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This paper represents a kinetic study of the binding of carbon monoxide by Lumbricus erythrocruorin. Observations on the quantum yield and the relaxation of the system both to equilibrium and to the steady state realized in the presence of constant illumination under various conditions are reported. The The results of the earlier paper (1), which represent an essentially static approach to the problem, may be summarized as follows:(i) The spectral measurements show the absence of any exact isosbestic points in the binding of CO to deoxyerythrocruorin such as would be expected in the case of a simple process.(ii) The shape of the binding curve (or Hill plot) is profoundly modified under the steady-state conditions produced by the stationary light, the cooperativity being reduced.(Mi) The quantum yield, as determined by the "pulse method," varies, though not greatly, with the saturation of the molecule with CO, passing through a maximum in the middle range of saturations. All these observations were shown to be phenomenologically consistent.The present study, which represents a kinetic approach to the same problem, describes observations on the quantum yield and the relaxation of the system both to the steady state and to equilibrium under various conditions. MATERIALS AND METHODS Earthworm erythrocruorin was prepared as previously described (3) Fig. 1. From an analysis of the data it appears that in case a the reaction was about 70% slow, with 1' = (1.9 4 0.6) X 105 M-1 sec-1, and 30% fast with 1' = (1.4 I 0.4) X 106. In case b, where the process was all fast, 1' = 1.5 X 106 M-1 sec-1, the same as in case a. 2. Action spectra Action spectra for the fast and slow phases of the reaction calculated from the oscillograph traces under conditions a are shown in Fig. 2 and are consistent with the lack of a true isosbestic point reported earlier (1). From Fig. 2 it is clear that the determination of the relative amounts of fast and slow components must depend on the observation wavelength. The figures, 70 and 30%, given above were based on measurements far removed from the point of crossing of the curves in Fig. 2. It was found that if the intensity of the flash was gradually decreased so that only partial dissociation was achieved, the results, both as to the relative amounts of the two components and their rate constants, were unaffected.