PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 189 



pH change is a relative measure of the progress of a reaction in which 

 hydrogen ions are produced. The method has limited application, since 

 the activity of a given system is usually dependent on pH and the linear 

 relationship of time and pH is of short duration. In addition, pH change 

 is not a simple function of progress of the reaction. This method has 

 been applied by Sable and Guarino (1952) to the purification of glucono- 

 kinase from yeast. 



The formation of acid during a reaction or during a sequence of reac- 

 tions may be followed manometrically, using essentially the same technics 

 mentioned above in the section on manometric technics. This method 

 takes advantage of the buffering capacity of the HCOa" + H+ :;:± CO 2 + 

 H2O system. The formation of each acid is accompanied by release of a 

 hydrogen ion into solution. As a result, CO 2 is evolved from the reaction 

 mixture and may be quantitatively measured. A complete account of 

 the buffer theory involved in this method, as well as a chart describing 

 the relationships of pH, temperature, and bicarbonate concentration, 

 may be found in Umbreit et al. (1949). 



The method is applicable to the fermentation or oxidation of substrates 

 which yield lactic or other acids as end products. If CO2 is also an end 

 product, a control flask is used containing a buffer other than bicarbonate 

 (e.g., phosphate) at the same pH, allowing the determination of the true 

 CO2 value. By difference, the CO2 evolved in the bicarbonate flask as a 

 result of acid formation is calculated. 



In an isolated reaction involving the reduction of di- or tri-phospho- 

 pyridine nucleotide, a hydrogen ion is produced; thus, with opaque sys- 

 tems which cannot be measured in a spectrophotometer (see below), the 

 reaction may be followed manometrically as acid production. In this 

 case, a stoichiometric quantity of the pyridine nucleotide must be added 

 or a second system must be included which is capable of oxidizing the 

 reduced pyridine nucleotide. For the second system, several reagents 

 are available which accomplish the oxidation of reduced pyridine nucleo- 

 tide, the best probably being K3Fe(CN)6. When coupled with the 

 ethanol-acetaldehyde system, for example, the reactions are written as 

 follows : 



CH3CH2OH -h DPN+ ^ CH3CHO -f- DPNH -F H+ 



2Fe(CN)6 + DPNH ^ 2Fe(CN)6 + H+ + DPN+ 



2H+ + 2HCO3- ^ 2H2O + 2CO2 

 Sum: CH3CH2OH -f- 2Fe(CN)6— + 2HCO3 



;=± CH3CHO + 2Fe(CN)6 + 2H2O + 2CO2 



Thus, under these conditions two molecules of CO2 are evolved for each 

 molecule of ethanol oxidized and two molecules of ferri cyanide are 



