Kinetic Evaluation of the Inhibition of Acetylcholinesterase for use as a biosensor

The release of pesticides into the environment has increased, and there is a lack of monitoring of these contaminants. Since the conventional methods of monitoring these contaminants are complicated, costly and time-consuming, mechanisms based on acetylcholinesterase inhibition have emerged as simple and rapid tools for such applications. However, theacetylcholinesterase’s effectiveness as a sensing element in such biosensor systems depend­s on the conditions selected to measure acetylcholinesterase activity and the concentration of substrate or inhibitor, which in turn affect the reaction rates. Therefore, in the present work, the factors affecting the acetylcholinesterase activity were investigated and inhibition experiments were carried out to evaluate the kinetic parameters. The inhibition rate constant for acetylcholinesterase Ki was found to be 1.9 ppm. The kinetic parameter Km was found to be 3.8mM and Vmax was found to be 1.3µM/min from the Eadie-Hofstee plot. The kinetic study using Lineweaver-Burk method showed mixed type of inhibition of acetylcholinesterase with carbofuran.

Ascorbate: antioxidant activity determined in a hydrocarbon phase using quaternary ammonium salts as phase-transfer agents

By shaking sodium ascorbate with tetra-n-butyl ammonium bromide in a two-phase system consisting of water and organic phases, such as 1,1,2,2-tetrachloroethane (TCE) or chlorobenzene, sufficient concentrations of the quaternary ammonium-ascorbate (quat-ascorbate) are transferred into the organic phase to be useful for antioxidant studies in non-polar solvents. The antioxidant activity of the quat-ascorbate was determined by measurement of the (inhibition) rate constant, kinh, for the inhibition of autoxidation of styrene initiated thermally by azobisisobutyrylnitrile (AIBN) at 30 °C. The kinh of ascorbate in styrene, found to be (9.63 ± 2.0) × 104 M−1 s−1, is very much lower than that found for ascor-byl-6-palmitate, kinh = (110 ± 11) × 104 M−1 s−1 under these conditions. The differences are discussed in terms of the relative effectiveness of hydrogen-atom transfer from the enolic hydroxyl at position 2 of the ascorbate ion compared with the more facile such transfer from position 3 of ascorbyl 6-palmitate in a nonpolar medium.

The relative molecular mass dependence of the anti-factor Xa properties of heparin

The effect of heparin fractions of various Mr, with high affinity for antithrombin III, on the kinetics of the reaction between factor Xa and antithrombin III have been studied using purified human proteins. Each of the heparin fractions, which varied between pentasaccharide and Mr 32,000, accelerated the inhibition of factor Xa although an increasing rate of inhibition was observed with increasing Mr. The chemically synthesized pentasaccharide preparation (Mr 1714) gave a maximum inhibition rate constant of 1.2 × 10(7) M-1 × min-1, compared with 6.3 × 10(4) M-1 × min-1 in the absence of heparin, and this rose progressively to 4.2 × 10(8) M-1 × min-1 with the two fractions of highest Mr (22,500 and 32,000). The 35-fold difference in inhibition rates observed with the high-affinity fractions was virtually abolished by the presence of 0.3 M-NaCl. The disparity in these rates of inhibition was shown to be due to a change in the Km for factor Xa when a two-substrate model of heparin catalysis was used. The Km for factor Xa rose from 28 nM for the fraction of Mr 32,000 to 770 nM for the pentasaccharide, whilst 0.3 M-NaCl also caused an increase in Km with the high-Mr fraction. These data suggest that the increased rates of inhibition observed with heparins of higher Mr may be due to an involvement of heparin binding to factor Xa as well as to antithrombin III.