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We examine the effects of concentration (aggregation), buffers, and ligation, under conditions of either constant ligand activity or limited total amount of ligand, upon thermal denaturation of macromolecules as measured by scanning calorimetry. In doing so we utilize and extend an earlier generalized allosteric treatment [S. J. Gill, B. Richey, G. Bishop, and J. Wyman (1985) Biophys. Chem. 21, 1-14], applicable to ligand binding, enthalpy changes, and volume changes in a macromolecular system. The approach is contrasted with formulations based on the idea of structural domains. We show how information from the full scanning calorimetric curves can be utilized in arriving at and testing appropriate models for observed behavior in selected examples.

An allosteric model is presented that provides a simple explanation for the low population of triply ligated species, relative to the other species, in the oxygenation of human hemoglobin tetramers as found in high-concentration studies [Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., & Robert, C. H. (1987) Biochemistry (preceding paper in this issue)]. The model is a quantitative interpretation of the Perutz mechanism [Perutz, M. F. (1970) Nature (London) 228, 726-739] and is based on a number of structural and thermodynamic findings so far reported in the analysis of hemoglobin properties. Human hemoglobin is assumed to exist in two quaternary states: the T or low-affinity state and the R or high-affinity state. An extreme chain heterogeneity in the T state is postulated so that oxygen binds only to the alpha chains. Nearest-neighbor interactions between the alpha chains may lead to cooperativity within the T state. The R state is noncooperative, and both the alpha and beta chains have equal oxygen affinity.

Complete thermodynamic characterization and comparison of ligand-binding reactions of hemocyanins is motivated by the obvious structural similarities and hierarchies in these molecules. We present here a study on the thermodynamics of oxygen binding to lobster (Homarus americanus) hemocyanin, a dodecameric protein made up of two identical hexamers, and we compare our results to those of the well-characterized free-hexameric hemocyanin of the spiny lobster [1]. Our methods are based on the unifying principles of generalized binding phenomena, which have been introduced in detail elsewhere [2]. For this study we conducted two types of experiments. The first type was a simple binding curve, in which we recorded changes in the average saturation of the macromolecule as a function of oxygen activity. From this data we obtained values of the equilibrium constants governing the binding of oxygen. The second type of experiment was a calorimetric measurement of the average-enthalpy change of a hemocyanin solution as a function of oxygen activity. Analysis of this data gave ΔH’s corresponding to the reactions involved in oxygen-binding.

The cooperative oxygen-binding behavior of the 24-subunit tarantula hemocyanin (Hc) has been difficult to quantify in the context of a simple allosteric model [1]. For this reason and because of the obvious structural hierarchies present in tarantula and other Hc’s, we have analyzed 02 binding data using hierarchies of allosteric equilibrium— a “nesting” model, originally conceived by Wyman [2]. The model can be easily generalized to account for a wide variety of structural observations as well as cooperative-binding data of Hc’s or other large macromolecular assemblies.

We measured the dry flux of nitric acid vapor to a pasture near Champaign, Illinois during June of 1982. The mass flux was determined by a modified Bowen-ratio method, employing the surface sensible heat flux and temperature and concentration variations with altitude. We found the daytime average deposition velocity to be 2.5 +- 0.9 cm/s. The average canopy (stomatal and adsorption) resistance averaged virtually zero. The measured June dry flux of HNO/sub 3/ was of the same magnitude as the wet flux measured nearby.