Cross-linking of protein subunits by 1,3,5-triacryloyl-hexahydro-s-triazine

Six difunctional and trifunctional derivatives of acrylamide were synthesized and investigated as potential protein lysine residue cross-linking agents. 1,3,5-Triacryloyl-hexahydro-s-triazine (TAT) was considered the best. The rate constants for the reactions of TAT with model nucleophiles in water solution at 25 degrees C were with the glycine anion amino group, 7.69 x 10(-3) M-1 s(-1); with the anionic form of the N-acetyl-L-cysteine thiol group, 5.54 M-1 s(-1); and with the N alpha-acetyl-L-histidine imidazole ring, 1.19 x 10(-5) M-1 s(-1) (at pH 9.0). The kinetics of modification of amino groups by TAT were studied for several proteins: alpha(1)-casein, bovine serum albumin, recombinant human growth hormone, recombinant human interferons-alpha 2b, and -gamma. The results indicate that if proteins are associated into oligomeric structures in water, their subunits are effectively cross-linked by TAT.