ISOELECTRIC-FOCUSING OF BIOTINIDASE AND LIPOAMIDASE WITH THE ADDITIONOF NONIONIC DETERGENT

Isoelectric focusing (IEF) of purified biotinidase and lipoamidase (si alylated glycoprotein enzymes) was found to be reproducible and reliab le 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 lip oamidase purifications were also successful; i.e. human serum biotinid ase was purified by IEF after Affi-Gel Blue chromatography in the fina l step, and microsomal biotinidase and lipoamidase from guinea pig liv er and kidney were purified by the preparative liquid IEF of Rotofor c ell with 1% NP-40 followed by the final gel-permeation chromatography with Sephadex G-200. Thus, this high-recovery IEF with non-ionic deter gent 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 othe r relatively hydrophobic proteins. (C) 1998 Elsevier Science B.V. All rights reserved.