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The rates of exchange of the C-2 protons of histidine residues in copper-zinc superoxide dismutase are substantially decreased by metal ion binding. This observation was used to distinguish between ligand and non ligand histidine residues in bovine and yeast copper-zinc superoxide dismutases; the effect was shown to depend only on metal ion co-ordination and not as a consequence of concomitant changes in protein structure. Selective deuteration of the zinc-only proteins at pH (uncorrected pH-meter reading) 8.2 and 50 degrees C resulted in the distinction between copper and zinc ligand resonances in the 1H n.m.r. spectrum of the enzymes. This method is proposed as a generally applicable technique for identifying histidine residues as ligands in metalloproteins.

The spectra of the α, β, and γ forms of zinc monoarsanilazotyrosine-248 carboxypeptidase A are indistinguishable. At pH 8.2 their crystals are yellow, while their solutions are red, λ max 510 nm. Absorption and circular dichroism-pH titrations of the modified zinc and apoenzymes demonstrate that the absorption band at 510 nm is due to a complex between arsanilazotyrosine-248 and the active-site zinc atom. Two pK app values, 7.7 and 9.5, characterize the formation and dissociation of this arsanilazotyrosine-248·Zn complex. On titrations of the apoenzyme, the absorption band at 510 nm is completely absent at all pH values. Instead, there is a single pK app , 9.4, due to the ionization of the azophenol, λ max 485 nm. Substitution of other metals for zinc results in analogous intramolecular coordination complexes with absorption maxima and circular dichroism extrema characteristic of the particular metal. Similar data and conclusions have been derived from studies of heterocyclic azophenol·metal complexes. The present studies demonstrate that the conformation of the crystals of all generally available α, β, and γ forms of the arsanilazoenzyme differs from that of their solutions. The spectra of the modified x-ray crystals, however, differ from those of all other carboxypeptidase forms and crystal habits studied. The internal consistency of their data, their interpretation, and the conclusions of Lipscomb and coworkers [ Proc. Nat. Acad. Sci. USA (1972) 69, 2850-2854] are examined. Dissimilar chemical modification or conformation is thought to underlie these differences. The arsanilazotyrosine-248·zinc complex is a sensitive, dynamic probe of environmental conditions. Its response to changes in pH and physical state of the enzyme suggest different orientation of the arsanilazotyrosine-248 side chain in solution from that in the crystal. This finding calls for reexamination of the basis of the substrate-induced conformation change which has been thought to be critical to the mechanism, postulated on the basis of the x-ray structure analysis performed at pH 7.5.

The hydrogen-deuterium exchange of diisopropyl phosphate-inhibited subtilisin (DIP-subtilisin) Carlsberg type and DIP-subtilisin Novo type has been measured in the pD interval 2–8. In the pD range 4.5–8 the native DIP-subtilisins demonstrated closely related exchange properties, especially with respect to the more slowly exchanging fractions of the peptide hydrogens, and this is taken as support for the hypothesis that the two subtilisin molecules have the same internal folding of the peptide chains. The subtilisins exhibit slower exchange rates than any other proteins thus far investigated. The acid denaturation of subtilisin Carlsberg from pD 2–4 is accompanied by the formation of β-structure. A similar transformation was not observed with subtilisin Novo.

The breakdown strength of rutile crystals has been measured ∥ and ⊥ to the optic axis. The dc breakdown is a thermal process occurring at a power level of 2 kw/cm3 for the particular sample size and test cell used here, with field strengths of 20 kv/cm ∥ to the axis and 120 kv/cm ⊥. Impulse measurements at 1‐μsec rise time required special techniques of sample and electrode preparation in order to prevent field distortion and surface breakdown. Breakdown strengths of 570 kv/cm ⊥ and 690 kv/cm ∥ to the optic axis were measured.