Section 25
Chapter 24,373

Complex formation of 1,10-phenanthroline with zinc ions and the zinc of alcohol dehydrogenase of horse liver

Vallee, B.L.; Coombs, T.L.

Journal of Biological Chemistry 234: 2615-2620


ISSN/ISBN: 0021-9258
PMID: 13840891
Accession: 024372790

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The complexes were studied by means of the method of continuous variations. The formation of [ZnOP1]++, [ZnOP2]++, and [ZnOP3]++ complexes has been confirmed and their extinction coefficients at 3275 A have been established. One mole of 1,10-phenanthroline interacts with each of the 2 Zn atoms of horse liver alcohol dehydrogenase to form a mixed complex [(LADH)Zn2.OP2] which has absorption maxima at 2972, 3287, and 3450 A. This mixed complex, demonstrated spectrophotometrically, represents the enzymatically inactive but dissociable enzyme-inhibitor complex postulated from kinetic studies. The extinction coefficient of the complex is 1700 + 100 and the dissociation constant is 3.3 + 0.5 x 10-5 [image]. This constant is numerically close to that which was derived from kinetic data and its magnitude supports the validity of the mechanisms of the interaction of 1,10-phenanthroline with the Zn atoms of alcohol dehydrogenase of hor6e liver previously suggested. Consequently, 1,10-phenanthroline can be employed simultaneously to measure the Zn of the enzyme and the directly correlated disappearance of enzymatic activity. The approach described here provides a means of establishing the intrinsic dissociation constants of such systems as these. With a series of chelating agents of different degree of binding strengths, the "true" value of the constant is susceptible to measurement by a series of successive approximations.

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