title: Optical biosensing of nitric oxide using the metalloprotein cytochrome c'.
abstract: The metalloprotein cytochrome c' was extracted and purified from the bacterium Paracoccus denitrificans in order to develop a specific biosensing system for nitric oxide (NO). The metalloprotein was encapsulated in a porous silicate 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 encapsulation, the cytochrome c' protein changed conformation in the locality of the haem moiety, possibly from a five to a six coordinate haem centre. Such conformational changes were also observed when the cytochrome c' was stored in solution, although over a 2-3 month period. The conformational changes occurring in the protein altered the spectral characteristics of the reduced, oxidised and nitrosyl complex of the cytochrome c' and appear to change the binding affinity of the protein towards NO. However, the encapsulated (reconformed) cytochrome c' was shown to retain its selectivity towards NO with good reproducibility (seven consecutive measurements of NO produced an intensity 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 working range of 10-400 mumol l-1.
authors.affiliation: School of Chemical Sciences, University of East Anglia, Norwich, UK. d.russell@uea.ac.uk
authors.forename: D J
authors.initials: DJ
authors.lastname: Blyth
authors.forename: J W
authors.initials: JW
authors.lastname: Aylott
authors.forename: J W
authors.initials: JW
authors.lastname: Moir
authors.forename: D J
authors.initials: DJ
authors.lastname: Richardson
authors.forename: D A
authors.initials: DA
authors.lastname: Russell
mesh_terms: D015374:Biosensing Techniques; D003574:Cytochrome c Group; D006801:Humans; D009569:Nitric Oxide / Q000032:analysis*
chemical_list: D003574:Cytochrome c Group; D009569:Nitric Oxide