Quantitative analyses of calcium-induced spectral changes in extrinsic Cotton effects of cobalt-substituted...

The visible cobalt circular dichroism (CD) of cobalt-substituted concanavalin A (Con A) is highly sensitive to Ca2+-induced conformational changes that occur in the metal binding region. The observed ellipticity is separately resolved into discrete conformational spectra with separate extrinsic bands. The conformational forms of the metal region are further delineated on the basis of their differential spectral response to the competitive removal of metals by ethylene-diaminetetraacetic acid (EDTA). The spectral forms sensitive to the effects of EDTA, cobalt-Con A (CPS, epsilon CPS470 - 215 M-1) and calcium-cobalt-Con A (CaCPS, epsilon CaCPS470 = 141 M-1), exhibit both unique extinctions and band shapes in the 400-500-nm region, as does the fully metalized EDTA-resistant species CaCPR (epsilon CaCPR470 = 54 M-1). Equations describing the time dependence of the observed ellipticity have been derived in terms of the kinetic scheme, CPS + Ca in equilibrium or formed from CaCPS in equilibrium or formed from CaCPR, in which the second equilibrium is slow compared to the first. The above assignments allow a more complete quantitative description of the changes in CD amplitudes and band shapes due to Ca2+ binding and thus facilitate the understanding of ca2+ interactions. Calcium binds to 0.93 Ca2+ site/25 500 Mr in CaCPS with a Kd for Ca2+ = 2.1 x 10(-3) M at pH 5.3 and 25 degrees C. The interaction of Ca2+ with CPS to form CaCPS occurs at two equivalent and noninteracting S2 sites each present on separate subunits of the Con A dimer. Furthermore, the rate constant describing the rate of formation of CaCPR was determined.