Preparation and properties of carp muscle parvalbumin fragments A (residues 1 leads to 75) and B (residues 76 leads to 108).
The calcium-binding protein (parvalbumin), isolated from carp (Cyprinus carpio) muscle, has been specifically fragmented into two polypeptides by tryptic hydrolysis at the single arginine residue at position 75. Fragment A contains residues 1 leads to 75 and fragment B is composed of residues 76 leads to 108. The fragments have been characterized according to size, amino acid composition, carboxyl- and aminoterminal analysis. Both fragments appear to be homogeneous by these criteria. The intact protein is known to bind 2 mol of calcium per mol of parvalbumin, and although each fragment alone contains all of the essential ligands for the coordination of one Ca2+, neither fragment displays calcium binding activity. Attempts to reconstitute the two fragments, under a variety of conditions, into a functional complex which can bind calcium have been unsuccessful. The side chain of Arg-75 is known to occupy an internal position in the crystalline structure of parvalbumin (Kretsinger, R.H. and Nockolds, C.E. (1973) J. Biol. Chem. 248, 3313), where it is stabilized by an intricate network of hydrogen bonding involving the side chain of Glu-81. Although this internal salt bridge is approx. 20 A from either calcium binding site, it has been suggested that this structural feature of the molecule plays an essential role in the reversible binding of Ca2+. That the side chain of Arg-75 likewise occupies an internal position in the solution structure is indicated by its unavailability for reaction with 1,2-cyclohexanedione under conditions of physiological pH and temperature. However, in the presence of EDTA and at pH 8, it is readily modified by cyclohexanedione. This modification is accompanied by a concomitant loss in calcium binding activity. Reversal of the modification by treatment with hydroxylamine is accompanied by restoration of calcium binding activity. The serum of these data support the hypothesis that Arg-75 plays a critical role in the structural organization and calcium binding activity of the molecule, and in addition, suggests that the integrity of the peptide bond between Arg-75 and Ala-76 may be necessary for establishing the proper micro-environment required for formation of the internal salt bridge between Arg-75 and Glu-81.