Monitoring of enzymatic activity in situ by EPR.
A novel approach using an EPR method for the determination of activity of enzymes, whose substrates or products are low molecular weight compounds containing SH-groups, is proposed. The approach is based on thiol-disulfide exchange of stable nitroxyl biradical containing a sulfur-sulfur bond with low molecular weight thiol. The method is applied to determine activity of acetylcholinesterase in a larvae bollworm (from the formation of thiocholine from acetylthiocholine). The method is characterized by high sensitivity (up to 2 x 10(-12)m thiocholine) and makes possible measurements in optically unclear (scattering and coloured) media and determination of enzymatic activity (ca. 1 min) directly in the homogenate obtained from the heads of individual larvae. Thus, the method can be recommended for the fast monitoring of many enzymatic systems (including glutathione-dependent) directly in biological tissues of warm-blooded animals and insects.
Khramtsov
V V
VV
Abteilung Spectroscopie, Max-Planck Institute für Biophysicalische Chemie, Göttingen, Germany.
Gorunova
T E
TE
Weiner
L M
LM
D000110:Acetylcholinesterase / Q000378:metabolism*; D000818:Animals; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007814:Larva; D007915:Lepidoptera / Q000201:enzymology; D013997:Time Factors
D000110:Acetylcholinesterase