Isolation of Hunan Growth Hormone Isopcrmones D and E in Milligram Amounts (II), Using Isoelectric Focusing on Polyacrylamide Gel

Abstract

Human growth hormone (hGH) isohormones D and E were prepared from a partial enzymatic hydrolyzate catalyzed by plasmin, using isoelectric focusing cn pclyacrylamide gel to fractionate the digest. Separation between the two lsohcrmone epecles was optimized by employing a maximally flattened p^H gradient between pH's 4.3 and 5.6. Gel slices containing isohormones D and E were extracted and concentrated by Steady-State Stacking on polyacrylamide gel. The extract was purified by gel filtration and lyophillzed. Overall yields of lyophllized isohormones 0 and E were 10.9 mg and represented a recovery of 51% relative to the densitometrically estimated isohormcne concentrations in the hGH digest. Purity of the products, based on Lowry analysis, U.V. absorbance and radioimmunoassay (RIA) was 60-70%. The isolated isohormone species were active in th'e rat tibia-line assay and in the specific RIA for hGH.