Immunopurification and characterization of human α-L-iduronidase with the use of monoclonal antibodies

P. R. Clements, D. A. Brooks, P. A G McCourt, J. J. Hopwood

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α-L-Iduronidase from human liver was purified by a three-step five-column procedure and by immunoaffinity chromatography with a monoclonal antibody raised against purified enzyme. Seven bands identified by staining with Coomassie Blue had molecular masses of 74, 65, 60, 49, 44, 18 and 13 kDa and were present in both preparations of the liver enzyme. However, relative to the immunopurification procedure, α-L-iduronidase purified by the five-column procedure was considerably enriched in the 65 kDa polypeptide band. The seven bands were identified by Western-blot analysis with two different monoclonal antibodies raised against α-L-iduronidase. The chromatographic behaviour of α-L-iduronidase on the antibody column was dependent upon the quantity of enzyme loaded. Above a particular load concentration a single peak of enzyme activity was eluted, whereas at load concentrations below the critical value α-L-iduronidase was eluted in two peaks of activity, designated form I (eluted first) and form II (eluted second). The following properties of the two forms of α-L-iduronidase were determined. (1) The two forms from liver were composed of different proportions of the same seven polypeptides. (2) When individually rechromatographed on the antibody column, each form from liver shifted to a more retarded elution position but essentially retained its chromatographic behaviour relative to the other form. (3) Forms I and II of liver α-L-iduronidase showed no difference in their activities towards disaccharide substrates derived from two glycosaminoglycan sources, heparan sulphate and dermatan sulphate. (4) The native molecular size of forms I and II of live α-L-iduronidase was 65 kDa as determined by gel-permeation chromatography. (5) Immunoaffinity chromatography of extracts of human lung and kidney resulted in the separation of α-L-iduronidase into two forms, each with different proportions of the seven common polypeptides species. (6) Lung forms I and II were taken up readily into cultured skin fibroblasts taken from a patient with α-L-iduronidase deficiency. Liver forms I and II were not taken up to any significant extent. Lung form II gave intracellular contents of α-L-iduronidase that were more than double those of normal control fibroblasts, whereas lung form I gave contents approximately equal to normal control values. We propose that all seven polypeptides are derived from a single α-L-iduronidase gene product, and that different proportions of these polypeptides can function as a single α-L-iduronidase entity. The separation of α-L-iduronidase into two forms by immunoaffinity chromatography may result from competition between affinity and self-association equilibria.

Original languageEnglish
Pages (from-to)199-208
Number of pages10
JournalBiochemical Journal
Issue number1
Publication statusPublished - 1989

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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