Co-purification of human serum lipoamidase and biotinidase: evidence that the two enzyme activities are due to the same enzyme protein

Abstract

A more than 20000-fold purification of human serum lipoamidase is described. This was accomplished by (NH4)2SO4 precipitation and chromatography on DEAE-Sepharose, Blue Sepharose CL-6B and phenyl-Sepharose CL-4B, followed by preparative isoelectric focusing (IEF) and finally by gel-permeation chromatography. Co-precipitation and co-chromatography of lipoamidase and biotinidase activities with equal yields and purification were obtained in all the purification steps, indicating that lipoamidase and biotinidase activities in human serum are due to the same enzyme protein. After preparative IEF, two fractions with both lipoamidase activity and biotinidase activity were found at pI 4.0 and pI 4.4 respectively. The molecular mass of the enzyme was found to be 76 kDa. When 2-mercaptoethanol was used instead of cysteine as stabilizer during the purification procedure, only one major form (pI 4.0) of the enzyme was obtained after preparative IEF. By addition of cysteine, this form was transformed to an enzyme with pI 4.4, indicating that this latter form is a cysteine adduct, produced during the IEF procedure.