Human PAI-1 NBD labeled at the reactive center loop
P9-NBD PAI-1 was created by mutagenesis of the P9 serine residue (Ser338) on the reactive center loop to cysteine. This then provided a free thiol group for incorporation of N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD), a fluorescent probe highly sensitive to changes in solvation and hydrophobic environment. The fluorescence emission of P9-NBD PAI-1 is enhanced 6-7 fold upon insertion of the reactive center loop into beta-sheet A following complex formation with proteinases, formation of the latent species, or cleavage by elastase. The incorporated probe is excited at 480 nm and displays a broad emission spectrum with a peak centered 542 nm with a resultant blue-shift to 520 nm following reactive center loop insertion. The modified PAI-1 is nearly as active as wt PAI-1 and is more resistant to the spontaneous latency reaction making this an excellent tool for monitoring reaction rates of PAI-1. P9-NBD PAI-1 has been utilized in a number of studies to determine the rates of loop insertion and SERPIN reaction mechanisms when reacted with various proteinases, inactivating antibodies and conformational changes imposed by the binding of vitronectin.