Human α-L-iduronidase

1. Purification, monoclonal antibody production, native and subunit molecular mass

Authors


Correspondence to J. J. Hopwood, Department of Chemical Pathology, The Adelaide Children's Hospital, King William Road, North Adelaide, South Australia, Australia 5006

Abstract

  • 1Human α-L iduronidase from liver was purified about 20 000-fold with a new rapid three-step, five-column procedure which consisted of a Concanavalin-A–Sepharose/Blue-A–Agarose coupled step, a CM-Sepharose/Bio-Gel HT coupled step followedby a cupric-ion-chelating Sepharose 6B step.
  • 2The behaviour of α-L-iduronidase on gel permeation chromatography was dependent upon both pH and ionic strength of the eluting buffer. The formation of species with enzyme activity which behaved as large-molecular-mass aggregates was favoured under conditions of low ionic strength and neutral pH. The amount of high-Mr species diminished as the pH decreased or the ionic strength increased to favour a single active species of Mr 65000.
  • 3A specific monoclonal antibody was generated against liver α-L-iduronidase. The antibody specifically immunoprecipitated enzyme activity from both crude and purified sources.
  • 4The subunit Mr of liver α-L-iduronidase was estimated to be 65000 using SDS-PAGE. Monoclonal antibody immunoprecipitation of radiolabelled enzyme was used to provide definitive confirmation of this subunit size.

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