Chemical and swelling evaluations of amino group crosslinking in gelatin and modified gelatin matrices.

PURPOSE: To determine the extent of amino group crosslinking in gelatin matrices by chemical assay, and to compare these results to crosslinking evaluations from swelling measurements. METHODS: Matrices crosslinked with a water soluble carbodiimide (EDC/G), glutaraldehyde (GTA/G), as well as a GTA crosslinked matrix prepared from gelatin modified to contain 230% greater crosslinking sites (GTA/Mod) were evaluated. Crosslinking extent, Xc, was determined by a UV assay of uncrosslinked amino groups before and after crosslinking, and was used to obtain crosslinking densities. Equilibrium swelling ratios, Qm, at 37 degrees C in isotonic pH 7.4 were used to calculate crosslinking degree from the Flory equation for swelling of ionic polymers for comparison to the chemically determined crosslinking densities. RESULTS: Of the original 33 x 10(-5) moles epsilon-amino groups/g gelatin, 91 to 95% were crosslinked in EDC/G and GTA/G. GTA/Mod lost 95% of the original 108 x 10(-5) moles amino groups/g gelatin. Crosslinking densities were 4.1 x 10(-4) and 4.2 x 10(-4) moles/mL for EDC/G and GTA/G, respectively. The value for GTA/Mod increased to 14.2 x 10(-4) moles/mL. Values of Qm followed the same trend. The Flory crosslinking degrees for both gelatin matrices were 12 x 10(-4) and 13 x 10(-4) moles/mL, respectively. The value for the more extensively crosslinked GTA/Mod was 280 x 10(-4) moles/mL. CONCLUSIONS: The swelling and chemical evaluations of crosslinking are in general agreement for matrices with the lower of two crosslinking levels. The chemical determination appears suitable for evaluating amino group crosslinking in gelatin and it may be suitable for other proteinaceous materials.

The determination of epsilon-amino groups in soluble and poorly soluble proteinaceous materials by a spectrophotometric method using trinitrobenzenesulfonic acid.

A procedure using 2,4,6-trinitrobenzenesulfonic acid (TNBS) for the determination of epsilon-amino groups in soluble and poorly soluble proteinaceous materials is presented. The major modification from previous procedures is an extended TNBS reaction time to allow a stoichiometric reaction with amino groups. In addition, autoclave hydrolysis is used to assure sample dissolution for spectrophotometric measurements. The assay accuracy was evaluated by determining epsilon-amino groups of insulin and bovine albumin. The determinations differed from literature values by < or = 3.3%. The epsilon-amino group content of Type B gelatin was found to be 33.0 mol/gelatin molecule of 1000 residues and is in agreement with similar source gelatins and collagen. The coefficient of variation for determinations on all three materials was < or = 5.3%. The assay should be applicable to a broad range of proteinaceous materials.