TRIAZINYLANILINE DERIVATIVES AS FLUORESCENCE PROBES .3. EFFECTS OF CALCIUM AND OTHER METAL-IONS ON THE STEADY-STATE AND TIME-RESOLVED FLUORESCENCE OF BOVINE BRAIN CALMODULIN LABELED AT LYSINE-75

The fluorescence characteristics of bovine brain calmodulin labelled a t lysine-75 by the reactive triazinylaniline (TA) dye p-Et(2)NC(6)H(4) C(3)N(3)(Cl)(2) reveal structural changes critically dependent on calc ium ion binding, Binding of two calcium ions to the C-terminal lobe ex poses a hydrophobic region to the TA fluorophore, enhancing the fluore scence yield four-fold. Addition of two further calcium ions, to the N -terminal lobe, decreases the TA probe fluorescence three-fold by a di splacement of the hydrophobic probe imposed by mutual interaction of t wo protein lobes/regions, The protein matrix induces circular dichrois m in the attached TA dye in a calcium-dependent manner, Auxiliary bind ing of divalent metal ions at mmol dm(-3) concentrations greatly enhan ces probe fluorescence, Despite the above changes the probe rotational relaxation times (8.0 ns) and limiting anisotropies (0.340) are insen sitive to the calcium status of the triazinylaniline-labelled calmodul in (TACaM), Close co-rotation of the probe with a single lobe of the c almodulin carrier is likely, but the possibility of the TA probe optic al transition axis lying parallel to a major axis of protein deformati ons is not excluded, Complex formation with melittin, and the potentia l utility of TACaM in the study of calmodulin-peptide interaction kine tics, are described.