A. Polson

A method of preparing agarose

Abstract 1. 1. Agarose has been prepared by fractional precipitation of agar with polyethylene glycol. 2. 2. Although relatively simple, the method ensures a product essentially free from sulphate and possessing a minimum of electroendosmotic and ion-exchange properties. 3. 3. Applications to tissue culture and physico-chemical methods are described.

Some physical characteristics of the agarose molecule

Abstract The physicochemical properties of agrasose and agaropectin were investigated. The techniques employed included ultracentrifugation, diffusion, intrinsic viscosity determination and electron microscopy. The molecular weight of agarose was found to be approx. 120 000. For this calculation three different methods were used involving diffusion coefficient and intrinsic viscosity, diffusion coefficient and selimentation coefficient, and sedimentation coefficient and intrinsic viscosity. The molecular weight of agaropectin is of the order of 12600. The width of the agarose molecule was found, by three methods, to lie between 10 and 16 A. The length to width ratio of the molecules was calculated by five methods, but agreement was less satisfactory. This disagreement could be due to the helical coiling of the agarose which has been demonstrated on electron micrographs. When agarose solutions are heated in the presence of air, but in the absence of buffering salts, rapid oxidation takes place with an accompanying lowering of pH and degradation of the polysaccharide molecules to a level where their ability to gel disappears. The degradation is less when oxygen is excluded and the solution is buffered.

Multi-membrane electrodecantation and its application to isolation and purification of proteins and viruses

1. 1. An electrodecantation apparatus is described in which the separation cell is subdivided into a large number of sections with the aid of thin “perspex” frames and “cellophane” membranes. 2. 2. Buffer cooling sections are employed through which chilled buffer from a reservoir is circulated. 3. 3. A temperature gradient and consequently a density gradient is established down the separation cell by appropriate sloping of the electrodes. Such a density gradient minimized convection currents in the cell. 4. 4. Separation of proteins is greatly accelerated if the apparatus is held in an inclined position. 5. 5. A continuous flow apparatus is described which enabled the continuous extraction of proteins from large volumes of solutions. 6. 6. Results are given of attempts at concentrating Poliomyelitis virus from mouse brain suspensions, purification of trypsin from bovine pancreas, and the extraction of γ-globulin from human sera using three different types of the apparatus.

DENSITY DETERMINATION IN A PREFORMED GRADIENT OF CAESIUM CHLORIDE.

A simple procedure for forming density gradients is described. By introducing the sample at a level close to the equilibrium position in a preformed gradient of CsCl, the time of centrifugation may be considerably shortened. The method applied to hemocyanin, MEF/sub 1/ poliovirus, and Rift Valley fever virus gave densities of 1.32 and 1.23 g/ml, respectively. (auth)

The isolation and purification of desoxyribonuclease by multi-membrane electrodecantation.

Abstract The iso-electric point of pancreatic DNase (desoxyribonuclease) was found to be pH 4·7 in acetate buffer. On the basis of this value the enzyme was purified by multi-membrane electrodecantation. The final product was homogeneous both in electrophoresis and ultra-centrifugution. The electrophoretic mobility and enzymic activity were identical with those reported for a crystalline preparation. A molecular weight of 40,000 was estimated from its sedimentation constant.

Particle Size of Soluble Antigen of Rabies Virus

Summary 1. A particle size of 12 m/μ has been determined by a method of diffusion coupled with biological assay for the soluble antigen of rabies extracted from infected suckling mouse brains. This value agrees with that calculated from the approximate sedimentation constant calculated from the sedimentation rate in the swinging bucket rotor of the Spinco ultracentrifuge. 2. No conclusions regarding the homogeneity of the soluble antigen can be drawn at this stage.

Investigations of Poliomyelitis Antigens.

ABSTRACT The complex nature of the antigens in suckling mouse-adapted MEFi virus was indicated by Selzer and Poison1, who found that the infectivity was associated with two components of approximate sedimentation coefficients 100 and 173 S respectively. In addition to these components a “soluble antigen” was found which had a sedimentation coefficient of about 22 S. These results, which were obtained by using a Spinco preparative centrifuge (Poison and Linder2), have now been confirmed in the Spinco analytical ultracentrifuge of the Nobel Institute, Stockholm, and other types of poliomyelitis virus have been studied in both instruments.

The calculation of molecular weights from difussion and viscosity data

Abstract The Scheraga and Mandelkern relationship of intrinsic viscosity diffusion coefficient and molecular weight has been tested on a number of proteins and other macromolecular substances. Extensive use was made of available data from the literature. It was found that the Scheraga and Mandelkern relationship is applicable provided the constant in the equation be increased from 6.58·10−16 to 7.36·10−16. It was shown how the equation could be modified in order to be applicable to substances of molecular weight less than 1000.

The diffusion constants of rabbit, guinea-pig and rat antibodies to a sincle common antigen

Abstract The diffusion constants of the precipitin antibodies prepared in rabbits, rats and guinea-pigs against the haemocyanin of Burnupena cincta have been determined by the gel-diffusion technique using the diffusion constant of the haemocyanin as reference standard. This work was done in an attempt to confirm the validity of the assumption in previous work, that the precipitating antibodies derived from the rabbit have the same diffusion constant as tetanus antitoxin derived from the horse. It was found that the diffusion constants of these precipitin antibodies are on the average appreciably higher than those given by other authors for gamma globulin and in this respect are confirmatory of the findings of Largier 4 if the diffusion constants determined by the present technique be regarded as being within 5% of the true value.

Diffusion osmotic equilibrium

Abstract A new method for measuring diffusion constants is described. In this method an equilibrium is established in a cell between upward diffusion and downward migration on to a semi-permeable membrane across which is established a strong osmotic gradient. The diffusion constant of bovine serum albumin determined by this method agrees well with the value obtained with the boundary spreading method. The method can also be used for testing the homogeneity of colloidally dispersed substances.