DETERMINATION OF THE PI OF HUMAN RHINOVIRUS SEROTYPE-2 BY CAPILLARY ISOLECTRIC FOCUSING

Capillary isoelectric focusing was applied to determine the pi value o f human rhinovirus serotype 2 (HRV 2), a picornavirus of about 8 500 0 00 Da in size. Using fused silica capillaries dynamically coated with hydroxypropylmethyl cellulose (added at 0.08% to the catholyte), the v irus zone failed to reach the steady state position in the pH gradient within times usually employed in focusing experiments, as the electro osmotic flow (EOF) pushed the analyte zone past the detector. Therefor e, the residence time of the zones in the separation capillary was ext ended by applying hydrodynamic pressure at the detector side during fo cusing, thus pneumatically counteracting the EOF. After completion of focusing, the zones were mobilized by pressure maintaining the high vo ltage. For calibration of the pH gradient, low molecular mass pI marke r substances were employed, Using the relation between the apparent pI value of the virus and the focusing time under counter pressure, the actual pI of HRV2 was determined as 6.8 by extrapolating to infinite t ime.