PORPHYREXIDE AND PORPHYRINDIN 667 



heat denaturation, titration gives results comparable to other methods for 

 total cysteine. This was confirmed by Greenstein (1938), who used urea and 

 guanidine for denaturation. The rapidity of SH oxidation was noted. Brand 

 and Kassell (1940), on the other hand, did not find good end-points with 

 denatured ovalbumin and, because of the pink color developed, felt that 

 tyrosine groups are also oxidized. This was criticized by Greenstein et al. 

 (1940) on the basis that far too much porphyrindin was used, and they em- 

 phasized that such high concentrations are probably not specific and should 

 be avoided. The method was somewhat improved (Greenstein and Edsall, 

 1940; Greenstein and Jenrette, 1942) by reducing the reaction time and 

 lowering the pH to 6.4-6.8 and, using myosin, seralbumin, ovalbumin, and 

 tobacco mosaic virus, it was believed that accurate titration of the SH 

 groups could be achieved with little interference from tyrosine oxidation. 

 Barron et al. (1941) treated scarlet fever toxin with 1 mM porphyrindin for 

 1 hr at pH 7 and found that the activity of the toxin, as determined by skin 

 tests, is abolished. Since other SH reagents do not inactivate the toxin, it 

 was felt that oxidation of groups other than SH is involved. However, the 

 SH reagents used (iodoacetate, iodoacetamide, hydrogen peroxide, alloxan, 

 and Cu++) are not the most satisfactory for the demonstration of SH groups, 

 so this evidence is not conclusive. In order to achieve specificity toward SH 

 groups it is advisable to (1) avoid alkaline conditions, (2) reduce the reac- 

 tion time with porphyrindin as much as possible, (3) use as low concentra- 

 tions of porphyrindin as possible, and (4) determine the disappearance of 

 SH groups by some secondary titration. 



Inhibition of Enzymes 



Results of treating enzymes with these oxidants are shown in Table 5-2; 

 one cannot help but be surprised that so little use has been made of these 

 substances, especially during the past few years. It is evident that quite 

 low concentrations are needed for those enzymes which have susceptible 

 SH groups and that porphyrindin and porphyrexide are among the most 

 potent oxidant inhibitors. 



Balls and Lineweaver (1939 b) attempted to titrate papain with por- 

 phyrindin but found that no clear end-point could be obtained at room 

 temperature, and at 2-3° no bleaching of the dye occurred during several 

 minutes when dilute concentrations were used. Higher concentrations pro- 

 duce a pink coloration, even at pH 4.6. The native papain SH groups are 

 thus not reactive with porphyrindin; neither are they reactive with nitro- 

 prusside. On the other hand, iodoacetate and iodoacetamide react and in- 

 hibit; papain treated with these alkylating agents still gives rise to the pink 

 coloration, indicating that tyrosine is oxidized, although not necessary for 

 enzyme activity. E. L. »Smith (1958) concluded that the SH group which is 



