G. S. ADAIR 



2 hours at 0°C to reach temperature equilibrium. The levels L, W 

 and B are then measured. 



A membrane containing protein which has been dialysed against the 

 buffer solution is then connected to the osmometer. Screw-clamps on 

 the tubes 7?j and R 2 may be used to keep the volume of buffer in the 

 bulb approximately constant, during operations of attaching and 

 detaching rubber tubes or connections to the membrane below the bulb. 



The rate of osmosis may be sufficient to eliminate 50 per cent of an 

 initial deviation from the equilibrium pressure within half an hour. 

 The osmometer is left overnight at 0°C to reach equilibrium. 



After equilibrium has been attained, the osmotic pressure of the 

 protein iz ti expressed in cm toluene, may be calculated by formula 2 

 from readings of L, W and B, The capillarity is redetermined at the 

 end of the experiment. 



7T, = (L - W) - {W - B) k + q - cap . . . .(2) 



k = (p b — p,) / (p, — pj. 9 b , p, and p a denote densities of buffer, 

 toluene and air. q = a correction term to allow for differences in 

 densities of buffer and of protein solution. Cap. = correction for 

 capillarity. 



The apparatus shown in Figure 2 can be modified by the addition 

 of U tubes and T pieces so that the manometer can be connected to 

 the dialysate by opening a screw clamp. 



Experimental data recorded in Tables I and 77 show to what degree 

 the measurements are reproducible. These data refer to carbon 

 monoxide haemoglobin prepared from horse blood and dialysed 

 against phosphate buffer Na 2 HP0 4 0-03M, NaH 2 P0 4 0-0 1M. The 

 final pH was 7-38 at 0°C. 



The third column in Table I gives molecular weights, computed by 

 formula 3. 



M = 170,330 (273 + //273) (C/w) <f> 

 <£ = 1/(1 -0-02853 C+0-0002109 C 2 ) = osmotic coefficient. .. .(3) 



/ = temperature. C = g haemoglobin per 100 ml solution.* 



The mean molecular weight is 66,400 ; the mean deviation is 400. 

 Table II shows the relationship between observed osmotic pressures 

 and values calculated on the assumption that the molecular weight is 

 66,400 in dilute solutions. 



Diffusion and ultracentrifugal measurements 11 have yielded evidence 

 for the dissociation of horse haemoglobin at concentrations below 

 0-8 per cent at a temperature of or above 20°C. The measurements 

 of osmotic pressure at either 0-1 °C or 1-0°C recorded in Table II show 



* The formula for the osmotic coefficient <f> was derived from unpublished data for 

 concentrated haemoglobin solutions by the method of Adair and Robinson. 10 



194 



