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The configuration of the copper complex of the glycopeptide bleomycin, CuBlm, is presumed to be pyramidal square planar from a previous X-ray structural determination of a fragment of cupric bleomycin. This study presents evidence for a difference in the ESR parameters for cupric bleomycin in the liquid as opposed to the solid state. A decrease in Aiso for CuBlm in the liquid state can be directly surmised from the low frequency S-band spectrum for which three of the four cupric hyperfine lines are partially resolved. Computer simulated spectra infer that the absolute value of All increases about 100 MHz and the value of Al may change sign for CuBlm in the liquid state. Simulations using a rotational correlation time of about 250 psec. indicate that CuBLM may not be spherical in the liquid phase. The fastest component for anisotropic motion could dominate and account for the well resolved cupric hyperfine structure. Furthermore, it is argued from an analysis of the cupric hyperfine coupling constants that the CuBlm structure opens up at room temperature and that the cupric ion is displaced from the square plane.

Diphtheria toxin (DT) induces the leakage of dipalmitoylphosphatidic acid (DPPA) membranes but not neutral dipalmitoylphosphatidylcholine (DPPC) membranes. Cholesterol incorporated into liposomes enhances the membrane leakage induced by DT in acidic DPPA membranes but not in neutral DPPC membranes. Membrane leakage was determined by assaying the release of TEMPOcholine, a cationic spin probe from the multilamellar vesicles by using electron spin resonance methods. The effect of DT on membrane leakage is noticeable at 3 micrograms/ml concentrations, and reaches a plateau of about 20% leakage at 20 micrograms/ml. This saturation phenomenon led to the postulation that DT binds to the first shell of DPPA membranes and induces the leakage of TEMPOcholine limited to this layer of DPPA multimellar vesicles.

The binding sites of purine bases in the presence of the Fe(I)(NO)-2 group were investigated on the basis of the nuclear hyperfine structure of the electron spin resonance spectra. Selective isotopic substitution with 15-NO was used to clarify co-ordindination. Coupling constants and different types of complexes were determined by means of computer-simulated ESR spectra. Comparison was made with the binding sites of nucleotides and it was concluded that N-7, in the imidazole ring, is the preferred binding site. A structure with two base molecules bonded to the iron atom was proposed. Finally 8-azaguanine, an antitumoral agent, was studied and the special behaviour of this antimetabolite was demonstrated in the biologically-interesting pH values.

We have used electron spin resonance (ESR) techniques to study the binding of divalent cations to diphtheria toxin (DT). Addition of DT to Mn(II) solution at a stoichiometry of 2:1 (DT:Mn) induces a 79% loss in the intensity of the ESR spectrum of Mn(II) suggesting a strong binding of Mn(II) to DT. Inclusion of Ca(II) at a ratio of 1:2:1 (Ca:DT:Mn) in the reaction mixture restores the intensity of the Mn(II) signal to 64%. This indicates that Ca(II) and Mn(II) share same binding site(s) in DT. The results presented in this communication suggest that DT is a Ca(II) binding protein.