270 RADIATION BIOLOGY 



conditions do not exist, serious errors may result. In measurements of 

 photosynthesis the intensity of Ught is changed, or the conditions of dark 

 and Hght are alternated. With each change there will be a change in 

 concentration of carbon dioxide and oxygen which requires time for 

 adjustment. 



In one experiment (Evans, 1951; Inouye, 1951) to test the time lag 

 directly, the manometers were allowed to reach equilibrium, and then a 

 little carbon dioxide was introduced into the manometer vessel by press- 

 ing the plunger of a hypodermic syringe. At the shaking rate of 150 

 oscillations per minute used by Warburg and his associates, it took 30 sec 

 to approach equilibrium conditions again within the limits of the accuracy 

 of the experiments. It took 200 sec when the shaking was reduced to 

 half the rate. 



Oxygen reaches equilibrium much faster than carbon dioxide, and seri- 

 ous errors of interpretation can arise if measurements are taken before 

 equilibrium of both gases is reached. The tendency has been to use short 

 periods of light exposure, so that the biological conditions will not have 

 time to change very much, and this emphasis on short exposures magnifies 

 any error resulting from lack of equilibrium following a change of con- 

 ditions. The time required for the attainment of equilibrium conditions 

 varies greatly from perhaps 0.5 to 2 or 3 min, and it should be deter- 

 mined for each type of experiment. Warburg, Burk, and Schade 

 (1951) urged a period of 5 min to remove the lag in one carbonate buffer 

 solution. 



The use of alkaline carbonate buffers simplifies the experimental con- 

 ditions and the calculations, but even here the time factor can introduce 

 complications. Carbon dioxide reaches equiUbrium with carbonate solu- 

 tions very slowly. In tissues and cells the enzyme carbonic anhydrase is 

 known to accelerate this reaction. A buffer frequently used contains 

 85 parts of 0.1 M sodium bicarbonate freshly mixed with 15 parts of 

 0.1 M sodium carbonate. This mixture has a pH of about 9 and at 

 20°C is in equilibrium with an atmosphere containing 0.5 per cent carbon 

 dioxide. The carbon dioxide content of the gas has been assumed to be 

 constant, and any change in pressure is due to oxygen. Equilibrium 

 is reached slowly. From this oxygen change it is possible to calculate the 

 rate of photosynthesis and respiration. Some species of algae do not 

 grow normally in carbonate buffers, but Chlorella cells seem to carry on 

 normal photosynthesis for hours in alkaline buffers (Emerson and Lewis, 

 1941; Nishimura et al., 1951). Warburg considered carbonate buffers 

 "unphysiological" (Burk and Warburg, 1950) but apparently accepts 

 them if the pH is under 8.8 (Warburg, Geleick, and Briese, 1951). 



The ratio 7 (see Sect. 1), which has a value of —1 if the reaction pro- 

 ceeds strictly as CO2 + H2O -^ (H2CO) + O2, is important in manometer 

 calculations. The value of 7 is best determined by direct chemical 



