Reconstitution of apoglucose oxidase with FAD conjugates for biosensoring of progesterone.
The reconstitution of Aspergillus niger apoglucose oxidase (apoGOx) with FAD conjugates for biosensoring of progesterone was investigated. ApoGOx prepared by partial unfolding of the protein under acidic conditions consisted of reconstitutable monomers (50+/-10%), reconstitutable dimers (20+/-10%) and irreversibly aggregated oligomers (30+/-20%). Incubation of monomeric apoGOx with FAD or N(6)-(6-aminohexyl)-FAD (ahFAD) restored glucose oxidase (GOx) activity and induced dimerization with stoichiometric incorporation of FAD. N(6)-(6-aminohexyl)-FAD progesterone conjugates also induced dimerization. However, holoenzyme reconstitution required relatively high concentrations of apoprotein and was dependent on the type of conjugate. Restoration to 25-50% of the original enzyme activity was obtained. Binding of the FAD-progesterone conjugates might hinder the closure of a protein lid needed for dimer formation. Our results illustrate the prospects of FAD conjugates in sensitive detection of progesterone in biological matrices in a biosensor based on the recombination of apoGOx with progesterone-conjugated FAD.
Posthuma-Trumpie
Geertruida A
GA
University of Groningen, University Medical Centre Groningen, School of Behavioural and Cognitive Neuroscience, Department of Psychiatry, Hanzeplein 1, 9713 EZ Groningen, The Netherlands. truus.posthuma@wur.nl
van den Berg
Willy A M
WA
van de Wiel
Dirk F M
DF
Schaaper
Wim M M
WM
Korf
Jakob
J
van Berkel
Willem J H
WJ
D001059:Apoproteins / Q000737:chemistry; D001234:Aspergillus niger / Q000201:enzymology* / Q000378:metabolism; D001681:Biological Assay; D015374:Biosensing Techniques; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002855:Chromatography, Thin Layer / Q000379:methods; D019281:Dimerization; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D005418:Flavodoxin / Q000737:chemistry; D005949:Glucose Oxidase / Q000737:chemistry*; D007118:Immunoassay / Q000379:methods; D007700:Kinetics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D011374:Progesterone / Q000737:chemistry*
D001059:Apoproteins; D005418:Flavodoxin; C026932:apoflavodoxin; D005182:Flavin-Adenine Dinucleotide; D011374:Progesterone; D005949:Glucose Oxidase