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The active sites of heme proteins are closely surrounded by protein side chains in a manner which provides steric hindrance to entering ligands.* 1•2 For example, hemoglobin binds isonitriles with affinities decreasing in the order EtNC > t-PrNC > t-BuNC,3 a series also found in hindered model compounds.43 Additionally, the observations that the Fe-OO bond is bent from the heme perpendicular in both heme proteins and unhindered model compounds5 whereas such distortion occurs only in heme proteins for FeII *1-C06"9 groups seem to support the suggestion10 that the proteins reduce the ratio of CO binding to 02 binding through distal side steric effects.2•11•12 Using two new cyclophane hemes having different amounts of steric hindrance to ligand binding, we find that CO and 02 are subject to the same magnitudes of steric effects and are thus not differentiated by distal side steric effects.Comparisons of affinities of CO and 02 for simple unhindered hemes with those of heme proteins have led to conflicting conclusions concerning distal steric effects.11•13"16 Such comparisons are subject to uncertainties because dioxygen affinities are sensitive to solvent polarity,17 and both carbon monoxide and dioxygen affinities are affected by structural changes which could prefer-

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to 200 ml of water containing 5.8 ml of acetic acid. After standing for 1 hr, the precipitate was collected and washed with cold water. The crude product (mp 319°, 62-74% yield) was crystallized from hot water (25-50% yield): mp 347°; ir (KBr) 3350 (broad), 3180 (broad), 2880 (broad), 1720, 1680,

We describe a simplified procedure for purification of Clostridium difficile toxin B. In this procedure, cytotoxicity is associated with a single protein band with a molecular mass of 230 kilodaltons. We used direct fluorescent staining of actin filaments to study the effect of this toxin on cultured cells. Morphologic changes were preceded by a decrease in the number and length of stress fibers followed by their disappearance with condensation of cellular actin around the nucleus. We then showed that cells treated with either cytochalasin B or toxin B had a significant increase in the monomeric actin pool as quantitated by DNase I inhibition. In contrast to the cytochalasins, toxin B had no direct effect on the rate or extent of actin polymerization or network formation in vitro. Cytoplasmic extracts of toxin B-treated cells had a significantly lower level of modulating activity on actin assembly and interactions in vitro compared with extracts of untreated cells. These results suggest that the action of toxin B on cells is due to direct or indirect effects on cellular proteins involved in controlling the state of actin assembly in the cells.