A colorimetric assay specific for gamma-carboxyglutamic acid-containing proteins: its utility in protein purification procedures.
A colorimetric method for the detection of gamma-carboxyglutamic acid (Gla)-containing proteins after reaction with 4-diazobenzenesulfonic acid is presented. Proteins can be visualized after electroblotting from polyacrylamide gels onto membrane supports, after dot-blotting onto membranes, or in solution as a red colored product with an absorbance maximum at 530 nm. The method is specific since other proteins without gamma-carboxyglutamic acid do not form a red color. The presence of other proteins does not inhibit or affect color production by gamma-carboxyglutamic acid-containing proteins. Application of the method for staining a Western blot of a crude extract of bone resulted in staining of only the gamma-carboxyglutamic acid-containing proteins. The usefulness of the method was verified when a second gamma-carboxyglutamic acid-containing protein, prothrombin, also resulted in red color production. A linear color response is seen up to 17 microM for the gamma-carboxyglutamic acid-containing protein bone Gla protein and up to 27 microM for the amino acid. The detection limit is down to 1 microgram of bone Gla protein or 0.17 nmol of the protein on electroblots or dot blots. The simplicity of the method allows rapid screening for gamma-carboxyglutamic acid-containing proteins or allows monitoring of purifications of these proteins in chromatographic or electrophoretic separations.
Nishimoto
S K
SK
Department of Biochemistry, University of Tennessee, Memphis 38163.
D015055:1-Carboxyglutamic Acid / Q000032:analysis*; D000818:Animals; D015153:Blotting, Western; D001842:Bone and Bones / Q000032:analysis; D003124:Colorimetry / Q000379:methods*; D003979:Diazonium Compounds; D004591:Electrophoresis, Polyacrylamide Gel; D015675:Osteocalcin / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification*; D051381:Rats; D013053:Spectrophotometry; D013425:Sulfanilic Acids
D003979:Diazonium Compounds; D011506:Proteins; D013425:Sulfanilic Acids; D015675:Osteocalcin; D015055:1-Carboxyglutamic Acid; C000469:diazobenzenesulfonic acid