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Purification and Measurement of HDL3-binding Proteins in Human Peripheral Blood Mononuclear Cells

Address correspondence to Hiroya Hidaka, Ph.D., Dept. of Biomedical Laboratory Sciences, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto, Nagano, Japan; tel & fax 81 263 37 2368; e-mail hiroyan{at}hsp.md.shinshu-u.ac.jp.

We previously identified a binding site for high density lipoprotein-3 (HDL3) on the surface of human peripheral blood monocytes. Here we describe the purification and measurement of HDL-binding proteins present on these cells. Purification of HDL-binding proteins was achieved by chromatography using DEAE ion-exchange, wheat germ lectin, and apoHDL3 affinity columns. Subsequent use of SDS-PAGE and ligand blotting lead to the identification of two major proteins with apparent molecular masses of 100 and 120 kDa, plus several smaller proteins that appeared to be degradation products. Analysis of purified HDL-binding proteins by reverse-phase HPLC showed that the two main proteins at 100 and 120kDa were eluted in 65 and 70% acetonitrile, respectively, indicating the proteins are strongly hydrophobic. To measure the amount of HDL-binding protein present in CHAPS-solubilized human mononuclear cells, a 96-well plate assay was developed that was based on the same principles as the purification method. The present study of HDL-binding proteins in mononuclear cells advances our understanding of the physiological roles and fate of HDL.

Keywords: HDL3, HDL-binding protein, peripheral blood mononuclear cells, RP-HPLC

Abbreviations: HDL, high density lipoprotein; PO, peroxidase; WG, wheat germ lectin; PBS, phosphate-buffered saline (10 mM Na₂HPO₄/NaH₂PO₄ (pH 7.4), 150 mM NaCl); CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate; Trizma-base, tris(hydroxymethyl)-amino-methane; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; TMB, tetramethylbendigin dihydrochroride; Tween 20, polyoxyethylene sorbitan monolaurate