Studies on the interaction of cibacron blue and procion red with dopamine β-monooxygenase

Abstract

1. 1. The interaction of Procion Red HE3B and three isomers of Cibacron Blue with the water-soluble form of the copper enzyme dopamine β-monooxygenase (3,4-dihydroxyphenylethylamine, ascorbate: oxygen oxidoreductase (β-hydroxylating), EC 1.14.17.1) was studied by enzyme inhibition, difference spectroscopy and binding of enzyme to the immobilized dyes. 2. 2. Cibacron Blue 3GA gave noncompetitive inhibition with both tyramine and ascorbate as the variable substrates (competitive inhibitor constants of 1–4 μM and uncompetitive inhibitor constants of 5–7 μM). 3. 3. Difference spectral titration of the apoenzyme (Cu-depleted) with Cibacron Blue 3GA indicated binding of four dye molecules per enzyme tetramer and gave Scatchard plots with upward curvatures, which implies that the dye either interacts at different classes of sites or that there is a negative cooperativity in the binding. The holoenzyme binds about eight dye molecules per tetramer. Addition of CuSO 4 to the apoenzyme indicated that the enzyme must attach five or slightly fewer copper atoms per tetramer to obtain the maximal binding of eight dye molecules per tetramer. The dissociation constant for the dye binding to the four Cu-dependent sites is in the same range as the competitive inhibitor constants, thus indicating that this binding of the dye is at the active site. These results indicate that dopamine β-monooxygenase contains four active sites per tetramer with one copper atom per active site. 4. 4. Different isomers of Cibacron Blue gave dissimilar difference spectra with dopamine β-monooxygenase, thus emphasizing the importance of using pure isomers of the dye when studying the interactions with proteins. 5. 5. The results presented indicate that Cibacron Blue can bind strongly to