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J.RADIOANAL.NUCL.CHEM.,LETTERS 137 [4[ 259-264 [1989/ ON THE HETEROGENEITY OF ] "25I-LABELLED PROTEINS USED AS TRACERS IN RADIOIMMUNOASSAY G. T6th Institute of Isotopes of the Hungarian Academy of Sciences, H-1525 Budapest, P.O.Box 77, Hungary Received 21 July 1989 Accepted 7 August 1989 The distribution of mono- poly- and unlabelled protein molecules in the radiolabelling mixture was calculated from probability point of view as a function of the radioiodine/protein ratio. INTRODUCTION In the majority of cases 125I-labelled compounds are used as tracers in radioimmunoassay /RIA/. When iodinating a protein the radioiodine label is incorporated via aromatic electrophilic substitution in one or several of the tyrosine residues at position 3 and/or 5 /Fig. i/. Even if the protein exhibits only a single tyrosine residue, which is a relatively rare case, the labelling reaction mixture contains, b e s i d e s unreacted free radioiodine, three components: unlabelled, mono- and dilabelled molecules. The relative amount of these c< nponents depends on the radioiodine/protein (I/P) ratio according to Eq. 2 /i/ /Ref. I/: 259 TOTH: HETEROGENEITY OF 12 s I-LABELLED PROTEINS OH OH C~IzH12gI ./" 'Z~I" //VVk NH--CH--CO,~ C,H, /VXANH--CH--CO,A~ ~A OH ~I@ 12sI /V~ANH--CH--CO/VVk Fig. 1. G e n e r a l scheme of the f o r m a t i o n of m o n o i o d o t h y r o s i n e r e s i d u e of p r o t e i n s m! n Cram) n Fn = n! w h e r e m stands /m-n/! re- n for the n u m b e r of resi- r for s u b s t i t u t i o n for the n u m b e r of r a d i o i o d i n e atoms degree, i.e., i n t r o d u c e d per pro- and F n for the f r a c t i o n of r a d i o a c t i v i t y in the t r a c e r c o n t r i b u t e d by m o l e c u l e s c o n t a i n i n g n ra- atoms. The s u b s t i t u t i o n degree, from the s t a r t i n g m o l a r labelling /i/ for the m a x i m u m n u m b e r of r e a c t a b l e dues a c t u a l l y reacted, dioiodine (l-r)m m-n r sidues per p r o t e i n molecule, tein m o l e c u l e and di- i.e., r can be c a l c u l a t e d ratio of the r e a c t a n t s mixture t a k i n g into a c c o u n t in the the e f f i c i e n c y of the l a b e l l i n g as well. The s u b s t i t u t i o n c u l a t e d is an average, w h i c h is s e l d o m a w h o l e number. The f r a c t i o n of m o n o - and p o l y l a b e l l e d labelled products Eq. /i/ a p p r o a c h e s degree thus cal- as well as un- can be o b t a i n e d by d i v i d i n g F n by n. the s u b s t i t u t i o n 2~ degree from a proba- TOTH: HETEROGENEITY OF 12 s I-LABELLED PROTEINS bility point of view and does not take the heterogeneity of the potential In fact, sites of substitution tyrosyl residues microenvironments reflected into account. in proteins exist in different and the unequal state of tyrosyls the distribution of the mono-, lecules more complicated by the use of Eq. poly- and unlabelled mo- as compared with that obtained /i/. The aim of this paper is to demonstrate position of the labelling average is in their reactivity with iodine 2. This renders h o w the com- reaction mixture and how the specific activity of the components vary by changing the radioiodine/protein (I/P) ratio. The specific activity of the tracer may influence the performance of the radioimmunoassay in two ways. Usually the higher the specific activity the higher the sensitivity of the assay. In this respect it seems to be advisable to increase the specific activity. other hand, polylabelled immunoreactivity and, proteins usually in addition, lose their with increasing specific activity the rate of radiolytic increases On the decomposition and thus the stability of the tracer decreases. RESULTS The distribution protein molecules of the mono-, poly- and unlabelled as a function of the I/P ratio in the labelling mixture was calculated by the use of Eq. [i/ for m = 2, m = 3, and m = 6 /Figs 2-4/. From Figure 2 it turns out that even in the case of two reactahle sites per m o l e c u l e [e.g., one tyrosyl residue - apart from the marginal cases I/P~O and I/P~2 - at are present any I/P ratio three components ling reaction mixture. 2" in the label- Since in the case of high molecular 261 TOTH: HETEROGENEITY OF a 2 s I-LABELLED PROTEINS 100 o/,. 80 60 4O 20 o I 1 0 I/P Fig. 2. The distribution of unlabelled /0/, monolabelled /i/ and dilabelled /2/ protein molecules as a function of radioiodine-protein ratio, m=2 100 ,/, 8O 6O 9 0 3 40 2O O: 0 Fig. 1 2 3 I/P 3. The distribution of unlabelled /O/, monolabelled /i/ and polylabelled /2,3/ protein molecules as a function of the radioiodine-protein ratio, m=3 262 TOTH: HETEROGENEITY OF 12 s I-LABELLED PROTEINS 0 0 Fig. 6 I/P 4. The d i s t r i b u t i o n of u n l a b e l l e d /O/, m o n o l a b e l l e d /i/ and p o l y l a b e l l e d /2,3,4,6/ protein m o l e c u l e s as a function of the r a d i o i o d i n e - p r o t e i n ratio, m=6 weight I proteins unlabelled, labelled 2 no separation mono- tracer technique can be p r o d u c e d and l a b e l l e d m o l e c u l e s sites 4 and d i l a b e l l e d For three r e a c t a b l e actable 3 in Fig. 5 can d i s t i n g u i s h molecules, no u n i f o r m l y at all. sites the d i s t r i b u t i o n is shown in Fig. of u n l a b e l l e d 3 and for six re- 4. CONCLUSIONS F r o m Figures most important active at all. 2-4 several However, molecules not be achieved. more than protein remain i.e., fact c o n s i d e r a b l e is also case, in the reaction 30% Of the protein m o l e c u l e s belled molecules can be p r o d u c e d formed. 263 i.e., one r a d i o i o d i n e If m=6 and I=P, but in spite of this than one re- in the l a b e l l i n g the o p t i m u m contains The a c t i v i t y of the t r a c e r can the I/P ratio from 0 to m, each protein m o l e c u l e unlabelled labelled the specific by changing action m i x t u r e can be drawn. is that in the case of m o r e site no u n i f o r m l y be v a r i e d conclusions tag and no mixture, I/P=l rewhen can- /see Fig. 4/ remain unlabelled, amount of p o l y l a - TOTH: HETEROGENEITYOF *25I-LABELLEDPROTEINS Since neither the increase of radiolytic decomposition nor the loss of immunoreactivity tion of more than one radioiodine molecule caused by the incorporaatom into the protein can be assessed on theoretical specific activity basis, the optimum is to be adjusted empirically. REFERENCES i. J.L. Oncley, /1957/ 3S. Chemical considerations, Fed. Proc., 2. E. Regoeczi, Iodine-labelled Plasma Proteins, Inc., Boca Raton, Florida, 1984. 264 16 CRC Press