Optical biosensing of nitric oxide using the metalloprotein cytochrome c '

The metalloprotein cytochrome c' was extracted and purified from the bacter ium Paracoccus denitrificans in order to develop a specific biosensing syst em for nitric oxide (NO). The metalloprotein was encapsulated in a porous s ilicate sol-gel glass to enable spectroscopic changes in the haem centre as a function of NO ligation to be quantified using absorption measurements. Spectroscopic evidence suggested that, between 2 and 4 d after encapsulatio n, the cytochrome c' protein changed conformation in the locality of the ha em moiety, possibly from a five to a six coordinate haem centre. Such confo rmational changes were also observed when the cytochrome c' was stored in s olution, although over a 2-3 month period. The conformational changes occur ring in the protein altered the spectral characteristics of the reduced, ox idised and nitrosyl complex of the cytochrome c' and appear to change the b inding affinity of the protein towards NO. However, the encapsulated (recon formed) cytochrome c' was shown to retain its selectivity towards NO with g ood reproducibility (seven consecutive measurements of NO produced an inten sity value with a relative standard deviation of 0.28%). An NO calibration curve, using the in situ release of NO from the donor diethylamine NONOate, was obtained for the encapsulated cytochrome c' with an approximate workin g range of 10-400 mu mol l(-1).