RADIOCHEMICAL AND RADIOBIOLOGICAL ALTERATIONS OF I131‐LABELED PROTEINS IN SOLUTION

Previous experieiice with 1131-labeled proteiiis has frequently raised qiiestioiis regarding their validity as tracers for the respective native proteins in biological studies. Uiipredictable beliavior in biological systems has been encouiitcred in certaiii instances. For iiistaiice, it has been ohserved that the iiietliod of iodination of serum albumin rnay influeiice significantly its rate of inetabolic degradation in viv0 in man1 and in the rabbit.2 Similarly, the rate of disappearance of protein-bound 1131 from plasma following iiitravenous administratioii of i1 i su l in-1~~~ showed a variability, at times, that is apparently unrelated to biological factors.3 The latter observations were correlated with the presence of Pl-labeled components that were bound to the plasina proteins. Attempts to indict tlie individual chemical siibstances employed in the iodiiiation procedure as the agents responsible for the production of these plasma protein-binding fractions of i1 i suI in-1~~~ were unsucc e c ~ f u l . ~ It was snbsequently appreciated tliat the extent of contaniination of insul i i1-1~~~ with these coiiiponents \?ras related to the time elapsed after preparation3 and to the concentration of present. I t thus appeared that the total dosage of radiation absorbed by the solutions might be a critical factor. and this possibility was confirmed in experiiiicnts employing external sources of irradiation? I t has been knowii for soiiie time that proteins are readily susceptible to the effects of ionizing radiation ; particularly in the case of serum albumin, Barron and his co-workers5 have shown changes in ionbinding capacity, spectral absorption in the ultraviolet range, and amino acid coniposition following X irradiation. These data should, perhaps, have already stimulated a formal investigation of the effects of radiation in proteiiis labeled with radioisotopes biit, in the absence of obvious denaturation or other alterations, it has not always seemed necessary to probe for every conceivable source of injury to the proteins. However, it has been observed that, even tliough the labeled protein may appcar to be identical to the native species by a nuiiiber of physicochemical criteria, such as ultraceiitrifugal sedinientation and electrophoretic mobilitv, its biological behavior rnay be altered comp1etely.l The studies on insulin-Ilsl and a 1 h ~ m i n T ~ ~ ~ described in this paper were therefore orieiited toward reactionc in botli biological and chemical systems. While tlie former may be more discriminatory, elucidation of some of the chemical clianges aids in interpreting anomalous biological behavior. In soiiie rcspccts, at least, the nature of the alterations produced by irradiation of the labeled proteins soliitions with X rays and with the seifcontained are ~ii i i i lar .~, o Thereiore. most of the radiation experiments clescribed here were performed with X rays since, aside from considerations