REACTION WITH PROTEIN SH GROUPS 703 



-S— S— R 

 R— SH ^R-S-= '"' > R— S— I — <)— coo" 



Reaction (1) would involve combination with another R — S-, while reac- 

 tions (2) and (3) would involve additional o-iodosobenzoate. Compounds of 

 the type R — S — I — R' are generally unstable, but on proteins such link- 

 ages may occasionally be more stable, as in the formatiom of sulfenyl thio- 

 sulfates (page 697). 



At neutrality, 25°, and 1-5 loM o-iodosobenzoate, only SH groups are 

 significantly oxidized; cystine, methionine, glucose, and ascorbate are not 

 oxidized appreciably under these conditions. However, ascorbate is oxi- 

 dized slowly by o-iodosobenzoate at pH 4.6 when both are present at 1 mM 

 in acetate buffer, half-reaction time being around 80 min (Caraway and 

 Hellerman, 1953). The nature of the buffer is important inasmuch as it is 

 an acid-catalyzed reaction. It is interesting that m- and p-iodosobenzoates 

 oxidize ascorbate almost instantaneously. NADH is not oxidized by o-iodo- 

 sobenzoate at pH 4.6 (Schellenberg and Hellerman, 1958). The oxidizability 

 of tyrosine phenolic groups by o-iodosobenzoate has not been thoroughly 

 examined but there is no evidence from work with proteins that this reac- 

 tion proceeds readily. The tyrosine groups of /5-amylase seem to be resistant 

 to o-iodosobenzoate since there is no change in absorption at 280 mju 

 (Englard et al, 1951). 



REACTION WITH PROTEIN SH GROUPS 



In order to titrate selectively protein SH groups with o-iodosobenzoate, 

 it is necessary to control the conditions carefuUy, as with all SH reagents. 

 It is usual in titrations with o-iodosobenzoate to add a slight excess of the 

 reagent and determine the amount not reduced by addition of KI and sub- 

 sequent titration of the released Ig with thiosulfate (Chinard and Heller- 

 man, 1954). The test is best run at pH 7 and between 15° and 25°. The 

 required reaction time varies with the protein tested but is usually less 

 than 30 min. Ovalbumin denatured with guanidine is titrated quite satis- 

 factorily and all of the SH groups are oxidized. Only a fraction of the SH 

 groups of native ovalbumin or other proteins is oxidized. 



The specific oxidation of protein SH groups to disulfide may be accom- 

 panied by changes in the protein structure, which could have important 

 bearing on the mechanism of enzyme inhibition. Evidence for such struc- 

 tural alteration is given by the increased water-binding capacity of gels 

 formed from serum proteins previously treated with o-iodosobenzoate (Jen- 

 sen et al., 1950). At about equimolar ratios, o-iodosobenzoate changes the 



