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The epitopes (antigenic determinants) recognized by four different monoclonal antibodies on horse cytochrome c have been partially characterized by differential acetylation of lysine residues of free and antibodybound cytochrome c. The degree of acetylation in the bound and free antigen molecule was assessed by a double-labeling procedure with [3H]acetic anhydride and [14C]acetic anhydride. Out of the 19 lysine residues of cytochrome c only very few were less reactive in the antigen-antibody complex, i.e. presumably located at the epitope for the antibody under study. The protection varied from 1.5-fold to over 20-fold lower reactivity in antibody-bound cytochrome c. The present results are complemented by previous data obtained by cross-reactivity analysis with cytochromes c from different species, with chemically modified cytochrome c derivatives, and by inhibition of proteolysis of cytochrome c in the presence of the antibodies. From the combined data we conclude that each of the four epitopes depends on the precise spatial folding of the antigen and contains residues which are brought together by the folding of the polypeptide chain. This work exemplifies that mapping of conformationdependent epitopes can be achieved by applying a combination of mapping procedures of which each by itself provides partial information.

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The kinetics of carbon monoxide binding to phenobarbital-induced cytochrome P-450 (P-450pB) and to its enzymatically inactive form P-420pB have been investigated by both stopped-flow and flash-photolysis spectrophotometry.When the simultaneous presence of both forms of the enzyme is taken into account, the binding of CO to these two proteins can be described in terms of two bimolecular processes with rate constants of 4.5 x 106 M-l s'1 and 4.7 x 10' M-ls'1 for P-450PB and 1.7 x 107 M-l s'1 and 1.5 x 106 M-l's'1 for P-420pB. From kinetic studies of the binding of CO to P 45OPBunder different experimental conditions, investigations of the homogeneity of our P-450pB preparations, and comparative kinetic investigations of P-450s from different sources, we conclude that CO binding to reduced P-450PB is a simple bimolecular process and that the observed biphasic traces are due to heterogeneity of the proteins. This conclusion is in contrast with previous reports of complex reaction mechanisms for the binding of CO to P-45OPB. ) and a fourth, very slow, monomolecular one (k4 = 0.3-0.6 s-1) when the measurements were done by stopped-flow techniques (9). From this rather confused picture, it is impossible to propose a reaction mechanism for the binding of ligands to ferrous P-450PB.However, the diversity of the results suggests that the preparations used by the different groups varied in their protein composition.One possible source of complication may be the ease by which P-450 converts to P-420 (12), a catalytically inactive form of the enzyme capable of binding CO and whose carbonyl derivative has an optical absorption spectrum with the Soret peak around 420 nm. For most of the previously reported kinetic studies on P-450PB, the presence of P-420PB cannot be excluded. In fact, only Debey et al. (7) and Galeotti, Dani, and Brunori (1975, personal communication) seem to have taken heed of this problem. Therefore, we have investigated the kinetics of CO binding to P-420PB and to P-450PB under a variety of experimental conditions, with the ultimate aim of learning whether a complex model is required to explain ligand binding to ferrous P-450 from liver microsomes (10) or whether a simple bimolecular mechanism is sufficient, as it is for other P-450s (13, 14). METHODSPreparation of P45OPB and P-420PB. P-450PB was purified from liver microsomes of phenobarbital-induced male Sprague-Dawley rats (150-170 g) as described (15). The P-450 content of the preparations was determined according to Gut et al. (15). Further purification of the isozymes P-450PB4 and P-450PB5 was achieved by hydroxylapatite chromatography (16). The P-450PB preparations, before and Abbreviations: P-450, cytochrome P-450; P-45OPB, phenobarbitalinduced P-450 from rat liver microsomes; CHAPS, 3-[(3-cholamido-propyl)dimethylammonio]-l-propanesulfonate.

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