REACTIONS WITH PROTEINS 763 



metric titration with Ag+ is still commonly used and is often useful when 

 combined with mercurial titration; one should consult Leach (1960) for a 

 discussion of some of the difficulties of this method. Amperometric titra- 

 tions at a rotating platinum electrode using Hg++ or MM have been shown 

 to be accurate and reliable in some cases (Saroff and Mark, 1953; Kolthoff 

 et al., 1954; Leach, 1960), but these technique have not yet been extensively 

 applied to enzymes. Leach has listed the requirements for an ideal SH re- 

 agent for titrations: It should (1) be specific for SH groups, (2) be highly 

 reactive, (3) have a small molecular size, (4) be preferably monofunctional, 

 (5) be devoid of charge or other reactive groups so that all protein SH 

 groups are equally favored despite their different environments, (6) be sol- 

 uble, stable, and reactive over a range of pH, and (7) show well-defined 

 reduction steps for amperometric use. MM fulfills most of these criteria and 

 perhaps it has been neglected in enzyme work. Past work on many pro- 

 teins has indicated that it is often well to use more than one method in 

 order to increase the reliability. 



The most commonly used method at present is the spectrophotometric 

 titration with p-MB developed by Boyer (1954), since it is convenient and 

 appears to be generally accurate. Furthermore, the sensitivity is as high 

 as with the amperometric methods, namely, around 0.01-0.1 ulM SH. 

 Reaction of p-MB with SH groups leads to an increase in the absorbancy 

 at 250 m// at pH 7 (Fig. 7-7); at pH 4.6 the maximal increment occurs at 

 255 m//, but it is usually preferable to titrate enzymes at the more physi- 

 ological pH of 7 to determine the number of reactive SH groups, unless 

 the mercaptide formation occurs too slowly. The increase in absorbancy is 

 a linear function of the SH groups reacted, for both simple thiols and pro- 

 teins (Fig. 7-8), but the absorbancy change is somewhat different for dif- 

 ferent SH groups, a fact of no importance in titrations. The p-MB may be 

 titrated with protein (as in Fig. 7-8), the end-point being the sharp break 

 between the two linear segments, or the protein may be titrated with p-MB; 

 in both cases one determines when all the reactive SH groups are transform- 

 ed into mercaptides. Although the details of the method may be found in 

 the original paper of Boyer (1954) and the excellent review of R. Benesch 

 and R. E. Benesch (1962), it may be useful in interpreting such titrations 

 applied to enzymes to note briefly certain precautions and difficulties. 



(1 ) The only mercurial which can be used is jj-MB, and since it is a rather 

 large molecule with a negative charge, steric or electrostatic factors may 

 reduce its reaction with certain SH groups. PM and 7)-MPS exhibit ab- 

 sorbancy shifts but at lower wavelengths where protein absorbs much more 

 strongly than at 250-255 m//. 



(2) The addition of excess p-MB occasionally results in a further small 

 increment in the absorbance, so that the flat portion of the curve may not 

 be exactly horizontal, and this may indicate reaction with non-SH groups 



