Publisher Summary This chapter explores that the changes that take place in the protein molecules during denaturation constitute one of the most interesting and complex classes of reactions that can be found either in nature or in the laboratory These reactions are important because of the information they can provide about the more intimate details of protein structure and function They are also significant because they challenge the chemist with a difficult area for the application of chemical principles The chapter reviews that the denaturation is a process in which the spatial arrangement of the polypeptide chains within the molecule is changed from that typical of the native protein to a more disordered arrangement The chapter also discusses the classification of protein structures: primary, secondary, and tertiary structures The primary structure is that expressed by the structural chemical formula and depends entirely on the chemical valence bonds that the classical organic chemist would write down for the protein molecule The secondary structure is the configuration of the polypeptide chain that results from the satisfaction of the hydrogen bonding potential between the peptide N-H and C=O groups The tertiary structure is the pattern according to which the secondary structures are packed together within the native protein molecule The term “denaturation” as used in this chapter is indented to include changes in both the secondary and tertiary structures

Some factors in the interpretation of protein denaturation.

Although electrophoresis is one of the most effective methods for the separation of ionic components of a mixture, the resolving power of different electrophoretic methods is quite variable. To separate two component ions, it is necessary to permit migration to continue until one of the kinds of ions has traveled at least one thickness of the volumes that it initially occupied (the starting zone) further than the other. However, the sharpness, and therefore the resolution, of the zones occupied by each ion diminishes with time because of the spreading of the zones as a result of diffusion. Remarkable resolution has been achieved when advantage is taken of the frictional properties of gels to aid separation by seiving at the molecular level (see Smithies’). A new method, disc electrophoresis, t has been designed that takes advantage of the adjustability of the pore size of a synthetic gel and that automatically produces starting zones of the order of 10 microns thickness from initial volumes with thicknesses of the order of centimeters. High resolution is thus achieved in very brief runs. With this technique, over 20 serum proteins are routinely separated from a sample of whole human serum as small as one microliter in a 20-minute run (see FIGURE 1) . Direct analysis of even very dilute samples becomes routine because the various ions are automatically concentrated to fixed high values at the beginning of the run just prior to separation. Preliminary laboratory studies and theoretic considerations provide evidence of the applicability of this technique to a wide range of ionic species for both analytic and large-scale preparative purposes. Theory has also provided the basis for a simple application of disc electrophoresis to the simultaneous determination of both the free mobility and the aqueous diffusion constant of a protein. This report will detail some mechanisms that provide a rationale for the resolution afforded by zone electrophoresis in many gels; will develop the theory of some new modifications of zone electrophoresis that have been designed to take maximum advantage of these mechanisms; and will provide some examples of the results that disc electrophoresis has produced.

Disc electrophoresis-i background and theory*

Adsorption of proteins from solution at the solid-liquid interface.

The specific binding of L-tryptophan to serum albumin.

Publisher Summary This chapter reviews that titration curves do not represent just another way of physically characterizing a protein molecule. More than most other physicochemical methods that are in common use, titration studies tend to emphasize individual differences among proteins, and this is reflected in the chapter. The chapter contains experimental procedures of general utility in the determination of titration data. The foundation for any study of hydrogen ion dissociation in proteins is the electrometric titration curve. To obtain such a curve, one begins with a protein solution of known concentration, at an arbitrary reference pH, adds to it varying amounts of a strong acid or a strong base, and then measures the new pH attained. In a separate experiment, or by means of calculations based on similar experiments, one determines how much acid or base is needed to take a solution, which does not contain protein, but otherwise has the same initial pH, ionic strength, and volume. The difference between the two amounts is the amount of acid or base that is bound to the protein in going from the reference pH to the final pH: a plot of this quantity versus the final pH is the desired titration curve. In plotting this curve, OH - ions bound are counted as H + ions dissociated a procedure, which is always permissible in aqueous solutions.

William S. Shore

Papers

Hydrogen Ion Equilibria of Bovine Serum Albumin1

Electrostatic Effects in the Combination of Cobalt with Amino Acids1,2

Removal of the Impurity in Bovine Serum Albumin and Its Effect on Zinc Binding