The optimum separation was achieved by potassium phosphate buffer
(20 mM, pH 7.25) at a flow rate of 0.40 mL/min.
The reaction mixture contained 50 mM potassium phosphate buffer
(pH 7.0), 0,5 mM ascorbic acid, 0,1 mM hydrogen peroxide and 0,1 mL of enzyme extract in a total volume of 1 mL.
A typical optimized assay mixture contained 1.0 mM ethoxyresorufin, 100 mM Tris-HCl buffer (pH 7.8), NADPH generating system consisting of 0.25 mM NADP+, 2.5 mM MgC[l.sub.2], 2.5 mM glucose-6-phosphate, 1.0 U glucose-6-phosphate dehydrogenase, and 14.2 mM potassium phosphate buffer
(pH 7.8) and 0.2 mg liver microsomal protein in a final volume of 1.0 mL.
After 18 h of growth, cells from experimental or control cultures were collected by centrifugation at room temperature (5 min, 5000g) and washed with 50 mM potassium phosphate buffer
MAO-A (0.05 mg/mL protein) was incubated with desmethoxyyangonin 6 (20 [micro]M and 100.0 [micro]M) and MAO-B (0.05 mg/mL protein) was incubated with desmethoxyyangonin 6 (1.50 [micro]M and 20.0 [micro]M) in an enzyme incubation mixture of 1 mL containing 100 mM potassium phosphate buffer
Casein solution (0.65 % w/v) was prepared in potassium phosphate buffer
(50 mM, pH 7.5).
The powder obtained was homogenized in 1.5mL of potassium phosphate buffer
400mM (pH 7.8) containing EDTA 10mM, ascorbic acid 200mM and PVP 1% (wt [vol.sup.-1]).
50 [micro]L sample of permeabilized cell suspension (0.05 mg) was mixed with 2 mL of 1.25 mM ONPG in 0.1 M potassium phosphate buffer
(pH 6.6) and incubated for 5 min at 37[degrees]C.
In the (MFO) assay, the three blank control wells received 80 [micro]l of potassium phosphate buffer
, and the three positive control wells received 60 [micro]l of potassium phosphate buffer
and 20 [micro]l of cytochrome C solution (0.01 mg/ml in 250 mM sodium acetate pH 5.0).
The incubation mixture (final volume 250 [micro]l, in 0.1 M potassium phosphate buffer
containing 3.3 mM Mg[Cl.sub.2], pH 7.4) consisted of an NADPH-generating system (1.3 mM NADP, 3.3 mM G6P, 0.4 units/ml G6PDH), 25 [micro]M dextromethorphan and 0.8 mg/ml pooled human liver microsomes.
The effect of pH and temperature for purified AChE was determined by measuring the enzyme activities at the different pH using an overlapping buffer system consisting of acetate buffer (pH 4 to 6), potassium phosphate buffer
(pH 6 to 8) and Tris-Cl (pH 7 to 9) and temperatures (15 to80C) for 10 min and enzyme activity was assayed withATC as previously described.
The reaction mixture in a total volume of 1 mL containing 1 mg calf thymus DNA was treated with [Fe.sup.3+] (10 mM), EDTA (10 mM), and [H.sub.2][O.sub.2] (2mM) without or with various concentrations of the extract (0.68-3.44 [micro]M) in potassium phosphate buffer
(20 mM, pH 7.4).