314 A. K. KRALL 



darkness under CO whose removal in light was hindered by the effect 

 of CO on respiratory, nonphotosynthetic reactions. 



This paper reports studies on isotopic phosphate incorporation 

 into phosphate esters involved in photosynthesis while the tissue was 

 exposed to air or to gas mixtures containing carbon monoxide under 

 various conditions of illumination. The object of the experiments was 

 to test the hypothesis that the activity of an enzyme analogous to 

 cytochrome oxidase is necessary for photosynthetic esterification of 

 phosphate. 



METHODS 



P*' was incorporated into weighed amounts of first leaves of barley 

 that were 6 to 9 days old by immersing the cut ends of the leaves in a 

 dilute aqueous phosphoric acid solution containing a known amount 

 of the isotope. The P^- was taken up in the transpiration stream dur- 

 ing a 1- to 2-hour exposure of the leaves to a gas mixture containing 

 the inhibitor or to a control gas. In some experiments the leaves took 

 up the isotope in air under white light for a long period of time (24 to 

 36 hours) before exposure to inhibitory conditions. 



The incorporation of P^- was terminated by dropping the leaves 

 into boiling 30% ethanol and extracting for 2 minutes. The extract, 

 after concentration in vacuo to 8 to 10 ml., was made alkaline with 

 dilute ammonia and fractionated on a Dowex-1-Cl column by a 

 modification of the method of Ivhym and Cohn (6), in which a 

 shorter column and continuous recording of the P'^ in the column 

 effluent permitted much more rapid analysis. The fractions were 

 then assayed for P^- with a dipping-tube counter. 



The carbon monoxide-carbon dioxide-oxygen mixtures used as 

 inhibitors were made up in 40-liter bottles by water displacement 

 and were forced over the leaves at the rate of 5 to 10 liters per hour 

 during an experiment. The leaves were ilkmiinated with a 150-watt 

 reflector flood lamp giving an intensity of about 2000 foot-candles at 

 the leaves or by the same type of lamp behind a Corning 2030 red 

 filter which gives a very much lower light intensity, probably equiva- 

 lent to no more than two to three times compensation level of light. 



A sodium arc lamp rated at 100 foot-candles was used as a source 

 of light for reversal of the carbon monoxide inhibition. Its effective- 

 ness in promoting photosynthetic uptake of C^*02 had previously been 

 found to be very low (3) . 



