Fig. 4. Transglutaminase activity of FXIIIA under different conditions.

A – Spectrophotometric kinetic assay. Glutamine donor K9 peptide was preincubated with the lysine mimic DMPDA at 37 °C for 5 min, and the crosslinking reaction was initiated by addition of 1 μM FXIIIA°^,high (green circle) or FXIIIA* (purple circle). The reaction was followed by an increase in absorbance at 278 nm due to formation of the anilide crosslinked product. The plot represents Michaelis-Menten fits of the initial reaction velocities as function of the K9 concentration. Apparent K_m and V_max resulting from the fits are shown in the table insert. Values are presented as mean ± SD (N=3).

B – MDC crosslinking SDS-PAGE based assay. Recombinant fibrinogen αC (233–425) was preincubated with 1 mM MDC at 37 °C for 5 min. The crosslinking reaction was initiated by addition of 100 nM FXIIIA°^,high or FXIIIA*. Reaction aliquots were withdrawn at 1 – 7 min and quenched by addition of reducing sample buffer and boiling. The time point samples were loaded on 15% SDS-PAGE side by side for FXIIIA* (triangles) and FXIIIA°^,high (circles). Two gels were run (1 – 4 and 5 – 7 min time points). The gels were aligned and photographed under UV light (panels i – iii). Panel iv – Coomassie Blue-stained gel pair from panel iii demonstrating absence of αC–αC conjugation. FXIIIA* was always preactivated in the presence of 4 mM CaCl₂, and FXIIIA°^,high was preactivated in the presence of 100 mM CaCl₂. Concentrations of αC and CaCl₂ in the crosslinking reaction mix are annotated on the right. The different MDC assay series were performed three independent times.