[12] Purification of Interstitial Retinol-Binding Protein from the Eye

S. L. Fong, Gregory I Liou, C. D.B. Bridges

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

This chapter describes the purification of interstitial retinol-binding protein (IRBP) from the eye. IRBP is synthesized and secreted by cells of the neural retina, which may therefore have an important function in regulating its amount in the subretinal space. Purified bovine IRBP binds approximately two molecules of all-trans-retinol and contains 8.4% by weight of carbohydrate that consists of sialic acid, neutral hexoses (mannose, fucose, galactose), and glucosamine in the molar ratio of approximately 1:3:2. The purification of IRBP to homogeneity by high-performance size-exclusion chromatography of crude bovine interphotoreceptor matrix (IPM) is described. Sufficient IRBP is obtained by this method to demonstrate that it consisted of a single concanavalin A (Con A)-binding polypeptide with apparent Mr of 140–145K by SDS–polyacrylamide gel electrophoresis. The anion-exchange chromatography on DEAE-cellulose (DE-52) followed by gel-filtration chromatography on Sepharose CL-4B can be used to obtain purified preparations of bovine and human IRBPs so permitting their partial characterization. For rat IRBP used, Con A-Sepharose can be followed by anion-exchange chromatography on DEAE-cellulose. The preparation of bovine IRBP in milligram quantities is described in the chapter.

Original languageEnglish (US)
Pages (from-to)102-111
Number of pages10
JournalMethods in Enzymology
Volume123
Issue numberC
DOIs
StatePublished - Jan 1 1986

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Purification
Chromatography
DEAE-Cellulose Chromatography
DEAE-Cellulose
Gel Chromatography
Anions
Gels
Hexoses
Fucose
Size exclusion chromatography
Glucosamine
N-Acetylneuraminic Acid
Mannose
Concanavalin A
interstitial retinol-binding protein
Electrophoresis
Vitamin A
Galactose
Retina
Rats

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

[12] Purification of Interstitial Retinol-Binding Protein from the Eye. / Fong, S. L.; Liou, Gregory I; Bridges, C. D.B.

In: Methods in Enzymology, Vol. 123, No. C, 01.01.1986, p. 102-111.

Research output: Contribution to journalArticle

Fong, S. L. ; Liou, Gregory I ; Bridges, C. D.B. / [12] Purification of Interstitial Retinol-Binding Protein from the Eye. In: Methods in Enzymology. 1986 ; Vol. 123, No. C. pp. 102-111.
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