Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases

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

Publisher Summary This chapter describes many of the techniques employed to study water–macromolecules interactions, their general usefulness, points out areas of mutual support and contradiction. The chapter also presents hydration of other macromolecules and the hydration of small molecules. Four broadly based approaches to operational definitions of hydration include—(1) preferential hydration, (2) hydrodynamic hydration, (3) structural hydration, and (4) low temperature hydration. The use of high resolution X-ray and neutron diffraction data on protein crystals provides an independent approach for estimation of hydration. The chapter presents several proposals for predicting protein hydration based on the amino acid composition of the protein; however, the two main questions concerned include—whether ionic groups are more hydrated than other polar groups, and whether the amide and peptide functions are hydrated or not. Low pH, and to a much smaller degree, high pH should dehydrate proteins. Intermolecular association via ionic mechanisms should be strongly dehydrating; whereas hydrophobic interactions should not have much effect on hydration levels. Although, there is a presumption that both the enthalpy and the entropy are negative for the process of adding water molecules to isolated proteins, which are maintained in their native conformations, but there is no unequivocal evidence for this.

Hydration of Proteins and Polypeptides

Separation of univalent fragments from the bivalent rabbit antibody molecule by reduction of disulfide bonds.

Publisher Summary The chapter discusses the proteins at interfaces and aims to present protein adsorption and its ramifications, such as the effects of adsorption on conformation and reactivity from a fundamental physicochemical approach Although many proteins are very soluble in water, adsorption leads to stable monolayers that are extremely difficult to desorb Adsorption may be followed at fluid/fluid interfaces by measuring changes in interfacial pressure, potential, or viscosity, using spread monolayers for calibration purposes Before a protein molecule can adsorb and exert its influence at a phase boundary or take part in an interfacial reaction, it must arrive at the interface by a diffusion process At fluid/fluid interfaces, it is established that proteins lose their tertiary structure One of the advantages of the fluid/fluid type interface is that the distribution between adsorbed (trains) and displaced segments (loops) may be quantitatively studied by carrying out measurements with a film balance This approach has been used for spread monolayers of protein The high free energy of adsorption of proteins results in all segments being at the interface at low interfacial pressures The conformational changes that accompany desorption from an interface have direct bearing on the important subject of protein biosynthesis The chapter also discusses the equilibrium aspects of adsorption, reactions at interfaces, and proteins at interfaces in biological systems

Proteins at interfaces.

Ferritin and apoferritin in the ultracentrifuge studies on the relationship of ferritin and apoferritin; precision measurements of the rates of sedimentation of apoferritin

Electrophoretic studies on purified crystalline ribonuclease showed the absence of any impurities differing in mobility from the bulk of material.

The isoelectric point of ribonuclease was found by electrophoresis to be at about pH 7.8.

Ultracentrifuge studies indicated fair homogeneity of ribonuclease in solution. Only one moving component has been observed.

The molecular weight of ribonuclease was found to be 12,700 from rate of sedimentation ( S 25 = 1.85 x 10–13 in 0.5 M (NH4)2SO4) and diffusion measurement ( D = 1.36 x 10–6 in 0.5 M (NH4)2SO4), in good agreement with the average value of 13,000 found from equilibrium measurements. This low value for the molecular weight of a protein would seem to discredit the value 17,600 as representing a universal unit weight for proteins in general.

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Molecular weight and electrophoresis of crystalline ribonuclease.

Immunological reactions between films of antigen and antibody molecules.

Films of several proteins, hen ovalbumin, horse and human serum albumins, and globulins were found to combine specifically with antibodies, showing that the reactions can take place independently of the structure which secures the compact shape of the protein molecule. Serum globulin films differed from ovalbumin in that they lost their reactivity when kept on the water surface. Species specific reactions were observed with films of serum albumin and suitably diluted antisera, and likewise in the customary precipitin tests in which immune sera for denatured hen ovalbumin were tested against ovalbumin of other species. Specific fixation was also observed upon exposing monolayers of purified antibodies for pneumococci of Types I and III to the corresponding polysaccharides.

https://rupress.org/jem/article-pdf/76/5/437/1182536/437.pdf

Alexandre Rothen

Papers

Ferritin and apoferritin in the ultracentrifuge studies on the relationship of ferritin and apoferritin; precision measurements of the rates of sedimentation of apoferritin

Molecular weight and electrophoresis of crystalline ribonuclease.

Immunological reactions between films of antigen and antibody molecules.

Serological reactions of protein films and denatured proteins

Optical activity and chemical structure