Sulfhydryl determination, alkylating

The molar ratios of the amino-acid residues were determined after erythrocuprein was subjected to acid hydrolysis at 110°C for 20 to 96 hours. The method of Moore and Stein was used throughout (88, 89). The sulfhydryl content of human erythrocuprein was determined using the spectrophotometric assay of Boyer (90). Total half-cystine was analysed as S-carboxy methyl cysteine in acid hydrolysates (91) of reduced and alkylated erythrocuprein, or as cysteic acid in acid hydrolysates oxidized with performic acid. The data are summarized in Table 2. 

If free sulfhydryl groups are present, they must be taken into account for these calculations. The number (Ash) of free sulfhydryl groups, if not known, is determined by following the same procedure but without performing the reduction step, and from the determined molecular mass of the alkylated protein... 

Chloroacetol phosphate alkylates the sulfhydryl group. ) The reaction mixture was cooled in an ice bath and then treated with a portion of the same stock solution of sodium [ Hjborohydride that was used in the reduction of the modified carboxylase (see above). The final borohydride concentration in the reaction mixture was 0.1 M. After 1 hr the mixture was acidified to pH 2.0 with 1 N HCl and chromatographed on a column (1.2 X 22 cm) of Dowex 50 (H ) equilibrated and eluted with 50 mAf HCl. The concentration of the glutathione derivative in the peak fraction was determined with the amino acid analyzer the radioactivity in the same fraction was also determined, thereby providing the specific radioactivity of the [ H] borohydride. 

It is known that aryl thiols are more reactive than alkyl thiols due to their lower p a values (5-7) [28]. Aromatic thiols, for example, have already been shown to increase the folding rate of disulfide-cOTitaining proteins more than aUphatic thiols [30, 31]. For a pharmaceutical excipient such as thiolated chitosans, the increased reactivity of the thiol group would further increase its beneficial properties over alkyl thiol chitosan and non-thiolated polymers. Examples of aryl sulfhydryl-bearing compounds covalently attached to chitosan via an amide bond are 6-mercaptonicotinic acid and 4-mercaptobenzoic acid. As mentioned for alkyl thiol chitosan, the amount of free and oxidized thiol groups can be determined by Ellman s method. Examples of developed thiolated chitosans and their degrees of modification are listed in Table 1. 

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