Purification of bovine bone morphogenetic protein by hydroxyapatite chromatography.

Abstract

Bovine bone morphogenetic protein (bBMP) induces differentiation of mesenchymal-type cells into cartilage and bone. bBMP has an apparent Mr of 18,500 ± 500 and represents <0.001% of the wet weight of bone tissue. A Mr 34,000 protein resembling osteonectin is separated by extraction with Triton X-100. A Mr 24,000 protein and about half of a Mr 22,000 protein are disassociated from bBMP by precipitation in 1.5 M guanidine hydrochloride. Aggregates of bBMP and a Mr 14,000 protein are insoluble in aqueous media; the bBMP becomes soluble when the Mr 14,000 protein is disassociated in 6 M urea and removed from the solution by ultrafiltration. Three separate molecular species with apparent Mrs 18,500, 17,500, and 17,000 are eluted at 0.10, 0.15, and 0.20 M phosphate ion concentrations, respectively, from a hydroxy-apatite column. The Mr 18,500 protein has the amino acid composition of acidic polypeptide and includes four halfcystine residues; the pI is 4.9-5.1. The Mr 22,000 component is a chromoprotein resembling ferritin. The NH12-terminal amino acid sequence of the Mr 17,500 protein simulates histone H2B.The Mr 17,000 protein may possess calmodulin activity. Aggregates of the Mr 18,500 and other proteins induce formation of large deposits of bone; the Mr 18,500 protein alone is rapidly absorbed and induces formation of small deposits. None of the other proteins induces bone formation.Under the influence of bone morphogenetic protein (BMP), perivascular mesenchymal-type cells (pericytes) differentiate into cartilage and woven bone. BMP is an acidic polypeptide (1, 2) embedded in a complex assortment of intra-and extracellular protein aggregates derived from dentin (3), bone (4, 5), and osteosarcoma tissues (6-9). We report here on the purification of BMP by means of a combination of differential precipitation, ultrafiltration, and hydroxyapatite chromatography. MATERIALS AND METHODSTen-kilogram batches of 1-year-old steer long bones were obtained from an abattoir. After the epiphyseal ends were cut away with a band saw, the diaphyses were mechanically scraped clean of soft tissues and extensively washed in cold water solution of 3 mM NaN3. The washed bone was frozen in liquid N2, ground in a Wiley mill to a particle size of 1 mm3, defatted in chloroform/methanol (1:1), and again washed in 10 liters of cold water (step 1). The bone particles were demineralized in 0.6 M HCl at 4°C for 48 hr and again extensively rewashed in NaN3 solution (step 2). The demineralized washed bone particles were chemically extracted to remove soluble noncollagenous protein (i.e., sialoproteins, plasma proteins, y-carboxyglutamyl proteins, and phosphoproteins), simultaneously converting the collagen to insoluble bone matrix gelatin (step 3) by previously described procedures (10). Ten kilograms of whole wet bone produced -'1.4 kg of freeze-dried insoluble bone matrix gelatin. The BMP was extracted from the insoluble bone matrix gelatin in an inorganic/organic solvent mixture of 0.5 M CaCl2 in 6 M urea at 28°C for 24 hr containing 10...

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