ABSTRACT
Purpose of the paper was the optimization of mobile phase in natrium chloride gradient in the chromatographic separation of butyrylcholinesterase from human plasma. MATERIALS/METHODS: Butyrylcholinesterase (BuChE) was isolated from human plasma using a diethylamminoethyl-cellulose column, by elution with 0.02M acetate buffer pH=4.0, gradually increasing NaCl percentage from 10% to 80%. The procedure lasted approximately 28 days. RESULTS: Absorbance of the successive collected fractions at 280 nm presented a maximum at 60 % NaCl concentration. Activity of obtained BuChE was maximum at the same concentration. Another observed effect of NaCl was the decrease of resistance of the column to flow of the elution fluid. In the absence of NaCl the flow rate was 7 mL/h. Increasing of NaCl concentration induced a continuous increase of the flow to a value of 21 mL/h at 60% NaCl solution. After this concentration the flow remained practically constant. The effect on the ionic exchange is essentially an effect on chromatographic partition coefficient, leading, as a rule, to a peak having a Gaussian form. Fitting separately of ascending and descending parts of the apparent peak, led to practically the same exponential coefficient. CONCLUSIONS: Separation of proteins and particularly of BuChE on a chromatographic DEAE Cellulose column can be considered as a method for separation and purification of BuChE from human plasma. Optimum concentration of NaCl is 60 %. Exponential fittings in the neighbourhood of maximum indicated a prevalence of effects of NaCl on the chromatographic partition face to effects on gel-sol equilibrium of stationary phase.
