766 



7. MERCURIALS 



given by Benesch and Benesch), and should be used in freshly made so- 

 lutions. 



(11) The presence of two or more SH groups close together on the protein 

 may prevent the reaction of each with jj-MB, as is the case with hemoglobin. 

 This will lead to low values for the total number of SH groups in proteins 

 or enzymes. 



A typical titration of an enzyme is shown in Fig. 7-9. The titration of 

 3-phosphoglyceraldehyde dehydrogenase at pH 4.6 presents a clear end- 

 point indicating a rapid reaction of the SH groups. This yields 10.3 SH 

 groups per molecule of enzyme (assumed molecular weight of 118,000). 



0.3 



4 5 



/i MOLES/ML iio' 



Fig. 7-9. Titration of 5 times recrystal- 

 lized 3-phosphoglyceraldehyde dehydro- 

 genase with p-MB at pH 4.6 and 0.03 

 /<mole/ml. (From Boyer and Segal, 1954.) 



The reaction of the SH groups occurs more slowly at pH 7 and a sharp 

 end-point was not obtained by incubations up to 15 min; however, longer 

 incubations would probably have given a sharp break in the curve. Here 

 the end-point yields 8.3 SH groups per enzyme molecule, suggesting that 

 2 SH groups become much more reactive when the pH is lowered. A value 

 of 10.7 half-cystines per molecule for this enzyme has been reported (Velick 

 and Ronzoni, 1948), so it is evident that most of these SH groups are free 

 and reactive. 



Colored Mercurials and Histochemical Determination of Protein SH Groups 



Various colored mercurials, usually azobenzene derivatives, have been 

 known for many years but were not applied to biological material until 

 Bennett (1948 a) studied the reaction of p-mercuriphenylazo-/5-naphthol 

 with tissue thiols. Direct visualization of thiol distribution in the tissues is 

 possible, but the dye has a very low solubility in water and at the usual 

 pH's so low a molecular extinction coefficient that its use is limited. How- 



