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 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 1,2, inactivating antibodies 2 and conformational changes imposed by the binding of vitronectin 4.

References:
1. Shore JD, et al.1995 J Biol Chem 270:5395-5398.
2. Lawrence D, et al.2000 J Biol Chem 275:5839-5844.
3. Verhamme IM, et al.1999 J Biol Chem 274:17511-17517.
4. Gibson A, et al.1997 J Biol Chem 272:5112-5121.



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Transport temperature: Dry ice
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