Preparative vertical and rotating horizontal (Rotofor)
ampholine column and
immobiline flat bed
polyacrylamide gel isoelectric focusing were evaluated for the isolation of the biologically active acidic form of
leukoregulin, a 50,000-Da
glycoprotein lymphokine with
tumor growth inhibitory activity.
Leukoregulin secreted by normal human lymphocytes was concentrated by 10,000 nominal molecular weight size exclusion ultrafiltration and by
DEAE anion exchange chromatography using step elution with 0.02 M Tris-HCl: 0.1 M NaCl, pH 7.4. Preparative isoelectric focusing was carried out in a 110-ml vertical column containing 1%
ampholines in a pH 4-6 gradient at 15 W constant power for 16-18 h, in a Rotofor 55-ml horizontal column containing 2%
ampholines in a pH 4-6 gradient at 12 W constant power for 4-6 h, or in an
immobiline pH 4.5-6.5 gradient within a 5%
polyacrylamide 120 X 110 X 5-mm flat bed gel at 3 W constant power for 16-18 h. Recovery of biologically active
leukoregulin from the vertical and horizontal
ampholine columns was similar. The pH 4.9-5.2 fractions from the Rotofor
ampholine column contained 4-7% and the fractions from the
immobiline gel contained 4% of the
leukoregulin activity applied to the electrofocusing column or gel, respectively. Analytical
immobiline isoelectric focusing of the
leukoregulin in the pH 4.9-5.2 fractions from the Rotofor column demonstrated that a single
silver staining band with a pI of 5.1 can be obtained by this rapid method of preparative isoelectric focusing.