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Formation constants of Gallium- and Indium-Transferrin
lacernor~~~nrrlJournal of Nuckur Mrdicinv und Biolny?: Vol. 6, pp. 138 TO 141 Pergamon Press Ltd 1979. Printed in Chat Britain
where (Ga3+), (L), and (Tf) refer to gallium, ligand and transferrin concentrations respectively, The first formation constant of gallium-transferrin refers to :
7’f + Ga 3+ = Tf - Ga
Formation constants of Galiiumand Indium-Traasferrin S. KULPRATHIPANJA,E
~i~;OWICH,*
KThis may be rewritten :
R. BEH and
Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A. and Massachusetts General Hospital, Boston, MA., U.S.A.
K1 =
K
now well established that unless administered into plasma or serum environments in a very stable chemical form such as the EDTA or DTPA chelate, gallium is rapidly and exclusively bound to transferrin proteins.“) Furthermore, there is adequate reason to believe that the same phenomenon occurs with indium, an element sharing many of the same chemical properties of gallium.‘2) A large formation constant for gallium- and indium-transferrin is necessary to explain these observations; however, these constants have not been previously reported.
Ga ,,u,Iide = (Ga,) = (Ga3+) + (L - Ga). In addition, equilibrium with the chelating agent (ligand) will be the same both .inside and outside the bag : (L - Ga) = (L) + (Ga3+)
*.Present address: University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, MA 01605, U.S.A.
Wh) - (Gad(~) KL [(Gail - (Gao)31 C(Gao)- (Ga3+)l’ (1)
Since. (Ga3’) may be ignored with respect to Gae, the terms in equation (1) are all known and K, may be calculated. The second formation constant of galliumtransferrin refers to : Tf - Ga + Ga3+ = Tf - Gar
The method chosen utilized equilibrium dialysis and was similar to that used by DAVIS et al. in the measurement of the iron-transferrin constants.‘3) The formation constant(s) for a metal-protein complex may be estimated by equilibrium dialysis of the metal-protein solution against a solution of a chelating agent with a known formation constant for the metal in question. At equilibrium, the concentrations of the solution inside and outside the dialysis bag are as follows:
+ (L - Ga) + (7’f - Ga) + (Tf - Ga2).
(L - W (L) (Ga’+)’
K1= l(Tf,) -
Materialsand Methds
Outside :
- Ga)
It may be assumed that if K, is not small with respect to K2 and if the gallium present is not sufficient to saturate both transferrin binding sites, then the term (Tf - Ga2) may be ignored with respect to (7’f - Ga). In this case, the expression reduces to
IT IS
Gain.idr = (Gai) = (Ga3’)
=
‘
Introduction
Tf,,,,, = (TA) = (Tf) + (Tf - Ga) + (Tf - Gal)
(L)(Tf - Ga)K, [(TX) - C(V - Ga) + (Tf - Ga,)l:(L
where
(Received 21 October 1978)
Inside :
Vf - W
’ - (Tf)(Ga3’)’
K2=(Tf
Vf - Gad - Ga)(Ga’+)’
The measurement of K2 requires a higher concentration of added Ga. We may therefore assume that (Tf) = 0 and that (Tf - Ga) > (Tf - Ga2) such that (Tfi) = (Tf - Ga) and K
=
(C(W) - (Gad1 - (Tft)} WI&
2 WXGao)
.
(2)
Therefore, the terms in equation (2) are all known and K2 may be calculated. The same procedure was used to calculate the constants for indium-transferrin. GalIium and indium citrate were prepared by dissolving known weights (about 5mg) of the metals (Sigma Chemical Corp., St Louis, MO.) in aqua regia, evaporating to dryness and redissolving in 1 ml of 1 M HCl. Stock solutions were prepared by diluting each solution to 100 ml with a sodium citrate solution (2 mg/ml) at pH 7.3. Carrier-free “‘Ga as the citrate and ’1‘In as the chloride complex were obtained from New England Nuclear Corp., Boston, MA. and were added as needed to aliquots of the stock solutions to yield the desired counting rate for solutions with the desired specific activity. Gallium- and indiumtransferrin were prepared by dissolving a known weight of human apotransferrin (Sigma Chemical Co, St Louis, MO.) in the 67Ga or ‘1iJn stock solutions. In both cases, a slight excess of the calculated amount 1138
ion (PM)
ion (PM)
Inin.idr (FM) In outride 01M) V&r (PM) Log KI Log K2
Indium Counter
Gauutridc 01M) Wk., (PM) Log KI Log Kz
Gaina, (PM)
Gallium Counter
8.90 0.0102 12.5
0.0
0.127 0.0076 15.52 -
0.0
TABLE 1. Results
6.44 0.059 12.5 29.2
lo3 EDTA
0.0804 0.0085 15.52 23.0 -
lo3 EDTA
of equilibrium
of gallium-
4.99 0.088 12.5 30.4
IO3 DTPA
1.35 0.159 11.5 23.1
IO3 EDTA
dialysis
26.6
10.18 0.017 9.54
lo3 EDTA
22.4
6.45 0.261 6.4
lo3 EDTA
solutions
3.62 0.116 12.5 29.5 -
IO4 EDTA
0.746 0.0087 15.52 23.9 -
lo4 EDTA
and indium-transferrin
3.01 0.128 12.5 32.0 -
lo4 DTPA
0.93s 0.167 11.5 23.9 -
24.4
-
9.63 0.086 9.54
IO4 EDTA
22.2
6.44 0.0382 6.4
lo4 EDTA
EDTA and DPTA
IO4 EDTA
against
ions
3.20 0.124 12.5 30.4
10’ EDTA
0.0210 0.0097 15.52 24.0
IO5 EDTA
as counter
0.306 0.182 12.5 31.4
IO5 DTPA
0.419 0.178 11.5 24.3
lo5 EDTA
140
Technical note
TABLE 2. Average values of gallium- and indiumtransferrin formation constants (units of M-r) Tf + Ga3+ = Tf-Ga Tf-Ga + Ga3+ = Tf-Ga, Tf+ In3+ = Tf-In
Tf-In + In’+ = Tf-In,
log log log log
and 27.65 M- ’ respectively, were used in the subsequent calculations.
Kr = 23.7 K2 = 22.3
Results and Discussion
K, = 30.5 Kz = 25.5
Transferrin is known to bind up to two atoms of Fe3+ or In3+ per molecule of protein.‘4J) Since gallium and indium ark both trivalent elements belonging to the same group of the periodic table, it is a reasonable assumption that up to two atoms of gallium will be bound as well. On this assumption, the formation constants with transferrin were calculated using the method described above. The results obtained are given in Tables 1 and 2. Because of the experimental difficulties in the measurement of the second formation constants, the log K2 values are considered to be less reliable than the log K, values. Since gallium and indium have similar electronic structures and similar chemical properties, it is not unreasonable that similar complex formation will occur with the same ligands. The formation constants for gallium and indium complexes with the same ligands have been reported@) and these values show that indium tends to form somewhat stronger complexes than gallium as was found in this study with transferrin. This may be seen in Fig. 1 where the literature values for log K, for these complexes and for transferrin are plotted. The formation constants for gallium-transferrin obtained in this work may be of particular significance in understanding the tumor localizing properties of 67Ga administered as the citrate. 67Ga citrate is generally regarded as the agent of choice for soft tissue tumor imaging”) although the mechanism of its localization is unknown. It is well established that 67Ga citrate dissociates in oivo such that 67Ga is
of gallium or indium needed to saturate the protein binding sites were used. The excess was then removed by passage through Sephadex G-50 (Sigma Chemical Corp., St Louis, MO.). The eluant was 0.04 M NaHCO,, pH 7.3. The concentration of gallium and indium citrates were 0.1 to 16,uM and the concentration of transferrin was 6-l 5 pM. 2ml aliquots of either gallium-transferrin or indium-transferrin solution were brought to the desired concentration of EDTA or DTPA (0.001-0.1 M) and securely tied into a dialysis bag with a 12,000 molecular weight cut-off (Arthur H. Thomas Co., Philadelphia, PA.). The dialysis bag was rinsed and placed in 100 ml of the EDTA or DTPA solution at the same concentration as that within the bag. All solutions were 0.04 M NaHCOs at pH 7.3. Equilibrium was achieved in about 24 h as determined by periodically removing aliquots of the external solution for counting; dialysis was allowed to proceed for an additional 24 h. The dialysis bags were then removed and rinsed. Samples of the protein solution and external solution were taken for counting in a NaI(Tl) well counter along with standards of the original solutions. From the known specific activity of 67Ga and “‘In, the molar concentrations of gallium and indium inside and outside the bags were determined. The log values of formation constants of SILLENand MAR~LL~‘) for Ga-EDTA. In-EDTA and In-DTPA of 20.25, 24.95 I
I
I
TRANSFERRIN
I
I
I
I
-----a
hi-
!l I
E I
z
IO
i
GALLIUM
LOG Kl
FIG. 1. Rot of log K1 indium vs log K, gallium for several chelates including
transferrin.
Technical Note
bound largely to transferrin moleculeso’ It has been shown recently that the presence of transferrin is required for appreciable uptake of “‘Ga into tumor cells growing in culture. (*) The suggestion has been made that the 67Ga is incorporated into tumor cells by a mechanism which involves, as a first step, the binding of gallium-transferrin to specific cell surface receptors, followed by intracellular incorporation of the gallium-transferrin complex.@) Use of the galliumtransferrin formation constants may help to establish the validity of these proposed mechanisms. Acknowledgements-This
by the U.S. Department EY-S-02-4115.
work was supported in part of Energy under contract
References
1. HARTMANR. E. and HAYESR. L. J. Pharmac. exp. Ther. 168, 193 (1969).
2.
141
3.
HOSAINF., MCINTYREP. A., POULOSEK., STERN H. S. and WAGNER H. N. Clinica. chim. Acta 24, 69 (1969). DAVIS B., SALTMANP. and BJZNS~NS. B&hem.
4.
biophys. Res. Commun. 8, 56 (1962). LAURELLC. B. In The Plasma Proteins (Edited
by F. W. PUTNAM),p. 349. Academic Press, New York (1962). 5. WELCHM. J. and WELCHT. J. In Radiopharmaceuticals (Edited by SUBRAMANIAN et al.), p. 73. Sot. Nucl. Med., New York (1975). 6. SILLENL. and MARTELLA. Stability Constunts of Metal Ion Complexes. Suppl. 1, Special Pubn No. 25. The Chemical Society, London (1971). 7. ANDREWSG. A. and EDWARDSC. L. J. Am. med. Ass. 233, 1100 (1975). 8. LARSONS. M., RA~EYJ. S. and ALLEN D. R. Proc. 2nd Int. Gong. World Federation Nuclear Medicine and Biology (1978).
of
where (Ga3+), (L), and (Tf) refer to gallium, ligand and transferrin concentrations respectively, The first formation constant of gallium-transferrin refers to :
7’f + Ga 3+ = Tf - Ga
Formation constants of Galiiumand Indium-Traasferrin S. KULPRATHIPANJA,E
~i~;OWICH,*
KThis may be rewritten :
R. BEH and
Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A. and Massachusetts General Hospital, Boston, MA., U.S.A.
K1 =
K
now well established that unless administered into plasma or serum environments in a very stable chemical form such as the EDTA or DTPA chelate, gallium is rapidly and exclusively bound to transferrin proteins.“) Furthermore, there is adequate reason to believe that the same phenomenon occurs with indium, an element sharing many of the same chemical properties of gallium.‘2) A large formation constant for gallium- and indium-transferrin is necessary to explain these observations; however, these constants have not been previously reported.
Ga ,,u,Iide = (Ga,) = (Ga3+) + (L - Ga). In addition, equilibrium with the chelating agent (ligand) will be the same both .inside and outside the bag : (L - Ga) = (L) + (Ga3+)
*.Present address: University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, MA 01605, U.S.A.
Wh) - (Gad(~) KL [(Gail - (Gao)31 C(Gao)- (Ga3+)l’ (1)
Since. (Ga3’) may be ignored with respect to Gae, the terms in equation (1) are all known and K, may be calculated. The second formation constant of galliumtransferrin refers to : Tf - Ga + Ga3+ = Tf - Gar
The method chosen utilized equilibrium dialysis and was similar to that used by DAVIS et al. in the measurement of the iron-transferrin constants.‘3) The formation constant(s) for a metal-protein complex may be estimated by equilibrium dialysis of the metal-protein solution against a solution of a chelating agent with a known formation constant for the metal in question. At equilibrium, the concentrations of the solution inside and outside the dialysis bag are as follows:
+ (L - Ga) + (7’f - Ga) + (Tf - Ga2).
(L - W (L) (Ga’+)’
K1= l(Tf,) -
Materialsand Methds
Outside :
- Ga)
It may be assumed that if K, is not small with respect to K2 and if the gallium present is not sufficient to saturate both transferrin binding sites, then the term (Tf - Ga2) may be ignored with respect to (7’f - Ga). In this case, the expression reduces to
IT IS
Gain.idr = (Gai) = (Ga3’)
=
‘
Introduction
Tf,,,,, = (TA) = (Tf) + (Tf - Ga) + (Tf - Gal)
(L)(Tf - Ga)K, [(TX) - C(V - Ga) + (Tf - Ga,)l:(L
where
(Received 21 October 1978)
Inside :
Vf - W
’ - (Tf)(Ga3’)’
K2=(Tf
Vf - Gad - Ga)(Ga’+)’
The measurement of K2 requires a higher concentration of added Ga. We may therefore assume that (Tf) = 0 and that (Tf - Ga) > (Tf - Ga2) such that (Tfi) = (Tf - Ga) and K
=
(C(W) - (Gad1 - (Tft)} WI&
2 WXGao)
.
(2)
Therefore, the terms in equation (2) are all known and K2 may be calculated. The same procedure was used to calculate the constants for indium-transferrin. GalIium and indium citrate were prepared by dissolving known weights (about 5mg) of the metals (Sigma Chemical Corp., St Louis, MO.) in aqua regia, evaporating to dryness and redissolving in 1 ml of 1 M HCl. Stock solutions were prepared by diluting each solution to 100 ml with a sodium citrate solution (2 mg/ml) at pH 7.3. Carrier-free “‘Ga as the citrate and ’1‘In as the chloride complex were obtained from New England Nuclear Corp., Boston, MA. and were added as needed to aliquots of the stock solutions to yield the desired counting rate for solutions with the desired specific activity. Gallium- and indiumtransferrin were prepared by dissolving a known weight of human apotransferrin (Sigma Chemical Co, St Louis, MO.) in the 67Ga or ‘1iJn stock solutions. In both cases, a slight excess of the calculated amount 1138
ion (PM)
ion (PM)
Inin.idr (FM) In outride 01M) V&r (PM) Log KI Log K2
Indium Counter
Gauutridc 01M) Wk., (PM) Log KI Log Kz
Gaina, (PM)
Gallium Counter
8.90 0.0102 12.5
0.0
0.127 0.0076 15.52 -
0.0
TABLE 1. Results
6.44 0.059 12.5 29.2
lo3 EDTA
0.0804 0.0085 15.52 23.0 -
lo3 EDTA
of equilibrium
of gallium-
4.99 0.088 12.5 30.4
IO3 DTPA
1.35 0.159 11.5 23.1
IO3 EDTA
dialysis
26.6
10.18 0.017 9.54
lo3 EDTA
22.4
6.45 0.261 6.4
lo3 EDTA
solutions
3.62 0.116 12.5 29.5 -
IO4 EDTA
0.746 0.0087 15.52 23.9 -
lo4 EDTA
and indium-transferrin
3.01 0.128 12.5 32.0 -
lo4 DTPA
0.93s 0.167 11.5 23.9 -
24.4
-
9.63 0.086 9.54
IO4 EDTA
22.2
6.44 0.0382 6.4
lo4 EDTA
EDTA and DPTA
IO4 EDTA
against
ions
3.20 0.124 12.5 30.4
10’ EDTA
0.0210 0.0097 15.52 24.0
IO5 EDTA
as counter
0.306 0.182 12.5 31.4
IO5 DTPA
0.419 0.178 11.5 24.3
lo5 EDTA
140
Technical note
TABLE 2. Average values of gallium- and indiumtransferrin formation constants (units of M-r) Tf + Ga3+ = Tf-Ga Tf-Ga + Ga3+ = Tf-Ga, Tf+ In3+ = Tf-In
Tf-In + In’+ = Tf-In,
log log log log
and 27.65 M- ’ respectively, were used in the subsequent calculations.
Kr = 23.7 K2 = 22.3
Results and Discussion
K, = 30.5 Kz = 25.5
Transferrin is known to bind up to two atoms of Fe3+ or In3+ per molecule of protein.‘4J) Since gallium and indium ark both trivalent elements belonging to the same group of the periodic table, it is a reasonable assumption that up to two atoms of gallium will be bound as well. On this assumption, the formation constants with transferrin were calculated using the method described above. The results obtained are given in Tables 1 and 2. Because of the experimental difficulties in the measurement of the second formation constants, the log K2 values are considered to be less reliable than the log K, values. Since gallium and indium have similar electronic structures and similar chemical properties, it is not unreasonable that similar complex formation will occur with the same ligands. The formation constants for gallium and indium complexes with the same ligands have been reported@) and these values show that indium tends to form somewhat stronger complexes than gallium as was found in this study with transferrin. This may be seen in Fig. 1 where the literature values for log K, for these complexes and for transferrin are plotted. The formation constants for gallium-transferrin obtained in this work may be of particular significance in understanding the tumor localizing properties of 67Ga administered as the citrate. 67Ga citrate is generally regarded as the agent of choice for soft tissue tumor imaging”) although the mechanism of its localization is unknown. It is well established that 67Ga citrate dissociates in oivo such that 67Ga is
of gallium or indium needed to saturate the protein binding sites were used. The excess was then removed by passage through Sephadex G-50 (Sigma Chemical Corp., St Louis, MO.). The eluant was 0.04 M NaHCO,, pH 7.3. The concentration of gallium and indium citrates were 0.1 to 16,uM and the concentration of transferrin was 6-l 5 pM. 2ml aliquots of either gallium-transferrin or indium-transferrin solution were brought to the desired concentration of EDTA or DTPA (0.001-0.1 M) and securely tied into a dialysis bag with a 12,000 molecular weight cut-off (Arthur H. Thomas Co., Philadelphia, PA.). The dialysis bag was rinsed and placed in 100 ml of the EDTA or DTPA solution at the same concentration as that within the bag. All solutions were 0.04 M NaHCOs at pH 7.3. Equilibrium was achieved in about 24 h as determined by periodically removing aliquots of the external solution for counting; dialysis was allowed to proceed for an additional 24 h. The dialysis bags were then removed and rinsed. Samples of the protein solution and external solution were taken for counting in a NaI(Tl) well counter along with standards of the original solutions. From the known specific activity of 67Ga and “‘In, the molar concentrations of gallium and indium inside and outside the bags were determined. The log values of formation constants of SILLENand MAR~LL~‘) for Ga-EDTA. In-EDTA and In-DTPA of 20.25, 24.95 I
I
I
TRANSFERRIN
I
I
I
I
-----a
hi-
!l I
E I
z
IO
i
GALLIUM
LOG Kl
FIG. 1. Rot of log K1 indium vs log K, gallium for several chelates including
transferrin.
Technical Note
bound largely to transferrin moleculeso’ It has been shown recently that the presence of transferrin is required for appreciable uptake of “‘Ga into tumor cells growing in culture. (*) The suggestion has been made that the 67Ga is incorporated into tumor cells by a mechanism which involves, as a first step, the binding of gallium-transferrin to specific cell surface receptors, followed by intracellular incorporation of the gallium-transferrin complex.@) Use of the galliumtransferrin formation constants may help to establish the validity of these proposed mechanisms. Acknowledgements-This
by the U.S. Department EY-S-02-4115.
work was supported in part of Energy under contract
References
1. HARTMANR. E. and HAYESR. L. J. Pharmac. exp. Ther. 168, 193 (1969).
2.
141
3.
HOSAINF., MCINTYREP. A., POULOSEK., STERN H. S. and WAGNER H. N. Clinica. chim. Acta 24, 69 (1969). DAVIS B., SALTMANP. and BJZNS~NS. B&hem.
4.
biophys. Res. Commun. 8, 56 (1962). LAURELLC. B. In The Plasma Proteins (Edited
by F. W. PUTNAM),p. 349. Academic Press, New York (1962). 5. WELCHM. J. and WELCHT. J. In Radiopharmaceuticals (Edited by SUBRAMANIAN et al.), p. 73. Sot. Nucl. Med., New York (1975). 6. SILLENL. and MARTELLA. Stability Constunts of Metal Ion Complexes. Suppl. 1, Special Pubn No. 25. The Chemical Society, London (1971). 7. ANDREWSG. A. and EDWARDSC. L. J. Am. med. Ass. 233, 1100 (1975). 8. LARSONS. M., RA~EYJ. S. and ALLEN D. R. Proc. 2nd Int. Gong. World Federation Nuclear Medicine and Biology (1978).
of