34] Purification of cellular retinol and retinoic acid-binding proteins from rat tissue

Abstract

This chapter discusses the purification of cellular retinol and retinoic acid-binding proteins from rat tissue. Cellular retinol-binding protein (CRBP) binds retinol (vitamin A alcohol) with high affinity and specificity. The cellular retinoic acid-binding protein (CRABP), binds retinoic acid (vitamin A acid) with high affinity and specificity. CRBP is present in most or all epithelial tissues of the rat, but the amount of binding protein present is small compared to total soluble protein. CRABP is present in fewer adult rat tissues than CRBP and is not found in liver. The best source, using criteria of the amount of tissue available and level of binding protein, appears to be testis. A 1.5 g testis contains about 4 μg of CRABP. Testis also contains CRBP (about 5.5 μg per testis). Because the behavior of CRABP and CRBP in a number of purification techniques is quite similar, the isolation of CRABP requires separation from CRBP. The procedures for the purification of CRBP from rat liver and CRABP from rat testes are quite similar in most part and are described together. All steps are carried out at 4° unless otherwise noted. However, determinations of pH for solutions used for column chromatography are at room temperature.