Estimates of the strength of iron binding to model phosphoproteins were obtained from equilibrium dialysis experiments. Iron-free phosvitin (chicken and frog) or alpha sl-casein (cow) was dialyzed against the iron(III) chelates of nitrilotriacetate (NTA), )ethylenedinitrilo)tetraacetate (EDTA), or citrate. Protein-bound metal was measured at equilibrium; competition of chelator and phosphoprotein for iron(III) was determined by reference to comprehensive equilibrium equations presented in the Appendix. Analysis of the iron-binding data for phosvitin suggested that clusters of di-O-phosphorylserine residues (SerP.SerP) were the most probable iron-binding sites. A stoichiometric equilibrium constant of 10(18.0) was calculated for the formation of the Fe3+(SerP.SerP) chelate. When comared on the basis of phosphate content, casein bound iron more weakly than phosvitin. However, if the stoichiometric equilibrium constant for the formation of the casein Fe3+(SerP.SerP) chelate (10(17.5) was adjusted to account for the fact that a smaller percentage of casein phosphoserines occurs in di-O-phosphorylserine clusters, the affinity of casein and phosvitin for iron was very similar. A theoretical comparison showed that the "strengths" of the ferric chelates can be ranked: EDTA greater than phosphoprotein di-O-phosphorylserine greater than citrate greater than NTA.

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The large intestinal flora of the leopard frog, Rana pipiens, was examined to determine whether differences existed between the nonhibernating and hibernating states of the animal and to determine the relative concentrations and proportions of potential frog pathogens. Hibernators had a logarithmic decrease of bacteria per milligram of intestine averaging one, and significantly greater proportions of facultative bacteria and psychrophiles relative to nonhibernators. The predominant anaerobic bacteria were gram-positive Clostridium species and gram-negative Bacteroides and Fusobacterium species. The predominant facultative bacteria were enterobacteria in nonhibernators but Pseudomonas species in hibernators. Many species of Pseudomonas are pathogenic for frogs, and thus the intestinal flora in hibernators may be a potential source of infectious disease.

The bacteria in the large intestines of 10 northern leopard frogs (Rana pipiens) were enumerated and partially characterized. Four nonhibernating frogs were collected in the summer, four hibernating frogs were collected in the winter, and two frogs ust emerged from hibernation were collected in the spring. All frogs had about 101 bacteria per g (wet weight) of intestinal contents and about 109 bacteria per g (wet weight) of mucosal scraping, although the counts from the winter frogs were slightly less than those from the other two groups of frogs. Another group of 14 summer frogs, after treatment to induce hibernation, showed a drop in bacterial counts accompanied by a change in the composition of the flora. In most frogs, Bacteroides was the dominant organism. Other bacteria repeatedly isolated at high dilutions were strict anaerobes, including butyrigenic and acetogenic helically coiled bacteria; fusobacteria; and acetogenic, small, gram-positive bacilli. These data indicate that the intestinal flora of frogs is similar to that of mammals and birds and that this flora can be maintained at temperatures close to freezing.