REACTIONS WITH PROTEINS 



765 



rapid at pH 4.6 than at 7, it is preferable, as mentioned above, to use as 

 physiological a pH as possible if the normal state of the protein or enzyme 

 is to be established. 



(7) Salt effects on the reaction of p-MB with proteins are also often of 

 some magnitude, so that attention must be given to the ionic composition 

 of the medium and the buffers used. It is preferable in most cases to use as 

 low concentrations of salt and buffer as possible, if maximal reactivity of 

 the SH groups is desired, but occasionally it is useful to add some salt, 

 such as KCl, to reduce the p-MB reactivity in order to eliminate non-SH 

 group effects. 



0.5 



0- LACTOGLOBULIN 



'0 5 



MilO 



Fig. 7-8. Titration of cysteine and proteins with p-MB at 



pH 4.6 in 330 mM acetate medium. The reaction times 



are: cysteine and ovalbumin 15 min, and lactoglobulin 



20 hr. (From Boyer, 1954.) 



(8) It is worth noting that Boyer found EDTA to interfere, presumably 

 due to a complex with p-MB, so it is advisable to omit this substance. 



(9) Masking of the reactive SH groups, e.g. with alkylating agents, abol- 

 ishes the absorbancy changes on adding p-MB, this providing evidence 

 that it is indeed the SH groups which are responsible for the changes. Ti- 

 tration of proteins or enzymes treated with various agents can thus provide 

 information on the disappearance of SH groups, 



(10) The p-MB must be pure, should be analyzed iodometricaUy or spec- 

 trophotometrically, should be standardized against glutathione (details are 



