Radioactive Labeling of Proteins with an Jodinated Amidination Reagent FREDERICK T. WOOD School of Dentistry, University of the Pacific, San Francisco, California 94115, USA
An iodinated phenolic imidoester has been synthesized for the labeling of proteins to high specific activities with radioactive iodine. The main advantage of this two-step labeling method is that it obviates direct contact of the protein with deleterious oxidizing agents, such as chloramine-T, pressent in direct methods of iodinating proteins. Several techniques for iodinating proteins have been developed based on the direct substitution of 1251 onto tyrosyl residues.1,2 These methods involve the use of oxidizing agents which in some instances lead to inactivation of the proteins, presumably by side reactions such as sulfhydryl oxidation. Recently, Bolton and Hunter3 and Rudinger and Ruegg4 reported the synthesis and use of an 1251-labeled acylating agent which is an efficient reagent for introducing radioactive iodine atoms into proteins while eliminating direct contact of the protein with the iodination oxidant. As a further advantage, this reagent allows the introduction of radioactive iodine into proteins and peptides not containing tyrosine, because of its high reactivity toward amines and other nucleophilic groups. We have independently synthesized a comparable reagent, a phenolic imidoester iodinated with 1251, which we report here as having the same advantages as the Bolton and Hunter compound, but which differs from their acylation reagent in the following ways: it is less reactive, and moderately stable in aqueous solutions, even at a pH of 9.5 and at 37. It is likely to be more specific in its reaction with proteins in This investigation was supported by National Institutes of Health Grant GM-19363. Predoctoral Hatton Award: 1st place. Sponsored by John C. Gerhart and Michael M. Wu.
forming protonated amidine linkages with £-amine groups of lysyl residues or a-amine groups of N-terminal residues, as judged from specificity studies with other imidoesters.5-8 In addition, it alters the charge on the protein by at most minus one unit per substitution, due to the di-iodo phenolate ion, rather than by minus two, as does the Bolton and Hunter reagent which introduces an equivalent phenolate and also removes the positive charge of the lysyl amine. We have used this imidoester to prepare proteins of high specific radioactivity. A similar iodinated imidoester has been reported,9 but the multistep synthesis of this compound precludes its ready use in radioactive work.
Materials and Methods Para-hydroxy benzonitrilea and reaction grade hydrogen chloride were obtained commercially and used without further purification. Absolute methanol and diethyl etherb were stored over molecular sievee to eliminate water. Other chemicals obtained commercially were as follows: carrier free 125I as Nal in 0.05 N NaOHd; chloramine-T, dimethyl sulfoxide (DMSO) (spectrograde), and /3-mercaptoethanole bovine plasma albumin (crystallized) f; dog serum albuming; and sodium borateh and sodium pyrophosphate.b Deuterium-labeled DMSO was used as a solvent in nuclear magnetic resonance studies. The radioactivity of samples containing a Eastman Organic Chemical Co., Rochester, NY. Mallinckrodt Chemical Works, Menlo Park, Calif. cUnion Carbide, San Francisco, Calif. d Schwarz-Mann Chemical Co., Van Nuys, Calif. eMatheson, Coleman and Bell, Norwood, Ohio. f Armore Pharmaceutical Co., Chicago, Ill. Sigma Chemical Co., St. Louis, Mo. h Allied Chemical Co., Morristown, NJ. BIORAD Laboratories, Richmond, Calif. b
-
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Vol1 54 1975
RADIOACTIVE IODINATION OF PROTEINS
125I was determined with a gamma counter.j Ultraviolet spectra were obtained with a spectrophotometer," and NMR spectra were obtained with a NMR spectrometer.' Synthesis of methyl para-hydroxy benzimidate HCl (MPHBIM) was accomplished by a modification of the method of Hunter and Ludwig.l1 In a 50 ml triple-necked round bottom flask (21 C) was placed 1.2 gm of para-hydroxy benzonitrile (0.01 M) to which was added 16 ml absolute methanol (0.4 Al), 10 ml of diethyl ether and five pellets of molecular sieve to reduce contamination by water. The flask was closed, fitted with a drying tube, and cooled to -20 C in an ice-salt bath, followed by saturation with dried (bubbled through H2S04) HCI gas. The solution was then J Mark II, Nuclear Chicago, Berkeley, Calif.
Cary 14, Varian Associates, Palo Alto, Calif. 'Varian T-60, Varian Associates, Palo Alto, Calif.
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allowed to warm up to 4 C and yielded orange needlelike crystals after 30 minutes at 4 C. The crystals were quickly filtered at 4 C, washed with cold methanol-ether (1:2) followed by a final ether wash, and subsequently stored at 0 C under vacuum desiccation (yield 90%). The product has been kept under these conditions for six months without measurable deterioration. The orange crystals eliminated gas on melting at 171 to 172 C, NMR peaks (D6DMSO) were found at 8 4.25 (s), weight 3, assigned to the O-CH3 protons, 8 6.98 (d), J = 9 cps, weight 2; 8 8.07 (d), J = 9 cps, weight 2, assigned to the aromatic protons. (Found: C, 49.5: H, 5.9: N, 7.7: Cl, 18.9, calculated for C8H10N02Cl (mol wt = 188 gm/mole): C, 51.3: H, 5.3: N, 7.5: Cl, 18.7%.) IODINATION OF MPHBIM.-The iodination of MPHBIM was performed on a micro-
(DNH2CIe (I)
HO
CN
+
CH3C)H
HCI HO+
\OCH (MPHBIM)
®NH,CP6
®DNH2CI® (2)
HO-
Q
oH3
(3)
HZ
HHO + 2NOI:125 Oxidizing
-C
\OCH3
Agent
+
(IIE)
+ CH30H
sI,125 OH FIG 1.-Synthesis and use of iodinated amidination reagent for labeling of proteins to high specific activity. Reaction 1, para-hydroxy benzonitrile is converted to methyl p-hydroxy-benzimidate hydrochloride (MPHBIM). Reaction 2, MPHBIM is iodinated to form methyl 3,5 diiodo-p-hydroxy benzimidate (IIE), the iodinated imidoester. Reaction 3, IIE adds to amino groups of protein via amidine linkage, thereby introducing radioactive iodine atoms into protein.
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scale by dissolving 3.7 mg of MPHBIM in 1 ml of 50 mM sodium borate buffer with a pH of 8.5 to obtain a 20 mM stock solution. Brief exposure to 37 C accelerated the dissolving rate. To 1.0 ml of the 20 mM solution of MPHBIM was added 1.0 ml of 40 mM NaI followed by 10 p,l of NaI125 solution (0 to 2 mCi depending on the desired specific activity). To this solution was added 1.0 ml of 40 mM chloramine-T with rapid mixing. A yellow color appeared and cleared within a few seconds. After 15 minutes at room temperature (20 to 22 C), 0.10 ml of 1.0 M ,B-mercaptoethanol was added to reduce the chloramine-T and residual iodine. The pH of the solution was subsequently lowered toward neutrality by adding 20 pul of 1.0 M acetic acid whereupon a flocculent white precipitate formed. Under these conditions unreacted MPHBIM, iodide, and chloramine-T remained soluble. The precipitate of the iodinated imidoester was collected by centrifugation at 10,000 rpm for 5 minutes, dissolved in 50 mM sodium borate buffer with a pH of 8.5 at 37 C for a few minutes, and was stored at 0 C, or frozen at -20 C. Frozen samples have been kept for seven days
6'
without detectable decomposition, as judged from spectral characteristics. The yield of methyl 3,5 di-iodo-p-hydroxy benzimidate (IIE) was estimated to be 50 to 75% on the basis of the fraction of input radioactivity recovered in the redissolved material. The product is considered to comprise primarily IIE on the basis of 1251 content, spectral characteristics at various pH values, and reactivity toward proteins, as discussed in the Results section of this article. AMIDINATION OF THE PROTEIN WITH IIE.The radioactive amidination of the protein was accomplished by mixing the IIE solution with the protein solution, under conditions described in the Results section of this article. Figure 1 summarizes the reaction sequence for the imidoester synthesis and iodination procedure.
Results ULTRAVIOLET SPECTRA.-The ultraviolet spectra of 1\IPHBIM and of IIE were obtained within the pH range of 2 to 12 as slhown in Figures 2 and 3. The ultraviolet spectrum of MPHBIM at a pH of 8.5 showed no detectable
.
R
(XiG5) tpH 8.6\
s4
220
240
~~~~\pH
/
260
\MPHBIM
755
280
300 A (nm)
320
340
360
FIG 2.-Ultraviolet spectra of methyl para-hydroxy benzimidate-HCI. A 10 mM stock solution (5.5 mg in 2.95 ml sodium borate; pH, 8.5) was prepared with brief warming at 37 C. Spectra were obtained at 21 C on dilution, 1 to 300, in the following solutions: pH 12, 0.01 M NaOH; pH 8.6, 40 mM sodium borate; pH 7.55, 40 mM sodium phosphate; pH 2.0, 0.01 N HCI. C, molar absorptivity, is given with units 10- M-1 cm71.
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Vo 1 54 1975
RADIOACTIVE IODINATION OF PROTEINS
A024 i0
220
~pH12
o2L;
\
C89
- 6- - - .N
L;~ ~ , H
250
300 350 X (nm) FIG 3.-Ultraviolet spectra of iodinated imidoester. Flocculent white material obtained by twice-repeated acid precipitation (see Materials and Methods section) was dissolved in 2 ml of 50 mM borate buffer (pH, 8.5) with brief warming to 37 C. Spectra were obtained at 21 C on dilution, 1 to 300, in the following solutions: pH 12, 0.01 M NaOH; pH 9.0, 40 mM sodium borate; pH 6.8, 40 mM potassium phosphate; pH 2.0, 0.02 N HCI. Absorptivity (A) is given, rather than E, since exact concentration and purity of IIE were not
determined.
change after five hours although considerable alteration had occurred after seven days at 21 C. The pH dependence of the MPHBIM spectrum suggests at least three ionizable forms of the compound are present over the pH range of 2 to 12. This is to be expected since the compound has two ionizable groups, the phenolic group and the imido group. At a pH of 7.7 the MPHBIM absorption peak at 326 nm reached a maximum molar absorptivity of 5,000 M-lcm-1. This absorption decreased to one-half value as the pH was raised to 9.0 or lowered to 6.4. In this neutral pH range, the major species exhibiting long wavelength absorption at 326 nm is presumably either the doubly-charged (zwitterion) intermediate or the uncharged intermediate, depending on the pK's of the phenolic and the imido groups. The IIE spectra resemble those of MPHBIM and also indicate the presence of at least three species in the pH range of
2 to 12. In the neutral pH range, an absorption maximum for IIE occurs at 337 nm and reaches its greatest molar absorptivity in the pH range of 5.5 to 6.5. The absorption maxima at 337 nm decreases to one-half of the maximal value as the pH is raised to 7.7 or lowered to 4. There is an acidic shift of the pH dependence of the absorption maxima for IIE by approximately two pH units as compared to MPHBIM. IIE possesses the two possibilities for ionization intermediates as does MPHBIM. The presence of an isoelectric intermediate (either uncharged or zwitterionic) in this pH range might be expected to decrease the solubility of IIE in polar solvents at neutral pH values and explain why IIE can be precipitated under such conditions and yet be dissolved in 5% trichloroacetic acid or at a high pH. EFFECT OF PH, TEMPERATURE, AND CONCENTRATION ON THE PROTEIN AMIDINATION.-The effects of the pH and the temperature on
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Dent Res Special Issue I~~~~~~~~~~~~~
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rate of reaction of bovine
plasma albumin illustrated in Figures 4 and 5. Figure 4 is a progress curve of the derivatization of bovine plasma albumin with IIE at a pH of 7.5 and 9.5 and at a temperature of 37 C. When the molar ratio of IIE to BPA was 14: 1, and BPA was present at a concentration of 20 mg/ml, the maximum rate of incorporation of radioactivity was I to 2% per hour, with a maximum incorporation of 30% of the 1251 input. These data illustrate the rather slow reaction rate of IIE. In tests of specificity of the reaction, it was found that 50 mM 8-mercaptoethanol had no detectable effect on the reaction rate, indicating the usefulness of this reaction' for protein which must be kept in the presence of sulfhydryl agents. Figure 5 indicates that the reaction yelocity increased by a factor of 3 as the
C
r') ful
with IIE are
pH 9.5
pH 8.4
pH 7.5
1OH~ vi
0.1
.oo.c
21,C
37'C
U.ul
II
%JAj
3.
3.2
3.3
3.4
3.5
3.6
3.7
T-1 XIO (0(O)
FIG 5 .-pH and temperature dependence of amidination of BPA with IIE. Reaction was performed as described in legend of Figure 4, with the following conditions: circles, pH 7.5, 100 mM sodium pyrophosphate; triangles, pH 8.4, 100 mM sodium borate; squares, pH 9.5, 100 mM sodium borate. Progress curves throughout 24 hours were obtained for each condition of temperature anid pH, arnd data were extrapolated back to zero time to evaluate initial velocity, V4, shown on ordinate. Units of V. are percent input counts incorporated per hour into protein (trichloroacetic acid-insoluble material) .
0
0. H-
z
"a
temperature increased by 10 C. Also, as the pH increased 1.4 units, the reaction velocity doubled. At this time it is not possible to evaluate the contribution to the pH dependence made by ionization of LIE, the protein amino groups, arid the LIE-amino
20 T
(hours)
4.-Progress curves for amidination of boplasma albumin with IIE. Reaction mixture (vol. 1.0 ml) contained 20 mg of albumin, 4 FIG
vine
mM
LIE
containing 5.6
x
107
cpm
I,
and
50
buffer
(sodium borate for pH of 9.5 and sodium pyrophosphate for pH of 7.5). Reaction was initiated by addition of IIE and allowed to proceed at 37 C. Samples of 0.1 ml vol were remM
moved
at
intervals
trichloroacetic acid
and mixed at 0
C.
with
1.0
ml
After 30 minutes
5%/,
(or precipitates were collected by filtration on Whatman GF/C filters. Radioactivity was determined by -y-solid crystal spectrometry. Recovery of 5.6 X 106 cpm per sample was considered to be 100% yield. The control was reacted in 50 mM sodium borate (pH, 9.5; at 37 C)
longer)
,
without BPA.
transition state.11 The dependence of reaction rate on the concentration of the two reactants was explored briefly under conditions in which each reactant was singly held constant and in large excess while the other was varied (pseudo-first order conditions) . As shown in Figure 6, the reaction velocity was linearly dependent on the variable reactant under these conditions, whichi suggests that the reaction is second order with respect to LIE and protein, together. There was no indication of the steep dependence on protein concentration mentioned by Bolton and Hunter3 for their acylating agent.
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RADIOACTIVE IODINATION OF PROTEINS
Vol 54 1975
[BPA][IrE], (mM)2
FIG 6.-Dependence of amidination rate on reactant concentrations. Reaction volume of 0.15 ml contained 50 mM sodium borate (pH, 8.5) and concentrations of BPA and IIE as follows: condition 1 (circles), BPA varied from 0 to 0.098 mM (6.6 mg/ml) while IIE remained constant at 1.07 mM and 107 cpm; or condition 2 (squares), IIE varied from 0 to 2.14 mM and 2 x 107 cpm, while BPA remained constant at 0.098 mM. Concentrations of IIE were estimated from fraction of input 125I radioactivity recovered (55%) after acid precipitation following iodination of MPHBIM and represent minimum estimates. Amidination reaction was initiated by addition of IIE, was allowed to proceed for one hour at 37 C, and was stopped by addition of 1 ml 5% trichloroacetic acid and chilled to 0 C. After 16 hours, precipitates were collected and counted as described in legend of Figure 4.
ANALYSIS OF THE AMIDINATED PROTEIN.Dog serum albumin was reacted with IIE (> 100 p.Ci/tLmole) for one day at 21 C and then dialyzed extensively against 0.15 M NaCl containing 5 mM sodium phosphate at a pH of 7.4. The specific activity of this preparation was approximately 10 ,uCi/mg protein. This material was analyzed by sodium dodecyl sulfate gel electrophoresis.12 It formed a sharp major band of radioactivity at the serum albumin position (67,000 daltons) and a minor band at the dimerized albumin position. Together the radioactivity at these positions comprised 80%9 of the input radioactivity. This result suggests that the radioactive imidoester had become attached to the protein by covalent linkage and not by non-covalent absorption. STABILITY OF IIE.-Since the reaction rate under pseudo-first order conditions pro-
C91
vided a means to measure IIE concentration on a relative scale, a test was made of the stability of IIE at a pH of 8.5 and at 21 C. Samples from a stock solution of IIE (approximately 1.1 mM) were removed at intervals and reacted with a standard amount of protein (10 mg/ml). The results indicated that after 24 hours of incubation the stock solution still contained greater than 80%o of the initial concentration of IIE. In addition, spectral studies with nonradioactive IIE have shown that solutions of IIE at a pH of 8.5 (50 mM sodium borate) undergo no detectable spectral change after a week of storage at -20 C. Thus, it should be possible to prepare and store the radioactive reagent for prolonged periods. Discussion The present article describes a method of introducing radioactive iodine into proteins by way of the iodinated imidoester, IIE. The parent imidoester, MPHBIM, is synthesized from para-hydroxy benzonitrile and methanol. The MlPHBIM is then reacted with NaI125 in the presence of an oxidant, chloramine-T, to form IIE which can be isolated by acid precipitation and stored frozen at -20 C for at least a week
without hydrolytic decomposition. This radioactive di-iodo compound can then be reacted with the protein in a pH range of 7.5 to 9.5 and between 21 and 37 C with a yield of 20 to 30% of the IIE incorporated into the protein in 24 hours. Preparations exceeding 104 cpm/pg protein have been obtained by this procedure. The main advantage from the use of IIE, as with the Bolton and Hunter iodinated acylating reagent,3 is that the protein need never be exposed to oxidizing agents such as chloramine-T used in active iodination, nor to the 1251 Nal commercial preparations which are thought to contain deleterious impurities.13 Furthermore, these reagents may be used to introduce radioactive iodine into protein lacking tryosine. On the basis of spectral changes, IIE was found to be a moderately acidic compound, undergoing ionization of its phenolic hydroxyl and imido group below a pH of 8. lodination is known to lower the pH of phenolic ionization by more than three units, from 10.1 in the case of tyrosine to 6.5 in the case of di-iodo tyrosine,14 and the same effect is apparent in the compar-
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ison of MPHBIM and IIE, as reported in the Results section of this article. As a consequence, the amidine derivative formed by the reaction of the IIE with protein probably bears a negative charge on its phenol group at slightly alkaline pH values. This charge alteration would also occur with the Bolton and Hunter reagent, as well as with di-iodo tyrosyl residues produced in proteins by direct iodination techniques.1,2 It is characteristic of imidoesters that the amidine group formed by their reaction with amino groups does preserve the positive charge of the amine, borne by the amidine nitrogens with pK values in the range 11.5 to 12.5.15 Therefore, the positive charge of E-amine group of lysyl residues or of a-amine groups of N-terminal residues will be preserved during the reaction with IIE, whereas these charges would be lost on reaction with the Bolton and Hunter acylating reagent. The author thanks Dr. S. Kent for his help in interpreting the NMR spectra, Ms. G. Pelatowsky for preparing the tables and graphs for publication, and Dr. M. Bothwell for suggesting acid-precipitation as a method of purification of IIE.
References 1. GREENWOOD, F.C.; IIUNTER, W.M.; and GLOVER,J.S.: The Preparation of "3'I-Labeled Human Growth Hormone of High Specific Radioactivity, Biochem J 89: 114-123, 1963. 2. MARCHALONIS, J.J.: An Enzymatic Method for the Trace lodination of Immunoglobulins and Other Proteins, Biochem J 113: 299305, 1969. 3. BOLTON, A.E., and HUNTER, W.M.: The Labeling of Proteins to High Specific Radioac-
tivities by Conjugation to a '15I-Containing Acylating Agent, Biochem J 133: 529-539, 1973. 4. RUDINGER, J., and RUEGG, U.: Preparation of
5.
6.
7. 8.
9.
10.
11.
12.
!3.
14.
N-Succinimidyl 3- (4-hydroxyphenyl) Propionate, Biochem J 133: 538-539, 1973. HAYNES, R., and FEENEY, R.E.: Transformation of Active Site Lysine in Naturally Occurring Trypsin Inhibitors. A Basis for a General Mechanism for Inhibition of Proteolytic Enzymes, Biochemistry 7: 2879-2885, 1968. PERHAM, R.M., and RICHARDs, F.M.: Reactivity and Structural Role of Protein Amino Groups in Tobacco Mosaic Virus, J Mol Biol 33: 795-807, 1968. REYNOLDS, J.H.: Acetimidation of Bovine Pancreatic Ribonuclease A, Biochemistry 7: 3131-3135, 1968. WOFSY, L., and SINGER, S.J.: Effects of the Amidination Reaction on Antibody Activity and on the Physical Properties of Some Proteins, Biochemistry 2: 109-116, 1963. RILEY, M., and PERHAM, R.N.: The Reaction of Protein Amino Groups with Methyl 5Iodopyridine-2-carboximidate: A Possible General Method of Preparing Isomorphous Heavy-Atom Derivatives of Proteins, Biochem J 131: 625-635, 1973. HUNTER, M.J., and LUDWIG, M.L.: The Reaction of Imidoesters with Proteins and Related Small Molecules, J Am Chem Soc 84: 3491-3504, 1962. HAND, E.S., and JENCKS, W.P.: Mechanism of the Reaction of Imido Esters with Amines, J Am Chem Soc 84: 3805-3514, 1962. WEBER, K., and OSBORN, M.: The Reliability of Molecular Weight Determination by Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, J Biol Chem 244: 4406-4412, 1969. KIRKHAM, K.E., and HUNTER, N.M. (eds): Radioimmunoassay Methods: European Workshop, Edinburgh: Churchill Livingstone, 1971, pp 3-23. ROCHE, J., and MICHEL, R.: Natural and Artificial lodoproteins, Adv Protein Chem 6: 253-297, 1951.
15. ALBERT, A.; GOLDACRE, R.; and PHILLIPS, J.: Study of the Organic Reactions of Imidoesters, J Chem Soc 2240-2249, 1948.
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An iodinated phenolic imidoester has been synthesized for the labeling of proteins to high specific activities with radioactive iodine. The main advantage of this two-step labeling method is that it obviates direct contact of the protein with deleterious oxidizing agents, such as chloramine-T, pressent in direct methods of iodinating proteins. Several techniques for iodinating proteins have been developed based on the direct substitution of 1251 onto tyrosyl residues.1,2 These methods involve the use of oxidizing agents which in some instances lead to inactivation of the proteins, presumably by side reactions such as sulfhydryl oxidation. Recently, Bolton and Hunter3 and Rudinger and Ruegg4 reported the synthesis and use of an 1251-labeled acylating agent which is an efficient reagent for introducing radioactive iodine atoms into proteins while eliminating direct contact of the protein with the iodination oxidant. As a further advantage, this reagent allows the introduction of radioactive iodine into proteins and peptides not containing tyrosine, because of its high reactivity toward amines and other nucleophilic groups. We have independently synthesized a comparable reagent, a phenolic imidoester iodinated with 1251, which we report here as having the same advantages as the Bolton and Hunter compound, but which differs from their acylation reagent in the following ways: it is less reactive, and moderately stable in aqueous solutions, even at a pH of 9.5 and at 37. It is likely to be more specific in its reaction with proteins in This investigation was supported by National Institutes of Health Grant GM-19363. Predoctoral Hatton Award: 1st place. Sponsored by John C. Gerhart and Michael M. Wu.
forming protonated amidine linkages with £-amine groups of lysyl residues or a-amine groups of N-terminal residues, as judged from specificity studies with other imidoesters.5-8 In addition, it alters the charge on the protein by at most minus one unit per substitution, due to the di-iodo phenolate ion, rather than by minus two, as does the Bolton and Hunter reagent which introduces an equivalent phenolate and also removes the positive charge of the lysyl amine. We have used this imidoester to prepare proteins of high specific radioactivity. A similar iodinated imidoester has been reported,9 but the multistep synthesis of this compound precludes its ready use in radioactive work.
Materials and Methods Para-hydroxy benzonitrilea and reaction grade hydrogen chloride were obtained commercially and used without further purification. Absolute methanol and diethyl etherb were stored over molecular sievee to eliminate water. Other chemicals obtained commercially were as follows: carrier free 125I as Nal in 0.05 N NaOHd; chloramine-T, dimethyl sulfoxide (DMSO) (spectrograde), and /3-mercaptoethanole bovine plasma albumin (crystallized) f; dog serum albuming; and sodium borateh and sodium pyrophosphate.b Deuterium-labeled DMSO was used as a solvent in nuclear magnetic resonance studies. The radioactivity of samples containing a Eastman Organic Chemical Co., Rochester, NY. Mallinckrodt Chemical Works, Menlo Park, Calif. cUnion Carbide, San Francisco, Calif. d Schwarz-Mann Chemical Co., Van Nuys, Calif. eMatheson, Coleman and Bell, Norwood, Ohio. f Armore Pharmaceutical Co., Chicago, Ill. Sigma Chemical Co., St. Louis, Mo. h Allied Chemical Co., Morristown, NJ. BIORAD Laboratories, Richmond, Calif. b
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Vol1 54 1975
RADIOACTIVE IODINATION OF PROTEINS
125I was determined with a gamma counter.j Ultraviolet spectra were obtained with a spectrophotometer," and NMR spectra were obtained with a NMR spectrometer.' Synthesis of methyl para-hydroxy benzimidate HCl (MPHBIM) was accomplished by a modification of the method of Hunter and Ludwig.l1 In a 50 ml triple-necked round bottom flask (21 C) was placed 1.2 gm of para-hydroxy benzonitrile (0.01 M) to which was added 16 ml absolute methanol (0.4 Al), 10 ml of diethyl ether and five pellets of molecular sieve to reduce contamination by water. The flask was closed, fitted with a drying tube, and cooled to -20 C in an ice-salt bath, followed by saturation with dried (bubbled through H2S04) HCI gas. The solution was then J Mark II, Nuclear Chicago, Berkeley, Calif.
Cary 14, Varian Associates, Palo Alto, Calif. 'Varian T-60, Varian Associates, Palo Alto, Calif.
C87
allowed to warm up to 4 C and yielded orange needlelike crystals after 30 minutes at 4 C. The crystals were quickly filtered at 4 C, washed with cold methanol-ether (1:2) followed by a final ether wash, and subsequently stored at 0 C under vacuum desiccation (yield 90%). The product has been kept under these conditions for six months without measurable deterioration. The orange crystals eliminated gas on melting at 171 to 172 C, NMR peaks (D6DMSO) were found at 8 4.25 (s), weight 3, assigned to the O-CH3 protons, 8 6.98 (d), J = 9 cps, weight 2; 8 8.07 (d), J = 9 cps, weight 2, assigned to the aromatic protons. (Found: C, 49.5: H, 5.9: N, 7.7: Cl, 18.9, calculated for C8H10N02Cl (mol wt = 188 gm/mole): C, 51.3: H, 5.3: N, 7.5: Cl, 18.7%.) IODINATION OF MPHBIM.-The iodination of MPHBIM was performed on a micro-
(DNH2CIe (I)
HO
CN
+
CH3C)H
HCI HO+
\OCH (MPHBIM)
®NH,CP6
®DNH2CI® (2)
HO-
Q
oH3
(3)
HZ
HHO + 2NOI:125 Oxidizing
-C
\OCH3
Agent
+
(IIE)
+ CH30H
sI,125 OH FIG 1.-Synthesis and use of iodinated amidination reagent for labeling of proteins to high specific activity. Reaction 1, para-hydroxy benzonitrile is converted to methyl p-hydroxy-benzimidate hydrochloride (MPHBIM). Reaction 2, MPHBIM is iodinated to form methyl 3,5 diiodo-p-hydroxy benzimidate (IIE), the iodinated imidoester. Reaction 3, IIE adds to amino groups of protein via amidine linkage, thereby introducing radioactive iodine atoms into protein.
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scale by dissolving 3.7 mg of MPHBIM in 1 ml of 50 mM sodium borate buffer with a pH of 8.5 to obtain a 20 mM stock solution. Brief exposure to 37 C accelerated the dissolving rate. To 1.0 ml of the 20 mM solution of MPHBIM was added 1.0 ml of 40 mM NaI followed by 10 p,l of NaI125 solution (0 to 2 mCi depending on the desired specific activity). To this solution was added 1.0 ml of 40 mM chloramine-T with rapid mixing. A yellow color appeared and cleared within a few seconds. After 15 minutes at room temperature (20 to 22 C), 0.10 ml of 1.0 M ,B-mercaptoethanol was added to reduce the chloramine-T and residual iodine. The pH of the solution was subsequently lowered toward neutrality by adding 20 pul of 1.0 M acetic acid whereupon a flocculent white precipitate formed. Under these conditions unreacted MPHBIM, iodide, and chloramine-T remained soluble. The precipitate of the iodinated imidoester was collected by centrifugation at 10,000 rpm for 5 minutes, dissolved in 50 mM sodium borate buffer with a pH of 8.5 at 37 C for a few minutes, and was stored at 0 C, or frozen at -20 C. Frozen samples have been kept for seven days
6'
without detectable decomposition, as judged from spectral characteristics. The yield of methyl 3,5 di-iodo-p-hydroxy benzimidate (IIE) was estimated to be 50 to 75% on the basis of the fraction of input radioactivity recovered in the redissolved material. The product is considered to comprise primarily IIE on the basis of 1251 content, spectral characteristics at various pH values, and reactivity toward proteins, as discussed in the Results section of this article. AMIDINATION OF THE PROTEIN WITH IIE.The radioactive amidination of the protein was accomplished by mixing the IIE solution with the protein solution, under conditions described in the Results section of this article. Figure 1 summarizes the reaction sequence for the imidoester synthesis and iodination procedure.
Results ULTRAVIOLET SPECTRA.-The ultraviolet spectra of 1\IPHBIM and of IIE were obtained within the pH range of 2 to 12 as slhown in Figures 2 and 3. The ultraviolet spectrum of MPHBIM at a pH of 8.5 showed no detectable
.
R
(XiG5) tpH 8.6\
s4
220
240
~~~~\pH
/
260
\MPHBIM
755
280
300 A (nm)
320
340
360
FIG 2.-Ultraviolet spectra of methyl para-hydroxy benzimidate-HCI. A 10 mM stock solution (5.5 mg in 2.95 ml sodium borate; pH, 8.5) was prepared with brief warming at 37 C. Spectra were obtained at 21 C on dilution, 1 to 300, in the following solutions: pH 12, 0.01 M NaOH; pH 8.6, 40 mM sodium borate; pH 7.55, 40 mM sodium phosphate; pH 2.0, 0.01 N HCI. C, molar absorptivity, is given with units 10- M-1 cm71.
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Vo 1 54 1975
RADIOACTIVE IODINATION OF PROTEINS
A024 i0
220
~pH12
o2L;
\
C89
- 6- - - .N
L;~ ~ , H
250
300 350 X (nm) FIG 3.-Ultraviolet spectra of iodinated imidoester. Flocculent white material obtained by twice-repeated acid precipitation (see Materials and Methods section) was dissolved in 2 ml of 50 mM borate buffer (pH, 8.5) with brief warming to 37 C. Spectra were obtained at 21 C on dilution, 1 to 300, in the following solutions: pH 12, 0.01 M NaOH; pH 9.0, 40 mM sodium borate; pH 6.8, 40 mM potassium phosphate; pH 2.0, 0.02 N HCI. Absorptivity (A) is given, rather than E, since exact concentration and purity of IIE were not
determined.
change after five hours although considerable alteration had occurred after seven days at 21 C. The pH dependence of the MPHBIM spectrum suggests at least three ionizable forms of the compound are present over the pH range of 2 to 12. This is to be expected since the compound has two ionizable groups, the phenolic group and the imido group. At a pH of 7.7 the MPHBIM absorption peak at 326 nm reached a maximum molar absorptivity of 5,000 M-lcm-1. This absorption decreased to one-half value as the pH was raised to 9.0 or lowered to 6.4. In this neutral pH range, the major species exhibiting long wavelength absorption at 326 nm is presumably either the doubly-charged (zwitterion) intermediate or the uncharged intermediate, depending on the pK's of the phenolic and the imido groups. The IIE spectra resemble those of MPHBIM and also indicate the presence of at least three species in the pH range of
2 to 12. In the neutral pH range, an absorption maximum for IIE occurs at 337 nm and reaches its greatest molar absorptivity in the pH range of 5.5 to 6.5. The absorption maxima at 337 nm decreases to one-half of the maximal value as the pH is raised to 7.7 or lowered to 4. There is an acidic shift of the pH dependence of the absorption maxima for IIE by approximately two pH units as compared to MPHBIM. IIE possesses the two possibilities for ionization intermediates as does MPHBIM. The presence of an isoelectric intermediate (either uncharged or zwitterionic) in this pH range might be expected to decrease the solubility of IIE in polar solvents at neutral pH values and explain why IIE can be precipitated under such conditions and yet be dissolved in 5% trichloroacetic acid or at a high pH. EFFECT OF PH, TEMPERATURE, AND CONCENTRATION ON THE PROTEIN AMIDINATION.-The effects of the pH and the temperature on
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Dent Res Special Issue I~~~~~~~~~~~~~
C90 WOOD WOOD
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rate of reaction of bovine
plasma albumin illustrated in Figures 4 and 5. Figure 4 is a progress curve of the derivatization of bovine plasma albumin with IIE at a pH of 7.5 and 9.5 and at a temperature of 37 C. When the molar ratio of IIE to BPA was 14: 1, and BPA was present at a concentration of 20 mg/ml, the maximum rate of incorporation of radioactivity was I to 2% per hour, with a maximum incorporation of 30% of the 1251 input. These data illustrate the rather slow reaction rate of IIE. In tests of specificity of the reaction, it was found that 50 mM 8-mercaptoethanol had no detectable effect on the reaction rate, indicating the usefulness of this reaction' for protein which must be kept in the presence of sulfhydryl agents. Figure 5 indicates that the reaction yelocity increased by a factor of 3 as the
C
r') ful
with IIE are
pH 9.5
pH 8.4
pH 7.5
1OH~ vi
0.1
.oo.c
21,C
37'C
U.ul
II
%JAj
3.
3.2
3.3
3.4
3.5
3.6
3.7
T-1 XIO (0(O)
FIG 5 .-pH and temperature dependence of amidination of BPA with IIE. Reaction was performed as described in legend of Figure 4, with the following conditions: circles, pH 7.5, 100 mM sodium pyrophosphate; triangles, pH 8.4, 100 mM sodium borate; squares, pH 9.5, 100 mM sodium borate. Progress curves throughout 24 hours were obtained for each condition of temperature anid pH, arnd data were extrapolated back to zero time to evaluate initial velocity, V4, shown on ordinate. Units of V. are percent input counts incorporated per hour into protein (trichloroacetic acid-insoluble material) .
0
0. H-
z
"a
temperature increased by 10 C. Also, as the pH increased 1.4 units, the reaction velocity doubled. At this time it is not possible to evaluate the contribution to the pH dependence made by ionization of LIE, the protein amino groups, arid the LIE-amino
20 T
(hours)
4.-Progress curves for amidination of boplasma albumin with IIE. Reaction mixture (vol. 1.0 ml) contained 20 mg of albumin, 4 FIG
vine
mM
LIE
containing 5.6
x
107
cpm
I,
and
50
buffer
(sodium borate for pH of 9.5 and sodium pyrophosphate for pH of 7.5). Reaction was initiated by addition of IIE and allowed to proceed at 37 C. Samples of 0.1 ml vol were remM
moved
at
intervals
trichloroacetic acid
and mixed at 0
C.
with
1.0
ml
After 30 minutes
5%/,
(or precipitates were collected by filtration on Whatman GF/C filters. Radioactivity was determined by -y-solid crystal spectrometry. Recovery of 5.6 X 106 cpm per sample was considered to be 100% yield. The control was reacted in 50 mM sodium borate (pH, 9.5; at 37 C)
longer)
,
without BPA.
transition state.11 The dependence of reaction rate on the concentration of the two reactants was explored briefly under conditions in which each reactant was singly held constant and in large excess while the other was varied (pseudo-first order conditions) . As shown in Figure 6, the reaction velocity was linearly dependent on the variable reactant under these conditions, whichi suggests that the reaction is second order with respect to LIE and protein, together. There was no indication of the steep dependence on protein concentration mentioned by Bolton and Hunter3 for their acylating agent.
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RADIOACTIVE IODINATION OF PROTEINS
Vol 54 1975
[BPA][IrE], (mM)2
FIG 6.-Dependence of amidination rate on reactant concentrations. Reaction volume of 0.15 ml contained 50 mM sodium borate (pH, 8.5) and concentrations of BPA and IIE as follows: condition 1 (circles), BPA varied from 0 to 0.098 mM (6.6 mg/ml) while IIE remained constant at 1.07 mM and 107 cpm; or condition 2 (squares), IIE varied from 0 to 2.14 mM and 2 x 107 cpm, while BPA remained constant at 0.098 mM. Concentrations of IIE were estimated from fraction of input 125I radioactivity recovered (55%) after acid precipitation following iodination of MPHBIM and represent minimum estimates. Amidination reaction was initiated by addition of IIE, was allowed to proceed for one hour at 37 C, and was stopped by addition of 1 ml 5% trichloroacetic acid and chilled to 0 C. After 16 hours, precipitates were collected and counted as described in legend of Figure 4.
ANALYSIS OF THE AMIDINATED PROTEIN.Dog serum albumin was reacted with IIE (> 100 p.Ci/tLmole) for one day at 21 C and then dialyzed extensively against 0.15 M NaCl containing 5 mM sodium phosphate at a pH of 7.4. The specific activity of this preparation was approximately 10 ,uCi/mg protein. This material was analyzed by sodium dodecyl sulfate gel electrophoresis.12 It formed a sharp major band of radioactivity at the serum albumin position (67,000 daltons) and a minor band at the dimerized albumin position. Together the radioactivity at these positions comprised 80%9 of the input radioactivity. This result suggests that the radioactive imidoester had become attached to the protein by covalent linkage and not by non-covalent absorption. STABILITY OF IIE.-Since the reaction rate under pseudo-first order conditions pro-
C91
vided a means to measure IIE concentration on a relative scale, a test was made of the stability of IIE at a pH of 8.5 and at 21 C. Samples from a stock solution of IIE (approximately 1.1 mM) were removed at intervals and reacted with a standard amount of protein (10 mg/ml). The results indicated that after 24 hours of incubation the stock solution still contained greater than 80%o of the initial concentration of IIE. In addition, spectral studies with nonradioactive IIE have shown that solutions of IIE at a pH of 8.5 (50 mM sodium borate) undergo no detectable spectral change after a week of storage at -20 C. Thus, it should be possible to prepare and store the radioactive reagent for prolonged periods. Discussion The present article describes a method of introducing radioactive iodine into proteins by way of the iodinated imidoester, IIE. The parent imidoester, MPHBIM, is synthesized from para-hydroxy benzonitrile and methanol. The MlPHBIM is then reacted with NaI125 in the presence of an oxidant, chloramine-T, to form IIE which can be isolated by acid precipitation and stored frozen at -20 C for at least a week
without hydrolytic decomposition. This radioactive di-iodo compound can then be reacted with the protein in a pH range of 7.5 to 9.5 and between 21 and 37 C with a yield of 20 to 30% of the IIE incorporated into the protein in 24 hours. Preparations exceeding 104 cpm/pg protein have been obtained by this procedure. The main advantage from the use of IIE, as with the Bolton and Hunter iodinated acylating reagent,3 is that the protein need never be exposed to oxidizing agents such as chloramine-T used in active iodination, nor to the 1251 Nal commercial preparations which are thought to contain deleterious impurities.13 Furthermore, these reagents may be used to introduce radioactive iodine into protein lacking tryosine. On the basis of spectral changes, IIE was found to be a moderately acidic compound, undergoing ionization of its phenolic hydroxyl and imido group below a pH of 8. lodination is known to lower the pH of phenolic ionization by more than three units, from 10.1 in the case of tyrosine to 6.5 in the case of di-iodo tyrosine,14 and the same effect is apparent in the compar-
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ison of MPHBIM and IIE, as reported in the Results section of this article. As a consequence, the amidine derivative formed by the reaction of the IIE with protein probably bears a negative charge on its phenol group at slightly alkaline pH values. This charge alteration would also occur with the Bolton and Hunter reagent, as well as with di-iodo tyrosyl residues produced in proteins by direct iodination techniques.1,2 It is characteristic of imidoesters that the amidine group formed by their reaction with amino groups does preserve the positive charge of the amine, borne by the amidine nitrogens with pK values in the range 11.5 to 12.5.15 Therefore, the positive charge of E-amine group of lysyl residues or of a-amine groups of N-terminal residues will be preserved during the reaction with IIE, whereas these charges would be lost on reaction with the Bolton and Hunter acylating reagent. The author thanks Dr. S. Kent for his help in interpreting the NMR spectra, Ms. G. Pelatowsky for preparing the tables and graphs for publication, and Dr. M. Bothwell for suggesting acid-precipitation as a method of purification of IIE.
References 1. GREENWOOD, F.C.; IIUNTER, W.M.; and GLOVER,J.S.: The Preparation of "3'I-Labeled Human Growth Hormone of High Specific Radioactivity, Biochem J 89: 114-123, 1963. 2. MARCHALONIS, J.J.: An Enzymatic Method for the Trace lodination of Immunoglobulins and Other Proteins, Biochem J 113: 299305, 1969. 3. BOLTON, A.E., and HUNTER, W.M.: The Labeling of Proteins to High Specific Radioac-
tivities by Conjugation to a '15I-Containing Acylating Agent, Biochem J 133: 529-539, 1973. 4. RUDINGER, J., and RUEGG, U.: Preparation of
5.
6.
7. 8.
9.
10.
11.
12.
!3.
14.
N-Succinimidyl 3- (4-hydroxyphenyl) Propionate, Biochem J 133: 538-539, 1973. HAYNES, R., and FEENEY, R.E.: Transformation of Active Site Lysine in Naturally Occurring Trypsin Inhibitors. A Basis for a General Mechanism for Inhibition of Proteolytic Enzymes, Biochemistry 7: 2879-2885, 1968. PERHAM, R.M., and RICHARDs, F.M.: Reactivity and Structural Role of Protein Amino Groups in Tobacco Mosaic Virus, J Mol Biol 33: 795-807, 1968. REYNOLDS, J.H.: Acetimidation of Bovine Pancreatic Ribonuclease A, Biochemistry 7: 3131-3135, 1968. WOFSY, L., and SINGER, S.J.: Effects of the Amidination Reaction on Antibody Activity and on the Physical Properties of Some Proteins, Biochemistry 2: 109-116, 1963. RILEY, M., and PERHAM, R.N.: The Reaction of Protein Amino Groups with Methyl 5Iodopyridine-2-carboximidate: A Possible General Method of Preparing Isomorphous Heavy-Atom Derivatives of Proteins, Biochem J 131: 625-635, 1973. HUNTER, M.J., and LUDWIG, M.L.: The Reaction of Imidoesters with Proteins and Related Small Molecules, J Am Chem Soc 84: 3491-3504, 1962. HAND, E.S., and JENCKS, W.P.: Mechanism of the Reaction of Imido Esters with Amines, J Am Chem Soc 84: 3805-3514, 1962. WEBER, K., and OSBORN, M.: The Reliability of Molecular Weight Determination by Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, J Biol Chem 244: 4406-4412, 1969. KIRKHAM, K.E., and HUNTER, N.M. (eds): Radioimmunoassay Methods: European Workshop, Edinburgh: Churchill Livingstone, 1971, pp 3-23. ROCHE, J., and MICHEL, R.: Natural and Artificial lodoproteins, Adv Protein Chem 6: 253-297, 1951.
15. ALBERT, A.; GOLDACRE, R.; and PHILLIPS, J.: Study of the Organic Reactions of Imidoesters, J Chem Soc 2240-2249, 1948.
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