Proteinaceous materials can be modified by crosslinking the amino groups present in the protein molecule. Presently, the physical presence of covalent crosslinking by heat dehydration treatment or chemical reagents has only been indirectly determined. A convenient assay specific to amino groups using 2,4,6-trinitrobenzenesulfonic acid (TNBS) is presented and can be used to directly evaluate crosslinking. The assay accuracy was evaluated by determining {dollar}epsilon{dollar}-amino groups of insulin and bovine albumin. The {dollar}epsilon{dollar}-amino group content of Type B gelatin was found to be 33.0 moles per gelatin molecule of 1000 residues and is in agreement with similar source gelatins and collagen.; Heat dehydration crosslinking in gelatin has been reported to occur by a condensation reaction between amino and carboxylic acid groups. The presence and extent of condensation crosslinking was evaluated by determining the number of {dollar}epsilon{dollar}-amino groups lost to this crosslinking reaction upon heat dehydration treatment (105{dollar}spcirc{dollar}C and 10 {dollar}mu{dollar} Hg pressure) using TNBS. Unchanging amounts of {dollar}epsilon{dollar}-amino groups indicate that the proposed condensation crosslinking reaction is unlikely to occur. The presence of condensation crosslinking was further tested by derivatizing the {dollar}epsilon{dollar}-amino groups to prohibit crosslinking upon heat treatment. Covalent crosslinking still occurred as evidenced by its poor solubility in 6 M guanidine hydrochloride at 37{dollar}spcirc{dollar}C. Other published data show decreases in gelatin solubility and equilibrium swelling upon heat dehydration treatment. The results suggest that the proposed condensation reaction is unlikely to occur in heat treated gelatin and that covalent crosslinking probably occurs by one or more yet unidentified mechanisms.; Crosslinked proteinaceous matrices have been investigated as devices for controlled release of conventional and macromolecular drugs. The number of amino group crosslinking sites in gelatin was enhanced 230% above original amounts by covalent modifying carboxylic acid groups. Crosslinked unmodified and modified gelatin matrices were prepared and loaded with the model macromolecule dextran (10,000 Da). Dextran release was evaluated using a polysaccharide colorimetric assay. The crosslinked matrices were also characterized by examination of crosslinking parameters. The release data was plotted with the empirical equation M{dollar}sb{lcub}rm t{rcub}{dollar}/M{dollar}sb{lcub}infty{rcub}{dollar} = kt{dollar}sp{lcub}0.5{rcub}{dollar} which suggested diffusion as the mechanism of dextran transport. Apparent diffusion coefficients for dextran were calculated as a function of time and then averaged. The release rate of dextran from the more extensively crosslinked modified gelatin matrix was faster than release of dextran from the crosslinked unmodified gelatin matrices. Possible explanations for the increased rate of dextran release were discussed.
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