INTERACTION OF ALBUMIN BINDING-PROTEINS WITH 2 FRAGMENTS OF BOVINE SERUM-ALBUMIN

In order to detect the binding sites for albumin binding proteins (ABP)*, albumin fragments were prepared by peptic digestion at pH 3.7 using a pepsin/albumin ratio of 1/1000 (1). A fragment of approximately 35 kDa and another of approximately 10 kDa were the principal products obtained. The purification of the approximately 35 kDa fragment was achieved in a two-step procedure: a) anionic exchange chromatography on a Q Sepharose Fast Flow column and b) chromatofocusing on a Polybuffer exchanger 94 column (2). The approximately 10 kDa fragment had a relatively high purity at a single-gel filtration step on a Sephadex G-50 column. We have tested the interaction of these two albumin fragments with ABP. The proteins from bovine pulmonary extract (BPE) were separated by SDS - PAGE, then electrotransferred to nitrocellulose membranes and incubated with albumin fragments coupled with 5 nm colloidal gold. The albumin fragments bound the 31 and 18 kDa pairs of bands accounting for ABP from BPE.