specific structural studies are required. Several procedures are available which differ from one laboratory to the other and Include chain separation by column chromatography, modification of the sulfhydryl groups through reaction with... 

Several precautions were taken to ensure the immobilization chemistry. First, the sulfhydryl groups containing the macromolecular fraction was spectrophotometrically determined according to the literature [15]. We found that every set of 150 base pairs contained approximately one disulfide group. Since the DNA fragment used has hundreds of base pairs, each DNA strand seems to have one disulfide as its terminal group. Next, we made IR spectral measurements in a reflection-absorption (RA) mode [14b]. A freshly evaporated gold substrate was immersed into the DNA solution for 24 h at 5°C. The substrate was carefully rinsed with deionized water, dried under vacuum and was immediately used for the measurements. An Au substrate treated with unmodified, native sonicated CT DNA solution was also prepared as the control measurement. The / -polar-ized radiation was introduced on the sample at 85° off the surface normal and data were collected at a spectral resolution of 4 cm with 2025 scans. 

Figure 19.21 The rate of reaction of cysteine with maleimide-activated BSA was determined using an Ellman s assay for remaining sulfhydryl groups after the reaction, according to Figure 19.20. Nearly all of the available maleimides are coupled with sulfhydryls within 2 hour.

Purify the thiolated toxin from unreacted Traut s reagent by gel filtration using 0.1M sodium phosphate, 0.15M NaCl, pH 7.5, containing lOmM EDTA. The presence of EDTA in this buffer helps to prevent oxidation of the sulfhydryl groups with resultant disulfide formation. The degree of —SH modification in the purified protein may be determined using the Ellman s assay (Chapter 1, Section 4.1). 

To determine how plants are injured, we studied changes in certain cellular constituents in plants exposed to ozone. The cells of these plants leak their contents and so it is probable that the initial damage is to cellular membranes. The normal functioning of these membranes depends on lipid constituents probably stabilized by sulfhydryl groups in associated proteins. 

The cyanide nitroprusside test determines the presence of free sulfhydryl or disulfide compounds in urine samples [1, 3,4]. During the first step of the assay, cyanide reduces any disulfides that are present to free sulfhydryl compounds. In the second step, a reddish color reaction results when the free sulfhydryl groups complex with nitroprusside. A positive result is most usually due to cystine in the urine. Familial cystinuria is among the most common aminoacidurias. Disulfides are also excreted in other metabolic disorders such as homo cystinuria and ji-m e reap lol ac la le - cy s lei ne disulfiduria. Both will also produce positive results according to the following reaction RSH + Na2Fe(CN)5NO (sodium nitroprusside) — chromophore + NO. 

The isoelectric point, determined by Sumner and Hand (50) to have a value of 5.0-5.1, has been redetermined by the electrofocusing technique (43). The value obtained was 4.8. The solubility is extremely small at this pH, but the urease can be located by its enzymic action. It is a point of interest that the solubility of the isoelectric species increases spectacularly if the sulfhydryl groups are substituted with A-ethylmaleimide (NEM) (43). 

As noted above, Sumner and co-workers were unable to determine the diffusion coefficient of urease unless they added Na2S03 and NaHSO-to the phosphate buffer (40) used. Nichol and Creeth, employing identical concentrations (60), measured both the sedimentation coefficient and the electrophoretic mobility of sulfite-modified urease. They concluded that sulfite contributed to the formation of -S-S03 groups attached to the (16n) species. Some of these groups they ascribed to the scission of intermolecular disulfide bonds of aggregated forms others, they suggested, arose from the 22 reactive sulfhydryl groups that react with 02 (air) to form transitory disulfides that can, in turn, react with sulfite. 

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