MEASUREMENT OF THE SPIN CONCENTRATION OF METALLOPROTEIN SAMPLES FROM SATURATION-MAGNETIZATION DATA WITH PARTICULAR REFERENCE TO CYTOCHROME-C-OXIDASE

A protocol for obtaining high-quality saturation-magnetization data fr om metalloprotein samples, employing a superconducting quantum interfe rence device (SQUID) magnetometer, has previously been reported [E. P. Day, T. A. Kent, P. A. Lindahl, E. Munck, W.H.Orme-Johnson, H. Roder and A. Roy (1987) Biophys. J, 52, 837-853 and E. P. Day (1993) Methods Enzymol. 227, 437-463]. Following studies of several dozen different metalloprotein derivatives, the methodology has been further refined, particularly in the area of sample preparation. The details of the sam ple-handling procedures now in use are described, and moreover, the cr itical issue of verifying that contamination by-paramagnetic impuritie s remains insignificant is considered. Importantly, it is shown that a n independent determination of the quantity of paramagnetic sample pre sent in the magnetometer is undesirable. Much more reliable parameters concerning the ground-state magnetic properties of the system under s tudy are obtained if enough saturation-magnetization data are collecte d to enable the spin concentration to be determined during the subsequ ent fitting procedure. As proof of the validity of this method, the re sults of magnetization studies on ferricytochrome c, ferrocytochrome c and the benzohydroxamic acid adduct of horseradish peroxidase are pre sented. The ability of saturation-magnetization measurements to routin ely determine spin concentration to within+/-4% of accepted values is firmly established; In addition, a saturation-magnetization study has been performed on resting and fully reduced derivatives of cytochrome c oxidase. These results provide an illustration of the usefulness of the technique in probing some systems which have proved difficult to s tudy by other methods. The increased difficulties inherent in obtainin g meaningful data from these cytochrome c oxidase and other integer sp in systems are delineated.