764 



7. MERCURIALS 



or less readily reacting SH groups. Such behavior seems to be rare and does 

 not seriously interfere with the determination of the end-point. 



(3) The time relations must be considered. It is common practice to in- 

 cubate the protein and p-MB for 10-15 min to allow reaction, but a decision 

 as to this depends on one's definition of a reactive SH groups. In some 

 proteins, additional SH groups are reacted when the incubation is prolonged; 

 is such cases one is not certain if these groups were initially exposed, or 

 if they arise during a progressive denaturation of the protein. 



Fig. 7-7. The spectral absorption curves for 



p-MB and its mercaptide with cysteine, at 



pH 7 in 60 raM phosphate buifer. (From 



Boyer, 1954.) 



(4) Both proteins and p-MB absorb significantly at 250-255 m/^ and the 

 appropriate controls must be run. For example, when protein is titrated 

 with p-MB, equivalent increments of the mercurial are added to the blank 

 cell. 



(5) Protein or enzyme solutions should be as pure as possible, since even 

 small amounts of certain impurities may cause large errors, and the solu- 

 tions should be clear so that light scattering is reduced. 



(6) Special consideration should be given to the pH since it has been 

 shown that both the rate and extent of reaction are markedly affected, as 

 in Boyer's experiments with ovalbumin. Although reaction may be more 



