Calcium binding to bone y-carboxyglutamic acid protein (BGB) from calf has been studied using 43Ca NMR. The temperature dependence of the 43Ca NMR signal has been used to calculate the calcium ion exchange rate, koff. The dependence of the 43Ca NMR band shape on the [Ca2+]/[BGP] ratio fits well to a chemical equilibrium model having a single Ca2+-binding site with an association constant in the range of 5 x lo3 -1 x lo5 M-I. The pH dependence of the 43Ca NMR line-width shows a single apparent pK, value of 5.1.Bone from mammals, birds and fishes contains a lowmolecular-mass non-collagenous protein, in which the unusual amino acid y-carboxyglutamic acid is found [l, 21. The protein, which has been named osteocalcin or bone y-carboxyglutamic acid protein (BGP), consists of a single polypeptide chain of about 50 amino acid residues, two or three of which are y-carboxyglutamic acid [3 -51. y-Carboxyglutamic acid, which is also found in some of the coagulation factors and related plasma proteins [6], is formed by a vitamin-K-dependent posttranslational carboxylation of certain glutamic acid residues in the protein polypeptide [7, 81. Most of the BGP is located in the extracellular bone matrix, adsorbed to the mineral crystals [9-121, but BGP also occurs in plasma at low concentrations [13- Abbreviations. Gla, 9-carboxyglutamic acid; BGP, bone Gla protein, osteocalcin; HPLC, high-performance liquid chromatography; SDS, sodium dodecyl sulphate.Note. The rotational correlation time ( 7 : ' ) of BGP can be estimated to approximately 2 ns at 20°C from the Debye-StokesEinstein equation [25]. We are thus able to determine an upper limit of 02, in our experiments: w t 2 <0.22 (w = 1.078. 10' s-' and ~~ rc <2 . 10-9 s~.technique, which has proven to be very useful in the study of various kinds of calcium-binding protein. BGP from calf bone was chosen because it is well characterized [3] and calf bone can easily be obtained in large quantities. A simple procedure for the large-scale purification of BGP from calf bone is also presented.
MATERIALS AND METHODS
Preparation and characterization of BGPBGP was isolated from calf bone by a large-scale purification procedure. In order to avoid any interference from a strong chelator (such as EDTA) in the NMR cation-binding studies [23] the bone was demineralized in acetic acid instead of using the more common EDTA procedure. The isolated protein was characterized by SDS polyacrylamide gel electrophoresis, HPLC, amino acid analysis and sequence determination. The isolated BGP was judged to be at least 95% pure and the analytical data, including Gla content, were in good agreement with published data on the protein [2,3]. The details of the purification procedure and the results of the characterization of the purified protein are given in the miniprint supplement.
BGP solutions for NMR measurementsCa2 + -free BGP was prepared by passing an aqueous solution of the protein through a column of Chelex-100 (BioRad Labs., Richmond, CA, USA) resulting in a protein containing less than 0.1 equ...
