Selective extractability of noncollagenous proteins from chicken bone.
Quantitative analyses of a wide variety of different solvents used for the extraction of several of the noncollagenous proteins of fully mineralized chicken bone powder were carried out to compare both the effectiveness of various procedures and the distribution of specific proteins which were solubilized. Extraction procedures included solutions of 6 M guanidine-HCl, pH 7.0, 0.5 M EDTA, pH 7.4, 0.3 N citric acid, 0.3 N HCl, 0.3 N formic acid, and 0.3 N acetic acid. Chelation of calcium ions by EDTA and dissolution of the mineral phase by acid extraction released 95% or more of the total calcium content of the bone powder by 48 hours, guanidine-HCl released less than 20% or less of the total calcium content even when extraction was carried out by 168 hours. Moreover, although guanidine-HCl solubilized a significant amount of collagen as gelatin, essentially none of the phosphoproteins, osteocalcin, or the proteoglycan decorin were solubilized, as detected by immunological techniques. In contrast, extraction of the mineralized bone powder by HCl and formic acid was very efficient in selectively solubilizing osteocalcin and osteopontin, while bone sialoprotein was selectively released by EDTA, and solubilized to a lesser extent by formic acid. Similarly, EDTA selectively removed decorin compared with HCl, formic, acetic, or citric acids. Only small amounts of osteopontin and osteocalcin were detected in the acetic acid extracts. These results provide methods for the selective solubilization of several different major, noncollagenous proteins from mineralized bone which should significantly aid in maximizing the amount of the specific protein recovered, and the ease with which the various proteins can be purified.(ABSTRACT TRUNCATED AT 250 WORDS)
Gerstenfeld
L C
LC
Department of Orthopaedic Surgery, Harvard Medical School, Children's Hospital, Boston, Massachusetts 02115.
Feng
M
M
Gotoh
Y
Y
Glimcher
M J
MJ
D000818:Animals; D015153:Blotting, Western; D001842:Bone and Bones / Q000737:chemistry*; D002645:Chickens; D003094:Collagen / Q000032:analysis; D058575:Decorin; D004492:Edetic Acid; D016326:Extracellular Matrix Proteins; D008903:Minerals / Q000032:analysis; D015675:Osteocalcin / Q000032:analysis; D053495:Osteopontin; D011509:Proteoglycans / Q000032:analysis; D012795:Sialoglycoproteins / Q000032:analysis*
D058575:Decorin; D016326:Extracellular Matrix Proteins; D008903:Minerals; D011509:Proteoglycans; D012795:Sialoglycoproteins; D015675:Osteocalcin; D053495:Osteopontin; D003094:Collagen; D004492:Edetic Acid