William P. Bryan

The thermodynamics of water-protein interactions.

Abstract Information about the effects of water on protein structure and function can be obtained from studies on freeze dried protein powders of varying water content. Sorption isotherms of water on proteins can be used to obtain thermodynamic quantities for water-protein interactions. Since such isotherms show hysteresis, there is doubt in regard to their interpretation. General expressions for the thermodynamic quantities of sorption are derived. If isotherms represent data at equilibrium, it is possible to calculate these thermodynamic quantities. There are two types of hysteresis, non-equilibrium hysteresis and equilibrium hysteresis. Absorption and desorption isotherms can show equilibrium hysteresis if different protein conformations, which are only slowly interconvertible, can be present. In this case valid thermodynamic quantities can be obtained. Experimental tests for equilibrium hysteresis are presented. More experiments are needed before definite conclusions can be drawn in regard to isotherms in the literature. If the protein conformation in a protein powder is similar to the protein conformation in aqueous solution, equilibrium data obtained from sorption isotherms can be used to approximate thermodynamic quantities for the interaction of water with proteins in aqueous solution. Examination of what experimental evidence is available indicates that the protein in powders prepared by desorption of water should have a conformation similar to that in solution. Further study of such samples will help to clarify the thermodynamics of water-protein interactions in aqueous solution.

Comparison of standards in the Karl Fischer method for water determination.

Abstract An accurate method for Karl Fischer titrations of water is presented. It involves continuous flushing of the titration cell with rigorously dried carbon dioxide and careful amperometric end-point determination. The water content of sodium tartrate dihydrate was determined, both by titration and drying at 150 °C, and is close to the theoretical value. This work does not support the contention that the dihydrate contains occluded water. Sodium tartrate dihydrate is a good primary standard for the Karl Fischer reagent.

Thermodynamics of water-biopolymer interactions: irreversible sorption by two uniform sorbent phases

In order to understand the mutual interactions between water and a biopolymer, thermodynamic analysis of sorption isotherms of water vapor by the biopolymer is necessary. These isotherms are irreversible and show sorption hysteresis. The reasons for such behavior are not established. As a continuation of previous work, general relationships for thermodynamic quantities of sorption are derived for the general case when the sorbent consists of two uniform phases. As in the case of a single sorbent phase, the Clausius–Clapeyron equation can be used to obtain differential entropies of sorption. Two special cases for the two‐phase situation—equilibrium hysteresis and partial equilibrium hysteresis—are plausible models for the irreversibility seen in water–biopolymer interactions. When differential entropy of sorption is plotted as a function of amount of water sorbed per mole of biopolymer, irregularities are generally seen. It is suggested that these irregularities reflect changes in conformation and/or dynamics of the biopolymer molecule.

Measurement of strongly held water of lysozyme

A method for the measurement of water that is strongly held by proteins is described. This water is slowly removed by vacuum drying of lyophilized protein preparations and can be titrated with the Karl Fisher reagent ( Mitchell & Smith, 1948 ). Drying curves for lysozyme show about 11 to 12 water molecules per molecule of protein, that are strongly held. About three to four of these water molecules are especially hard to remove and may correspond to the three buried water molecules observed in the X-ray analysis of the crystal structure of the protein.

The blue dextran excluded volume of the human erythrocyte membrane.

Permanent holes were produced in hemoglobin-free human erythrocyte ghosts by treatment with lysolecithin. A measured amount of the large probe molecule Blue Dextran 2000 was added to a known amount of the ghost suspension and the mixture made up to a known final volume. The blue dextran in the supernatant was determined and the ghost membrane volume excluded from blue dextran calculated. A plateau in the excluded volume versus amount lysolecithin per ghost curve was observed. This can be taken to indicate complete equilibration of blue dextran into the membrane interior. The excluded volume of 8.7±0.5 μm3 is consistent with a membrane thickness of about 600 A. This large value might be explained by the presence of a gel or low protein density structure at the membranes inner face. Other evidence consistent with such a structure is summarized.