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The applicability of the methodology of disc electrophoresis to the determination of the component molecular weights of complex protein mixtures has been investigated. To this end, variable factors in the formation of polyacrylamide gel columns have been studied, along with their influence on the determination of relative mobilities. The effect of gelling time was demonstrated as a major factor in this regard, and the influence of catalyst and monomer concentration on this parameter defined. Migration of proteins of established amino acid composition at different pH's has been shown to vary in a parallel fashion to the alteration in charge of the protein. As a result of these investigations, the adoption of a standardized set of conditions which allow for the accurate estimation of the molecular weights of the protein components of biological systems has been made possible, a method that seems capable of wide application in assessing protein interrelationships.

A detailed study of the chromatographic fractionation of baleen S-carboxymethyl-kerateines has been made, using a system of automated step-wise elution. A comparison of the total kerateine preparations from baleen cells of widely differing inorganic content suggests a variation in protein proportions with the degree of mineralisation. The type of chromatographic system used is described and its reliable reproducibility suggests it to be suitable for quantitative analyses of complex protein mixtures.

It has now become of interest to study the structure of the synthetic polypeptides as they form simple models to use in the study of the more complex protein structure. In a very wide field, this has been done from the point of view of the helical and extended forms of the α and β polypeptide structures, and many polypeptide configurations have been characterized. In this paper, studies are made of the α, β and ω forms of the poly-β-benzyl- l -aspartates and their structural transformations on heating, including the types of energy transforms (endothermic or exothermic) accompanying these modifications. These latter changes have also been followed by X-ray and i.r. studies which have enabled us to characterize the forms unequivocally. In one of the poly- β-benzyl- l -aspartates, the transformation on heating has been shown to be α → ω → β; α → ω transformation is exothermic and the ω → β endothermic in character.

Lp(a)-lipoprotein of human serum, when isolated by ultracentrifugation and gelfiltration on Sepharose 4 B, may disaggregate into several components either spontaneously or by mild treatment with detergents

Elford, Grabar and Fischer1 have reported that the “antibody activity” of antipneumococcal horse-serum is associated with the “larger complex protein in the serum.” Thus it was estimated by the method of optimal proportions that 75% of the antibody passed a 140mμ membrane, rather less than one percent passed an 80mμ membrane, while the filtrate from a 54mμ membrane showed no trace of antibody-activity.The membranes used in the present experiments were prepared by the method of Elford2 with certain modifications described by Bauer and Hughes.3 For filtration, Type I antipneumococcal horse-and rabbit-sera were diluted 1:5 in broth. Concentrated antipneumococcal horse-serum was diluted 1:10 in the same reagent. For evaluating the amount of antibody in the filtrate the quantitative precipitation method of Heidelberger, Sia, and Kendall4 was used. Because of the difficulties in securing large amounts of filtrates complete analyses over a wide range of amounts of polysaccharide were impossible. Therefore an arb...

Publisher Summary This chapter focuses on dietary proteins and synthesis of tissue proteins. The synthesis of a complex protein molecule is dependent on the presence of all the essential amino acids. The importance of the time factor has been demonstrated in two types of experiments. Firstly, the intravenous injection of tryptophane to dogs previously fed on acid hydrolysate of casein, deficient only in this essential amino acid, can bring about nitrogen retention, which otherwise will not take place. However, the supply of tryptophane must not be unduly delayed. Secondly, it was shown that if animals were offered two deficient proteins simultaneously, which can adequately supplement one another for growth requirements, such a mixture can be readily utilized for the synthesis of body tissues. However, if the second protein were given long after the first one had been ingested, the animal received no benefit from the mixture. The hypothesis that the synthesis of tissue proteins is dependent on the presence of a nutritional factor or factors receives a further support in that dogs so severely depleted of their protein reserves as to cause a sharp decrease in the total circulating alpha globulins, cannot utilize nitrogen from an otherwise adequate casein hydrolysate, and will ultimately end in death.

Publisher Summary This chapter reviews that the starch-gel electrophoretic method can be applied to the investigation of many complex protein mixtures in addition to serum proteins, for example, enzymes, hormones, and tissue extracts. One aim of this chapter is to describe the current technical problems associated with starch-gel electrophoresis and to consider their possible solutions. A second aim is to summarize the information already obtained by application of the method to studies of serum proteins and their inheritance, and to put this information into perspective in relation to future work. The chapter discusses the experimental procedures used for starch-gel electrophoresis and focus on the technical details is important for the realization of the best starch-gel separations. It provides a summary of the data relevant to the specific identification of serum components demonstrated by starch-gel electrophoresis. The chapter also reviews the results and significance of recent studies of serum proteins using the method. The nature of the physical factors governing the electrophoretic separations obtained in starch gels is discussed. The hypothesis is considered that molecular size plays a particularly important role in the separation processes, and the evidence in support of this is presented. Evidence concerning the validity of the results obtained in starch gels is also reviewed.

A continuous preparative electrophoresis apparatus is described. It has proven extremely useful as a practical instrument for the physical isolation of electrophoretically homogeneous subfractions of complex protein systems in quantities sufficient for extensive analytical and immunological studies. The procedure, developed specifically for the study of antigen-antibody systems in parasitic diseases, is also suitable for preparative fractionation of lower molecular weight substances. The advantages of the instrument especially in minimizing denaturation of labile reactants and eliminating their chemical alteration by reagents is discussed.

The possible uses of discontinuous buffer systems in cellulose acetate electrophoresis have been investigated. Several such systems allowed improved separations of complex protein mixtures, although the results obtained depended on the type of cellulose acetate used. In addition one discontinuous buffer system gave rise to approximately constant voltage and current conditions throughout the electrophoresis. The advantages of discontinuous buffers are discussed and some applications described.

WE have recently been engaged on the determination of the velocities of the chemical reactions of haemoglobin. These are of interest both to the physiologist because of the important part played by this pigment in respiration, and also to the physical chemist because this pigment is an almost unique example of a large complex protein molecule which combines with gases in a simple chemical manner. Some of the results that we have obtained and the methods we have used may therefore be of interest to readers of NATURE.

IT is now clear that intraspecific serum protein variation is widespread in mammals at least, and that the technique of starch-gel electrophoresis developed by Smithies1 is a powerful tool for revealing these differences. Complex protein patterns have been obtained with sera from horses, due to concomitant variation in several protein systems.

Excerpt I. INTRODUCTION The substance, amyloid, is now considered to be a complex protein, the exact chemical composition of which has not yet been definitely determined. The disease, secondary amy...