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

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Abstract
The visible cobalt circular dichroism (CD) of cobalt-substituted concanavalin A (Con A), a metallolectin isolated from the Jack bean, Canaualia ensiformis, 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 ethylenediaminetetraacetic 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 400-600 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 .dblarw. CaCPS .dblarw. CaCPR, in which the 2nd equilibrium is slow compared to the first. The above assignments allow a more complete quantitative description of the changes in CD amplitudes and bnad shapes due to Ca2+ binding and thus facilitate the understanding of Ca2+ interactions. Ca binds to 0.93 Ca2+ site/25,500 MW in CaCPS, with a Kd for Ca2+ = 2.1 .times. 10-3 M at pH 5.3 and 25.degree. C. The interaction of Ca2+ with CPS to form CaCPS occurs at 2 equivalent and noninteracting S2 sites, each present on separate subunits of the Con A dimer. The rate constant describing the rate of formation of CaCPR was determined.