Download I-labelled proteins used as tracers in radioimmunoassay

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