Appl. Radial. Isor. Vol. 41, No. I, pp. 6347, Int. .I. Radiat. Appl. Instrum. Part A
0883-2889/90 $3.00 + 0.00
1990 Copyright
cc’ 1990 Pergamon
Press plc
Printed in Great Britain. All rights reserved
Labeling of Human Serum Albumin with 105Rh-Cysteine Complexes J. M. LO,* M. R. A. PILLAI,? Department
of Chemistry,
C. S. JOHN1
University
(Received 12 December
of Missouri,
and D. E. TROUTNER
Columbia,
1988; in revisedform
MO 65211, U.S.A.
20 March 1989)
The conjugation of a complex formed by reacting RhCl, with cysteine to human serum albumin has been investigated. Approximately 50% of the rhodium (labeled with “‘Rh) was converted to the complex. Conjugation of the complex to HSA via the ECDI method resulted in yields of -40% of the total rhodium or -80% of the Rhxysteine complex. No conjugation was observed in the absence of the ECDI. At approximately equal molar concentrations of rhodium and HSA, an average of -0.4 rhodium atoms per HSA molecule was achieved.
immunoassay using the cobalt radioisotope 57Co which has a half-life of 270 d and a y-energy of 122 keV. It is believed that Rh(III), which has the same oxidation state as Co(III), can also form particularly strong complexes with cysteine or the mixed multiamine ligands. According to the previous work (Malcolme-Lawes, 1980), the Co(II1) complex with cysteine showed higher protein conjugation yields than the mixed multiamine ligands. In the present work, we chose only cysteine as the bifunctional agent to complex “‘Rh and then to conjugate it with protein. Human serum albumin was used as a model protein. The significant difference in using “‘Rhcysteine instead of “Cocysteine for labeling protein is that it may be possibly developed as a potential radioimmunotherapeutic agent.
Introduction “‘Rh can be developed as a potential radiotherapeutic radioisotope. It emits moderate-energy prays (560 keV, 70%; 250 keV, 30%) suitable for radiotherapy, associated with a y-ray (3 19 keV, 19%) suitable for simultaneous imaging when used in nuclear medicine. The half-life of “‘Rh is 35.5 h, favorable for clinical use. The radioisotope can be produced in general research reactors with high specific activity. An appropriate procedure for producing “‘Rh using Ru-metal as a target has been reported (Grazman and Troutner, 1988). Another procedure using ruthenium acetylacetonate as a by the target from which “‘Rh was separated SzilardChalmers process has also been developed (Wong et al., 1988). Recently, we have been utilizing multidentate bifunctional agents for complexing with “‘Rh followed by coupling it to antibodies for possible use in radioimmunotherapy (John et al., 1988). Generally, complexes of rhodium are kinetically inert and may be treated as stable molecules or ions to conjugate to proteins. Malcolme-Lawes (MalcolmeLawes, 1980) has reported that complexes of Co(II1) with cysteine or mixed hgands with ethylenediamine and triethylenetetramine can be conjugated to bovine serum albumin. This protein labeling work was to develop a new diagnostic tool in radio-
Experimental Reagents “‘Rh was supplied as [‘05RhCl,(H,0), r]m(’ 3’ in saline by the University of Missouri Research Reactor (Grazman and Troutner, 1988). The solution was diluted with inactive rhodium(II1) chloride solution to a suitable specific activity for experiments in the open laboratory. L-Cysteine and RhCI, 3H20 were purchased from Aldrich Chemical Company. Human serum albumin (HSA) A-1653, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (ECDI) and Sephadex G-75 were from Sigma Chemical Company. Trichloroacetic acid was from Fisher Scientific Company. Magnesium oxide (USP. a light powder) and flexible silica gel TLC plates, 7.5 x 2.5 cm, were from J. T. Baker Chemical Company. All other materials or solvents were reagent grade. Saline used
Permanent addresses: *Institute of Nuclear Science, National Tsing Hua University, Hsinchu, Taiwan 30043, R.O.C. tRadiopharmaceuticals Division, Bhabha Atomic Research Centre, Bombay 400 085, India. fDepartment of Radiology, Division of Nuclear Medicine, University of Pennsylvania Hospital, Philadelphia, PA 19104, U.S.A. 63
J. M. Lo er al
64
m these studies was prepared NaCl in 1 L of double-distilled
by dissolving water.
9 g of
(-5
x 10m5M)
incubated Prepurution
of ‘“‘Rh-qtsteine
complexes
Conjugation
To 1 mL of RhCl, (2.5 x 10m4 M or 2.5 x lo-’ M) in saline was added 25 PL of “‘Rh (4 MBq). The pH of the “‘5RhClj solution usually was found to be z 334. The solution was heated in a boiling water bath for - 15-20 min. Then 0.1 mL of cysteine (7.5 x 10 ‘M or 7.5 x IO-‘M) in saline was added and the mixture heated in a boiling water bath for 1 h. By this procedure. the cysteine to rhodium molar ratio for reaction either at lower concentrations (i.e. - IO ’ M) or higher concentrations (i.e. -IO-’ M) was (‘N 3 : I. Another batch of ‘“SRh-cvsteine complex was prepared in basic solution. . One mL of RhCI, (2.5 x 10mJM) in saline was mixed with 25pL of “‘5Rh (4 MBq), heated in a boiling water bath, and then cooled. The solution was again heated from -40 to 100 C after 0.2 mL of 0.5 M NaHCO, was added until a clear yellow solution was obtained. On heating at 100 C for longer than 5 min a yellow precipitate appeared. Therefore, care was taken to avoid heating for longer times. Finally 0.1 mL of cysteine (7.5 x lo- ’ M) in saline was added and the mixture was heated in a boiling water bath for I h. The cysteine to rhodium molar ratio for complexation in basic medium was also cn 3: I. Characterixtion
of’ ‘“Rh-c)asteine
complexes
Thin luyer chromutographr. About 5 PL of complex solution was spotted about 1.5 cm from the end of the silica gel TLC plates and developed ascendingly in saline to the top of the plate, a 6-cm migration. The plate was dried in air and cut into 10 0.75 cm sections. The sections were placed in counting tubes and counted in a well type NaI(T1) counter. Activities at both R,= 0 and R, = I were integrated and compared with the total activity (-3-5 kBq) applied in the plate. Magnesium o.yide adsorption. To 0.4 mL of the complex solution (or O.lmL of the complex solution and 0.3 mL of the medium solution, if the sample was less). approx. 50 mg of MgO was added. The solution was mixed well with MgO for about 1 min over a vortex mixer and then centrifuged. An amount of 0.2 mL of the supernate solution was withdrawn and counted. The magnesium oxide fraction containing 0.2 mL of solution was also counted. Both samples were in 13 x 75 mm tubes and therefore were essentially in the same geometry. The yield of complex was calculated as below %complex
Non -spec$c
=
2 x activity of supernate activity of supernate + activity of fraction
in
pH4
saline.
ECDI
(0.1 mL)
(- 3 x IO-* M) in saline was added. The mixture was
x 100
with MgO
binding
HSA solution (0.4 mL) ( - 6 x 10m5 M) in saline was mixed with 0.4mL of ‘“‘RhCl, solution
at room temperature
for 24 h.
of ““Rh comp1e.u with HSA
Conjugations using different molar ratios of Rh complex/HSA were performed. Usually a solution of HSA in saline (3 mgjml) was used but a solution of HSA in pM 5 acetate was also tested. The lu5Rh complex solutions obtained from the preparation in acidic medium were added either directly to the HSA solution or added after dilution to suitable concentrations with saline. The complex solution prepared from basic medium was added to the HSA solution after adjusting from pH 9 to pH 4.5 by dropwise addition of 0.1 M HCI. The solution of ECDI in saline (ca IOO-fold excess in quantity) was subsequently added in the mixture of complex and HSA. Conjugation yield was estimated after incubating at room temperature for a period of time for 0.5524 h. Corresponding blanks were usually run along with the conjugated samples by incubating the mixture of complex and HSA in the absence of ECDI. Estimation
qf’coqjugution yield
Trichloroacetic ucid precipitation method. The protein was denatured by trichloroacetic acid and precipitated so that the fraction of ““Rh complex conjugated with HSA could bc estimated from the radioactivity associated with the precipitate. To 0.3 mL of the conjugating solutions was added 0.1 mL of 15% trichloroacetic acid. The solution was mixed well for about 1 min over a vortex mixer and centrifuged. Supernate solution (0.2 mL) was withdrawn and counted. The precipitate fraction containing 0.2 mL of solution was also counted. The yield of conjugation was calculated as below % conjugation
=
activity in precipitate total activity
activity of fraction with precipitate -activity of supernate =p activity of fraction with precipitate + activity of supernate
x 100
x 100.
Sephude.x G- 75 column chromutogruph~~. A I .4-cm glass column was packed to a height of 30cm with Sephadex G-75 which had been stored in saline. About 0.3 mL of the conjugated solution was loaded in the column and eluted with IOOmL saline. TwomL fractions were collected and the radioactivity of each measured. The labeled HSA was collected from the elution volume between 14 and 24 mL while the unconjugated complex and/or free rhodium RhCI, was from 34 to 60 mL. EDTA
challenge
The stability of lnSRh in the labeled HSA was estimated by challenge experiments with EDTA. Two mL of the purified labeled protein from a Sephadex
Labeling of HSA with ‘OSRhxysteine
65
G-75 column was mixed with 0.1 mL of an EDTA solution to give at least a lOO-fold excess with respect to rhodium, and incubated for 24 h at -25’C. The radioactivity associated with the protein was measured by the trichloroacetic acid precipitation method. Due to the low concentration of HSA in the purified labeled HSA solution, 0.2 mg of HSA was added as unlabeled carrier before adding 0.1 mL of 15% TCA.
Results and Discussion Table 1 summarizes the results for complexation and conjugation of “‘Rh
0883-2889/90 $3.00 + 0.00
1990 Copyright
cc’ 1990 Pergamon
Press plc
Printed in Great Britain. All rights reserved
Labeling of Human Serum Albumin with 105Rh-Cysteine Complexes J. M. LO,* M. R. A. PILLAI,? Department
of Chemistry,
C. S. JOHN1
University
(Received 12 December
of Missouri,
and D. E. TROUTNER
Columbia,
1988; in revisedform
MO 65211, U.S.A.
20 March 1989)
The conjugation of a complex formed by reacting RhCl, with cysteine to human serum albumin has been investigated. Approximately 50% of the rhodium (labeled with “‘Rh) was converted to the complex. Conjugation of the complex to HSA via the ECDI method resulted in yields of -40% of the total rhodium or -80% of the Rhxysteine complex. No conjugation was observed in the absence of the ECDI. At approximately equal molar concentrations of rhodium and HSA, an average of -0.4 rhodium atoms per HSA molecule was achieved.
immunoassay using the cobalt radioisotope 57Co which has a half-life of 270 d and a y-energy of 122 keV. It is believed that Rh(III), which has the same oxidation state as Co(III), can also form particularly strong complexes with cysteine or the mixed multiamine ligands. According to the previous work (Malcolme-Lawes, 1980), the Co(II1) complex with cysteine showed higher protein conjugation yields than the mixed multiamine ligands. In the present work, we chose only cysteine as the bifunctional agent to complex “‘Rh and then to conjugate it with protein. Human serum albumin was used as a model protein. The significant difference in using “‘Rhcysteine instead of “Cocysteine for labeling protein is that it may be possibly developed as a potential radioimmunotherapeutic agent.
Introduction “‘Rh can be developed as a potential radiotherapeutic radioisotope. It emits moderate-energy prays (560 keV, 70%; 250 keV, 30%) suitable for radiotherapy, associated with a y-ray (3 19 keV, 19%) suitable for simultaneous imaging when used in nuclear medicine. The half-life of “‘Rh is 35.5 h, favorable for clinical use. The radioisotope can be produced in general research reactors with high specific activity. An appropriate procedure for producing “‘Rh using Ru-metal as a target has been reported (Grazman and Troutner, 1988). Another procedure using ruthenium acetylacetonate as a by the target from which “‘Rh was separated SzilardChalmers process has also been developed (Wong et al., 1988). Recently, we have been utilizing multidentate bifunctional agents for complexing with “‘Rh followed by coupling it to antibodies for possible use in radioimmunotherapy (John et al., 1988). Generally, complexes of rhodium are kinetically inert and may be treated as stable molecules or ions to conjugate to proteins. Malcolme-Lawes (MalcolmeLawes, 1980) has reported that complexes of Co(II1) with cysteine or mixed hgands with ethylenediamine and triethylenetetramine can be conjugated to bovine serum albumin. This protein labeling work was to develop a new diagnostic tool in radio-
Experimental Reagents “‘Rh was supplied as [‘05RhCl,(H,0), r]m(’ 3’ in saline by the University of Missouri Research Reactor (Grazman and Troutner, 1988). The solution was diluted with inactive rhodium(II1) chloride solution to a suitable specific activity for experiments in the open laboratory. L-Cysteine and RhCI, 3H20 were purchased from Aldrich Chemical Company. Human serum albumin (HSA) A-1653, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (ECDI) and Sephadex G-75 were from Sigma Chemical Company. Trichloroacetic acid was from Fisher Scientific Company. Magnesium oxide (USP. a light powder) and flexible silica gel TLC plates, 7.5 x 2.5 cm, were from J. T. Baker Chemical Company. All other materials or solvents were reagent grade. Saline used
Permanent addresses: *Institute of Nuclear Science, National Tsing Hua University, Hsinchu, Taiwan 30043, R.O.C. tRadiopharmaceuticals Division, Bhabha Atomic Research Centre, Bombay 400 085, India. fDepartment of Radiology, Division of Nuclear Medicine, University of Pennsylvania Hospital, Philadelphia, PA 19104, U.S.A. 63
J. M. Lo er al
64
m these studies was prepared NaCl in 1 L of double-distilled
by dissolving water.
9 g of
(-5
x 10m5M)
incubated Prepurution
of ‘“‘Rh-qtsteine
complexes
Conjugation
To 1 mL of RhCl, (2.5 x 10m4 M or 2.5 x lo-’ M) in saline was added 25 PL of “‘Rh (4 MBq). The pH of the “‘5RhClj solution usually was found to be z 334. The solution was heated in a boiling water bath for - 15-20 min. Then 0.1 mL of cysteine (7.5 x 10 ‘M or 7.5 x IO-‘M) in saline was added and the mixture heated in a boiling water bath for 1 h. By this procedure. the cysteine to rhodium molar ratio for reaction either at lower concentrations (i.e. - IO ’ M) or higher concentrations (i.e. -IO-’ M) was (‘N 3 : I. Another batch of ‘“SRh-cvsteine complex was prepared in basic solution. . One mL of RhCI, (2.5 x 10mJM) in saline was mixed with 25pL of “‘5Rh (4 MBq), heated in a boiling water bath, and then cooled. The solution was again heated from -40 to 100 C after 0.2 mL of 0.5 M NaHCO, was added until a clear yellow solution was obtained. On heating at 100 C for longer than 5 min a yellow precipitate appeared. Therefore, care was taken to avoid heating for longer times. Finally 0.1 mL of cysteine (7.5 x lo- ’ M) in saline was added and the mixture was heated in a boiling water bath for I h. The cysteine to rhodium molar ratio for complexation in basic medium was also cn 3: I. Characterixtion
of’ ‘“Rh-c)asteine
complexes
Thin luyer chromutographr. About 5 PL of complex solution was spotted about 1.5 cm from the end of the silica gel TLC plates and developed ascendingly in saline to the top of the plate, a 6-cm migration. The plate was dried in air and cut into 10 0.75 cm sections. The sections were placed in counting tubes and counted in a well type NaI(T1) counter. Activities at both R,= 0 and R, = I were integrated and compared with the total activity (-3-5 kBq) applied in the plate. Magnesium o.yide adsorption. To 0.4 mL of the complex solution (or O.lmL of the complex solution and 0.3 mL of the medium solution, if the sample was less). approx. 50 mg of MgO was added. The solution was mixed well with MgO for about 1 min over a vortex mixer and then centrifuged. An amount of 0.2 mL of the supernate solution was withdrawn and counted. The magnesium oxide fraction containing 0.2 mL of solution was also counted. Both samples were in 13 x 75 mm tubes and therefore were essentially in the same geometry. The yield of complex was calculated as below %complex
Non -spec$c
=
2 x activity of supernate activity of supernate + activity of fraction
in
pH4
saline.
ECDI
(0.1 mL)
(- 3 x IO-* M) in saline was added. The mixture was
x 100
with MgO
binding
HSA solution (0.4 mL) ( - 6 x 10m5 M) in saline was mixed with 0.4mL of ‘“‘RhCl, solution
at room temperature
for 24 h.
of ““Rh comp1e.u with HSA
Conjugations using different molar ratios of Rh complex/HSA were performed. Usually a solution of HSA in saline (3 mgjml) was used but a solution of HSA in pM 5 acetate was also tested. The lu5Rh complex solutions obtained from the preparation in acidic medium were added either directly to the HSA solution or added after dilution to suitable concentrations with saline. The complex solution prepared from basic medium was added to the HSA solution after adjusting from pH 9 to pH 4.5 by dropwise addition of 0.1 M HCI. The solution of ECDI in saline (ca IOO-fold excess in quantity) was subsequently added in the mixture of complex and HSA. Conjugation yield was estimated after incubating at room temperature for a period of time for 0.5524 h. Corresponding blanks were usually run along with the conjugated samples by incubating the mixture of complex and HSA in the absence of ECDI. Estimation
qf’coqjugution yield
Trichloroacetic ucid precipitation method. The protein was denatured by trichloroacetic acid and precipitated so that the fraction of ““Rh complex conjugated with HSA could bc estimated from the radioactivity associated with the precipitate. To 0.3 mL of the conjugating solutions was added 0.1 mL of 15% trichloroacetic acid. The solution was mixed well for about 1 min over a vortex mixer and centrifuged. Supernate solution (0.2 mL) was withdrawn and counted. The precipitate fraction containing 0.2 mL of solution was also counted. The yield of conjugation was calculated as below % conjugation
=
activity in precipitate total activity
activity of fraction with precipitate -activity of supernate =p activity of fraction with precipitate + activity of supernate
x 100
x 100.
Sephude.x G- 75 column chromutogruph~~. A I .4-cm glass column was packed to a height of 30cm with Sephadex G-75 which had been stored in saline. About 0.3 mL of the conjugated solution was loaded in the column and eluted with IOOmL saline. TwomL fractions were collected and the radioactivity of each measured. The labeled HSA was collected from the elution volume between 14 and 24 mL while the unconjugated complex and/or free rhodium RhCI, was from 34 to 60 mL. EDTA
challenge
The stability of lnSRh in the labeled HSA was estimated by challenge experiments with EDTA. Two mL of the purified labeled protein from a Sephadex
Labeling of HSA with ‘OSRhxysteine
65
G-75 column was mixed with 0.1 mL of an EDTA solution to give at least a lOO-fold excess with respect to rhodium, and incubated for 24 h at -25’C. The radioactivity associated with the protein was measured by the trichloroacetic acid precipitation method. Due to the low concentration of HSA in the purified labeled HSA solution, 0.2 mg of HSA was added as unlabeled carrier before adding 0.1 mL of 15% TCA.
Results and Discussion Table 1 summarizes the results for complexation and conjugation of “‘Rh
