Osmotic pressures and sedimentation velocity of Gastrophilus methaemoglobin.

In the preceding paper, Keilin & Wang (1946) have described the preparation and properties of the haemoglobin obtained from the tracheal cells of the larva of Gastrophilus (the bot fly). Samples from three of their preparations in the form ofmethaemoglobin were used for measurements of osmotic pressure. The amount of material available was small and the results recorded must be regarded as provisional. METHODS An account of the manufacture of collodion membranes and the method of measurement of osmotic pressures has been published elsewhere (Adair, 1925). Small cylindrical membranes of 0-6 cm. diameter were used, the permeability being about one-tenth that which had been found suitable for the serum proteins, as the more permeable membraneswere found to adsorb Gatrophilus haemoglobin. The equilibrating solution used was a sodium phosphate buffer mixture of ionic strength 0-1 and pH 7-4 (0-03 MNa2HPO4 and 0-01 M-NaH2PO4). Previous investigations on sheep haemoglobin showed that an ionic strength of 0-1 is sufficient to reduce the pressure due to the unequal distribution ofdiffusible ions (symbolizedpi) to a very low value over the range from the isoelectric point to pH 7-4. The slightly alkaline reaction reduces the adsorption of protein by the membrane and the tendency of the protein to precipitate. The protein concentrations were determined refractometrically. The determiniation of the specific refraction increments of the serum proteins in the presence and absence of salts has been described by Adair & Robinson (1930a). Unpublished experiments on CO-haemoglobin prepared from ox blood gave a specific refraction increment of 0-001932 for aqueous solutions and 0-001930 for solutions equilibrated with a sodium phosphate buffer mixture ofionic strength 0-1 and pH 7-4. The quantity of Ga8trophilws methaemoglobin available was insufficient for exactdeterminations of the refraction increment. A preliminary estimate ofthe value was made by measuring the difference between the refractive indices of water and of a dilute aqueous solution of the protein, and dividing the result obtained by the content of dry matter of, the protein solution (0.48%) as determined by Keilin & Wang. A refraction increment of 0-00204 was obtained. In this paper, protein concentrations have been calculated by dividing the difference between the refractive indices of the protein solution and of the diffusate by the factor 0-00204. The estimation of protein content of the material used in Table 1, Exp. 6, agreed with a value calculated from a spectroscopic determination of the haematin content of the same solution made by Keilin & Wang.