Open AccessHuman α‐ L ‐iduronidase
Author(s) -
CLEMENTS Peter R.,
BROOKS Doug A.,
SACCONE Gino T. P.,
HOPWOOD John J.
Publication year - 1985
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1985.tb09158.x
Subject(s) - monoclonal antibody , chemistry , sepharose , molecular mass , chromatography , protein subunit , ionic strength , immunoprecipitation , enzyme , agarose , biochemistry , microbiology and biotechnology , antibody , biology , aqueous solution , gene , immunology
1 Human 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. 2 The 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‐ M r species diminished as the pH decreased or the ionic strength increased to favour a single active species of M r 65000. 3 A specific monoclonal antibody was generated against liver α‐ L ‐iduronidase. The antibody specifically immunoprecipitated enzyme activity from both crude and purified sources. 4 The subunit M r 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.