4. Effect of macromolecular crowding on the rate of acetyl cholinesterase
4.2. Materials and Methods
Alkaline phosphatase (from bovine intestinal mucosa), Acetylcholine esterase (from electrophorus electricus, electric eel), were purchased from Sigma-Aldrich Chemicals Pvt. Ltd., India. 2-Naphthyl acetate, 3-indoxyl acetate, Dextran (from Leuconostoc mesenteriodes) of molecular weight 15, 40, 70, 200, 500 and 2000 kDa were purchased from Fluka. The polydispersities of the dextrans were typically less than 2.0 as reported by the manufacturer. Glycine, disodium hydrogen phosphate and glycerol (98% purity) were obtained from Merck. p-nitro phenyl phosphate disodium salt was bought from Sisco Research Laboratories, India. All other chemicals employed were of analytical grade.
(a) Hydrolysis of PNPP by Alkaline phosphatase:
A typical reaction mixture contained alkaline phosphatase (2 µM) and PNPP of desired concentration dissolved in an aqueous solution of 100 mM glycine buffered at pH 9.5. The substrate concentration was kept at 1 mM, which is well above the measured Km under the reaction conditions employed (0.25 mM). The ratio of molar concentration of substrate to enzyme is higher. This is in agreement with similar ratios inside living cells.
However, to rule out situations, such as substrate being trapped/bound in dextrans, a few
experiments were also carried out with 20 mM PNPP. These experiments revealed that the profile observed with 20 mM PNPP against increasing dextran size is similar to that observed with (data not shown) 1mM PNPP. The concentration of dextran in the medium was varied between 0 and 30% (w/w). The total weight of the reaction medium was kept constant at 1.0 g.
The reaction was initiated by forcefully mixing the enzyme (typically ~50 µL in buffered aqueous medium) with an aqueous buffered mixture containing PNPP and crowding agent (typically ~950 µL) in an eppendorf tube using a syringe. This mixture was vigorously vortexed for 30 seconds. Immediately after, the mixture was transferred to a cuvette and the progress of the reaction was conveniently monitored using a spectrophotometer by recording the absorbance of the product p-nitro phenol at 450 nm after a dead time of 30 seconds. The above procedure ensured efficient mixing of substrate and enzyme in the midst of crowding agents.
(b) Hydrolysis of 2-Naphthyl acetate by Acetyl choline esterase:
A typical reaction mixture contained Acetyl choline esterase (61.7 nM) and 2- Naphthyl acetate of desired concentration in an aqueous solution of 10 % methanol and 20 mM phosphate, buffered at pH 7.5. The substrate concentration was kept at 1.5 mM which is lesser than Km (2.3 mM). This is because we observe formation of precipitate upon mixing high concentrations (> 2 mM) of substrate with enzyme in the presence of crowding agent (dextran). The concentration of dextran in the medium was varied between 0 and 30% (w/w). The total weight of the reaction medium was kept constant at 1.0 g. The reaction was initiated by mixing the enzyme (typically ~50 µL in buffered aqueous medium) with an aqueous buffered mixture containing 2-Naphthyl acetate and crowding agent (typically ~950 µL) in an identical procedure as described for AP reaction above. Subsequently the progress of the reaction was conveniently monitored using a spectrophotometer by recording the absorbance of the product 2-Naphthol at 327 nm.
(c) Hydrolysis of 3-Indoxyl acetate by Acetyl choline esterase:
A typical reaction mixture contained Acetyl choline esterase (61.7 nM) and 3- Indoxyl acetate of desired concentration in an aqueous solution of 10 % methanol and 20 mM phosphate buffered at pH 7.5. The substrate concentration was kept at 5 mM which
is near to the Km (5.1 mM). This is because we observe formation of precipitate upon mixing high concentrations (> 5.5 mM) of substrate with enzyme in the presence of crowding agent (dextran). The concentration of dextran in the medium was varied between 0 and 30% (w/w). The total weight of the reaction medium was kept constant at 1.0 g. The reaction was initiated by forcefully mixing the enzyme (typically ~50 µL in buffered aqueous medium) with an aqueous buffered mixture containing 3-Indoxyl acetate and crowding agent (typically ~950 µL) as described for AP reaction. The progress of the reaction was conveniently monitored using a spectrophotometer by recording the absorbance of the product 3-hydroxy indole at 385 nm.
In all the reactions above, the completeness of the mixing was ensured and supported by the following observations: 1) the absorbance of product formed initially increased steadily with time from the start, maintaining a linear (monophasic) profile. ii) The initial slope of the absorbance/time plot obtained above was reproducible when the experiment was repeated subsequently multiple times under identical conditions.
(d) Calculation of Normalized rate of reaction:
The initial velocity, V, was obtained by linear regression of the first 20 s of the recorded absorbance/time data, so that inhibition from appreciable build up of the product is negligible. The initial velocity observed under identical conditions, but in the complete absence of the crowding species, was referred to as V0. The normalized rate, Vnorm, was calculated from the following equation:
Vnorm = 100 (V/V0)
The points depicted in the figures are the averages of at least three independent experiments done on different days. Blank solutions containing a mixture of 25% (w/w) of the crowding agent employed and the corresponding substrate showed negligible change in the absorbance in the complete absence of the enzyme under identical conditions, proving that all of the crowding agents employed in the study are indeed chemically inert. All samples were made in deionized water. All the experiments were carried out at 25 0C.
(e) Calculation of rate of reaction at different viscosity:
The dependence of the rate of the above enzymatic reactions on solution viscosity are studied by using glycerol water mixtures. The method adopted to calculate the rate is
same as above. The relative viscosity of aqueous solutions of glycerol (at 25 0C) as a function of concentration expressed in weight percent were obtained from literature (Borchers, 1955). For some concentrations (15, 25, 35, 45 % w/w) of glycerol, the relative viscosity values are not given in the literature. These values are calculated using interpolation from the plot of known values of relative viscosity against glycerol concentration (0 - 60% w/w). The fitted points yielded an R2 = 0.985.