Reactive glutamines in αC (233-425) are crosslinked by FXIIIa in wild-type αC and following single Q to N substitutions. (A) Reaction scheme for FXIIIa-catalyzed crosslinking reaction of ¹⁵N-GEE with reactive glutamines in αC (233-425) as monitored by NMR spectroscopy. (B) 2D ¹H-¹⁵N HSQC NMR spectrum for the reactions between αC (233-425) and ¹⁵N-GEE. The 3 NMR peaks observed correspond to successful crosslinking reactions with the 3 glutamines Q237, Q328, and Q366. Each NMR peak corresponds to a proton (HN) covalently attached to an ¹⁵N-labeled nitrogen. (C) FXIIIa-catalyzed reactions between ¹⁵N-GEE and the individual αC mutants Q237N, Q328N, and Q366N as monitored by 2D ¹H-¹⁵N HSQC NMR. In αC Q237N, peaks corresponding to Q328 and Q366 were seen (top). Q237 reacted fastest following either Q328N (middle) or Q366N (bottom) substitutions.