284 



Arthur L. Koch 



were particularly interested in the possibility of interchange under strong 

 aqueous acid conditions that are present during protein hydrolysis. In our 

 laboratory this system has been used to study the effects of strong anhydrous 

 acid media (7). In the former case interchange was found and this could be 

 repressed with thiol compounds; in the latter case no interchange occurs. 



The system consists of a mixture of /?/5-2,4-dinitrophenyl-L-cystine {bis- 

 DNP cystine) and L-cystine. If the interchange occurs, the reaction product, 

 mono-2,4-dinitrophenyl-L-cystine (mono-DNP cystine) may be readily measured 

 by removing the bis compound by acid ether extraction, or by chromatography 

 in a solvent system consisting of aqueous 5 per cent Na2HP04 overlaid with 

 isoamyl alcohol. The spots can be visualized by observation with near ultra- 

 violet hght. By a combination of these two techniques additional sensitivity can 

 be obtained. 



Dry Irradiation 



Using this system it was quickly established that even with doses as large 

 as 3 X 10'^ r of Co^° gamma rays no detectable interchange product was produced 

 in the radiation of dry films of mixed cystine and its /?w-dinitrophenyl derivative. 

 As very small amounts could be detected by the combination of the extraction 

 and chromatographic techniques, it is felt that disulfide interchange cannot 

 be of importance in the denaturation of dry protein samples, as certainly much 

 less than one interchange per 1000 disulfide bonds could have been detected. 



E^ect oj OH Radicals 



In aqueous solution the experimental situation is quite different. We first 

 investigated the effects of hydroxyl radicals by themselves. Experiments (Table 

 I) with Fenton's reagent (a mixture of Fe++ and H2O2 prepared as described 



Table I. The Absence of Interchange Produced by OH Radicals 



The complete system contains 1 x 10^ M cystine; 1.25 x 

 10-* M 6w-DNP cystine; 1.2 x 10"^ M phosphate buffer, pH 

 7.9; 5 X 10-5 M pe++ (^s FeSOi); 5 x 10^ M H2O2. For 

 analysis, 0.5 ml aliquots were added to 2.0 ml of 1 N HCl. 

 The solution was then exhaustively extracted with ether and 

 read at 350 m/t in the Beckman spectrophotometer. 



Complete 



Minus Fe++ 



Minus H2O2* 



Minus Fe++ and H2O2* 



If interchange is complete 



(calculated) 



Increase in optical density 



5.5 hours 



48 hours 



0.048 



0.062 



0.004 



-0.006 



0.052 

 0.052 

 0.084 

 0.100 

 0.489 



* The interchange observed at 48 hours in absence of H2O2 

 is caused by thiol compounds produced by the hydrolysis of the 

 disulfide (6). 



