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Effect of anions chaotropes and phenol on the attachment of fad to phenol hydroxylase ec 1.14.13.7



Effect of anions chaotropes and phenol on the attachment of fad to phenol hydroxylase ec 1.14.13.7



Biochemistry 22(3): 580-584



Monovalent anions, ethylene glycol or Mg2+ inhibits the activity of phenol hydroxylase [found in Trichosporon cutaneum and Candida tropicalis] to a degree essentially compatible with the relative degree of their chaotropic power. All these agents affect the spectrum of phenol hydroxylase in a way indicating changes in the hydrophobic interaction between FAD and the enzyme. All agents, except fluoride, abolish the characteristic shoulders on either side of the maximum at 443 nm, bringing the spectrum of phenol hydroxylase closer to that of free FAD. The effect of fluoride is opposite; the shoulders become more accentuated, indicating a more hydrophobic interaction between FAD and the protein than that in the native enzyme. This interpretation is supported by the results of fluorometric measurements. The fluorescence of enzyme-bound FAD is .apprx. 10-fold smaller than that of free FAD. In the presence of several monovalent anions the fluorescence of the enzyme increases significantly, whereas in the presence of fluoride it decreases. Displacement of FAD, at 0.015-0.030 M monovalent anions, not giving easily perceptible changes in the primary spectrum, is indicated by difference spectra in the presence of these agents. Absorption spectra of protein eluates from Sephadex G-25 columns, equilibrated with 0.25-1.0 M azide, cyanide or thiocyanate, indicate complete removal of FAD. The removal of FAD is, to a varying degree, counteracted by low concentrations of phenol. This protective effect of phenol is discussed with view of its known dual function as both effector and substrate of phenol hydroxylase. Spectrophotometric titration of the binding site(s) for phenol reveals 1 binding site of high affinity (Ks .simeq. 10-6 M) and additional binding site(s) of much lower affinity (Ks .simeq. 10-3 M).

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