Discussion 35 



serum albumin has been irradiated in dilute solution, the u.v. absorption 

 peak at 2,800 increases and this has been interpreted by Barron and 

 others as a reaction by the free radicals with the tyrosine residues in the 

 protein molecule. In reality this increase is due to aggregation and not 

 due to a change in the actual light-absorbing groups. On irradiation the 

 protein molecules form aggregates which scatter more light. The amount 

 of light scattered varies inversely as to the fourth power of the wave- 

 length, and a solution which does not appear cloudy in visible light may 

 scatter a great deal at 2,8QP A. We proved that the increase in absorp- 

 tion at the 2,800 peak is entirely due to aggregate formation, by measur- 

 ing the amount of light scattered in a special instrument. In the case of 

 lysozyme which contains much tryptophan, the position is slightly 

 different: one does first of all get a decrease in the absorption peak and 

 this is due to the destruction of the tr^T)tophan. With higher doses the 

 absorption goes up, but this increase is due to aggregation. 



Dale: I should like to answer the first part. I have no experience of 

 the second part, which is actually a communication of your experiments 

 rather than a question. I think it would be rather unfortunate to choose 

 this criterion of aggregation, because it is a very common experience 

 with all colloids that the particle size increases on standing. I should 

 like to ask whether you have examined these solutions after irradiation 

 has been finished, and whether there are after-effects of aggregation 

 or not. 



Alexander: Not after the first 30 minutes, which was the shortest 

 period in which we were able to look at it after irradiation. 



Dale: When you precipitate colloids with various precipitins, with 

 various salts, you find continuous aggregation leading eventually to 

 flocculation. You must have had a similar phenomenon because you 

 mentioned the effect of addition of salts. I don't know whether you 

 varied valency of the salts using divalent ions and trivalent ions and so 

 on and whether you had a negative or a positive colloid, but the very 

 fact, as you say, that the enzymatic activity is not necessarily changed 

 does not bring this phenomenon within these experiments, because what 

 we measured is the effect on the activity of the enzyme rather than 

 on aggregation. I think the aggregations are rather non-specific changes 

 which with bigger doses probably also point to denaturation and I think 

 it does not affect activity measurements with radiation, which are 

 strictly quantitatively what you would expect, that doubling the dose 

 or making the dose 100 times as great has 100 times greater effect, apart 

 from the region where you have wide separation of solute molecules 

 with recombination of radicals. 



Forssberg: When speaking about colloids and irradiation it may be 

 relevant to recall that J. A. Crowther and others, some twenty years 

 ago, studied the changes produced by very small doses on, for example, 

 colloidal gold and graphite, but also on proteins. It would seem that 

 irradiation causes cyclic changes both in particle size and in charge. 

 These changes sometimes proceed even after the irradiation is finished, 

 which implies that they are a function of the time of assay. It is not 

 known whether similar effects occur in vivo. 



