714 6. O-IODOSOBENZOATE 



dehydrogenases. At the present time, o-iodosobenzoate is more useful in the 

 study of pure enzymes as an indicator of SH groups than in cellular systems, 

 but has been little investigated in the latter and may possess potentialities 

 as a metabolic blocker if applied properly. 



Titration of Enzyme SH Groups 



Muscle phosphoglyceraldehyde dehydrogenase contains 11 cysteine resi- 

 dues and reacts rapidly with 1 1 moles of ^^-chloromercuribenzoate per mole 

 of enzyme. Segal and Boyer (1953) reported that 7.3-7.45 moles of o-iodo- 

 sobenzoate react with each mole of this enzyme, indicating 14.6-14.9 re- 

 ducing groups. Theoretically one would expect 5.5 moles of o-iodosobenzoate 

 to be reduced by each mole of enzyme, assuming that all the SH groups 

 are oxidized to the disulfide level. Segal and Boyer thus suggested that some 

 of the SH groups may be oxidized beyond the disulfide stage. Actually only 

 10 of the 11 SH groups could form intramolecular disulfide bonds, so the 

 extra SH group must either remain unoxidized, form a disulfide link with 

 another molecule of the enzyme (which is unlikely), or be oxidized to some 

 state other than the disulfide. Since oxidation to the S — 0~ or SOg" state 

 would require 2 molecules of o-iodosobenzoate for each SH group, 7 moles 

 of o-iodosobenzoate would react with each mole of enzyme if oxidation of 

 the extra SH group occurred in this way. Rafter (1957) investigated this 

 problem further and, under his conditions, found 10-11 moles of o-iodoso- 

 benzoate to react with each mole of the enzyme, indicating 20-22 reducing 

 equivalents. Furthermore, the o-iodosobenzoate-treated enzyme still pos- 

 sesses 30% of its initial SH groups, as determined by reaction with p-chloro- 

 mercuribenzoate. These results point to reaction of o-iodosobenzoate with 

 groups other than SH groups, or to oxidation of a fraction of the SH groups 

 beyond the disulfide stage. There is no evidence for reaction with other 

 groups and the enzyme is completely reactivated by cysteine. Thus one 

 might assume that 4 SH groups are oxidized to the disulfide level, 4 are 

 oxidized beyond this, and 3 remain unreacted; this would require 10 moles 

 of o-iodosobenzoate per mole of enzyme. Another possibility is that some 

 of the SH groups form E — S — I — cp — C00~ residues. It is interesting that 

 although o-iodosobenzoate abolishes the usual phosphoglyceraldehyde dehy- 

 drogenase activity and the arsenolysis reaction, it simultaneously increases 

 phosphatase activity 6-fold, this phosphatase activity being dependent on 

 NAD. The esterolytic activity with p-nitrophenylacetate as substrate is in- 

 hibited completely in 10 min when 4-5 moles of o-iodosobenzoate have react- 

 ed per mole of enzyme (Olson and Park, 1964). No substrate protection was 

 observed. Yeast phosphoglyceraldehyde dehydrogenase contains fewer SH 

 groups than the muscle enzyme — 2-4 per molecule — and reacts with 6 

 molecules of o-iodosobenzoate, so that here too an anomalous effect is seen. 

 Barron and Levine (1952) report 11.9 SH groups in yeast alcohol dehydro- 



