Isoelectric focusing of biotinidase and lipoamidase with the addition of non-ionic detergent Original Research Article

Isoelectric focusing (IEF) of purified biotinidase and lipoamidase (sialylated glycoprotein enzymes) was found to be reproducible and reliable when non-ionic detergent, Nonidet P-40 (NP-40), was added at 1% (v/v). Applications of the IEF containing 1% NP-40 on biotinidase and lipoamidase purifications were also successful; i.e. human serum biotinidase was purified by IEF after Affi-Gel Blue chromatography in the final step, and microsomal biotinidase and lipoamidase from guinea pig liver and kidney were purified by the preparative liquid IEF of Rotofor cell with 1% NP-40 followed by the final gel-permeation chromatography with Sephadex G-200. Thus, this high-recovery IEF with non-ionic detergent of NP-40 at 1% is shown to be a practical and reproducible method applicable to analysis and purification for several biotinidases and lipoamidases, and also expected to be generally applicable to the other relatively hydrophobic proteins.