Imidazoles as well as thiolates in proteins bind technetium-99m

Paul O. Zamora, and Buck A. Rhodes. Bioconjugate Chem. , 1992, 3 (6), pp 493–498. DOI: 10.1021/bc00018a005. Publication Date: November 1992...
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Bioconlugete Chem. 1992, 3, 493-498

Imidazoles as Well as Thiolates in Proteins Bind Technetium-99m Paul 0. Zamora' and Buck A. Rhodes RhoMed Inc., 4261 Balloon Park Road NE, Albuquerque, New Mexico 87109. Received April 13, 1992 9gmTcis widely thought to directly bind proteins through thiolate groups of cysteine residues, resulting in Tc-cysteinyl-protein bonds. Chemical reduction of disulfide bonds in proteins is widely used to binding. This strategy is used because most proteins generate thiolates with the goal of increasing 99mT~ contain no thiolates, but many do contain disulfide bonds. In this study, we have evaluated the hypothesis that imidazole groups of histidine are also involved in direct 99mTcbinding to proteins. Human y-globulin was used as the model protein in these studies. The immunoglobulin was used (a) without reduction or was (b) treated with stannous ions to reduce disulfide bonds thereby increasing thiolate concentration. These proteins were used to evaluate the hypothesis that imidazole as well as thiolate groups bind Tc. The proteins were evaluated by (a) using free amino acids to compete with proteins for 9gmTcand (b) by chemical modification of amino acid side chains. In addition, peptides known to contain either cysteine or histidine, but not both, were also successfully directly labeled with 9gmTc. These results indicate that in proteins (and peptides) imidazole-containing groups as well as thiolate-containing groups bind Tc.

INTRODUCTION

Technetium-99m is the most widely used radionuclide in nuclear medicine and considerable effort has been focused on directly binding it to proteins for diagnostic imaging of human diseases (Eckelman & Steigman, 1991; Rhodes, 1991). Clinical-grade Tc is normally produced from a 99Mo/99"Tc generator as sodium pertechnetate (NaTcOd); in that form it does not bind to proteins. Tc can bind to proteins when the pertechnetate [Tc(VII)I ion is chemically reduced to a lower oxidation state (Steigman & Eckelman, 1992). In most proteins, endogenous Tc-binding capacity is easily saturated, and consequently the Tc-binding efficiency is low. To increase the Tc-binding efficiency, a number of investigators have chemically treated one class of proteins, immunoglobulins, with reducing agents (stannous ions, dithiothreitol, 2-mercaptoethanol, and the like) with the goal of generating reactive thiolates for subsequent Tc binding (see Rhodes, 1991 for specific references). This strategy is based on the well-known ability of reduced Tc to bind to thiolate-containing molecules (cysteine, penicillamine, mercaptoacetyl triglycine) (Dewanjee, 1990). Stannous ions reduce the disulfide bonds in proteins and generate cysteine thiolates. Excess tin ions presumptively bind thiolates, forming Sn-cysteinyl-protein bonds, and thereby minimizing disulfide bond reformation. On addition of pertechnetate, stannous effects reduction of the pertechnetate, and the Tc then undergoes a replacement reaction with the bound tin, forming strongly bound Tc-cysteinyl-protein (Hawkins et al., 1990). As applied to immunoglobulins, this approach results in immunoreactive antibody preparations which are radiolabeled with high efficiency in a single step, using a methodology suited to diagnostic imaging. In spite of the attention focused on direct binding of Tc to thiolates in proteins, little is known about the environment of thiolate binding site(@and the extent to which other binding sites contribute to overall Tc binding. That

* Address correspondence to Dr. Paul 0. Zamora, RhoMed Inc., 4261 Balloon Park Road NE, Albuquerque, NM 87109. Telephone: (505)344-7200. Telefax: (505)344-9460.

proteins contain multiple types of Tc-binding sites has been inferred by a number of investigators (Steigman et al., 1975; Lanteigne & Hnatowich, 1984;Paik, et al., 1985; 1986). Since Tc is a transition metal (group VIIb), it might be expected to share some of the protein-binding characteristics of other transition metals such as iron, cobalt, copper, zinc, molybdenum, and ruthenium. We have pointed out the similarity of Cu and Tc binding in immunoglobulins (Zamora et al., 1992). In general, proteins form stable coordination complexes with transition metals (including Cu) by interactions not only with thiolates in cysteine but alsowith imidazolesof histidine (Arnold & Haymore, 1991). For example, ruthenium(III),which is adjacent to Tc on the periodic chart, forms stable coordination complexes with peptides containing histidine (Ghardiri & Fernholz, 1990). This report describes results which validate the hypothesis that Tc can directly bind to proteins via at least two different types of binding sites. Human IgG was used in these studies as a model protein and was selected, in part, due to the wide interest in direct labeling of antibodies for clinical diagnostic imaging. The results show that one type of binding site involves thiolates of cysteine, resulting in Tc-cysteinyl-protein bonds. Another type of binding site, however, involves imidazoles of histidine, resulting in Tc-histidinyl-protein bonds. This report appears to be the first description of Tc binding to imidazoles of histidine in proteins. EXPERIMENTAL PROCEDURES

Preparation of Immunoglobulin Kits for 9SmTc Labeling. Human y-globulin (Gamimune N, Cutter Biological,Elkhart, IN) was used as a source of immunoglobulin and was used without additional purification. Two types of immunoglobulin labeling kits were used. One type of kit was prepared using unmodified immunoglobulin, and is referred to as an IgG kit. The other type of kit was prepared using stannous ion-reduced immunoglobulin and is referred to as an IgG-r kit. The IgG and the IgG-r kit contents were the same except that the immunoglobulin in the latter was pretreated with stannous ions to increase the thiolate concentration. All immunoglobulin prepa0 1992 Amerlcan Chemlcal Soclety

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rations were prepared aseptically using nitrogen-purged solutions, and whenever feasible under an atmosphere of nitrogen. To prepare IgG labeling kits, the immunoglobulin was diluted to a final concentration of 1.4 mg/mL in chilled, nitrogen-purged 10mM tartrate/40 mM phthalate buffer, pH 5.6 (P/T buffer). The antibody solution was mixed (7:3) with P / T buffer containing 1.25mM stannoustartrate and excipients. Aliquota (typically 0.5 mL containing 500 pg of antibody) were then dispensed into individual vials and lyophilized. To prepare IgG-r labeling kits, immunoglobulin was treated with stannous ion to increase the amount of thiolates (Rhodes et al., 1986; Hawkins et al., 1990). To do this, the immunoglobulin was diluted to 8.3 mg/mL in P / T buffer and the resulting solution mixed (3:2) in an amber vial with P / T buffer containing 5 mM stannous tartrate. The head-space gas was purged with nitrogen, the vial sealed, and the reaction allowed to proceed for 21 h at room temperature. At the end of the incubation period, the solution was filtered through a 0.22-pm filter and chromatographed over Sephadex G-25 pre-equilibrated in P/T buffer, thereby removing uncomplexed tin ions. Protein concentration was determined by use of a commercially available dye binding assay (CommassieBlue G-250) using unmodified human y-globulin as the standard. After determining the protein concentration, the IgG-r was mixed (7:3) with P / T buffer containing 1.25 mM stannous tartrate and excipients. Aliquots of 0.5 mL were then dispensed into individual vials and lyophilized. fiTc Labeling. To radiolabel, the lyophilizedkits were reconstituted with 0.5 mL of water (or 0.9 % saline); each rehydrated kit contained 0.5 mg of IgG, 40 mM phthalate, 10 mM tartrate, and 22 pg of stannous tartrate. The labeling reaction was accomplished by the addition of 0.52.0 mCi of g g m T(sodium ~ pertechnetate in saline). Radiochemical analysis began 30 min after the introduction of the pertechnetate. Radiochemical Analysis by High-PerformanceLiquidchromatography. To determine the relative amount of 99mTcbound to a given antibody preparation, aliquots of the %"Tc-labeled preparations were analyzed by HPLC at a flow rate of 1mL/min phosphate-buffered saline (0.01 M phosphate, pH 7.0, containing 0.15 M NaC1) using a 7.5 X 300 mm TSK G3000SW column with a TSK-SW 7.5 X 7.5 mm guard column (TosoHaas, Philadelphia, PA) and a UV and radioisotope detector in series. Additional information on analysis of radiolabeled antibodies by this method can be found elsewhere (Hawkins et al., 1990). The following points are noted with regard to this analytical system: (a) free pertechnetate was quantitatively recovered, (b) reduced Tc bound to carrier molecules (chelators, proteins) was recovered in high yield, and (c) uncomplexed reduced Tc was quantitatively adsorbed onto the column and could not be subsequently eluted. According to information supplied by the manufacturer, the HPLC separation and guard columns are packed with a porous, hydrophilic silica support housed in stainless steel columns. It is not known what component of the chromatography system binds uncomplexed, reduced Tc. Thin-LayerChromatography (TLC). TLC was used to measure the amount of protein-bound (and unbound) ggmT~. ITLC-SG (Gelman Sciences, #61886) chromatography paper was cut into 1.5 X 10 cm strips and activated by heating for 30 min at 110 "C, as per the manufacturer's instructions. After heating, the strips were stored at room temperature until use.

Zamora and Rhodes

Protein-bound 9gmTc in the radiolabeled antibody preparations was measured using TLC in an organicsolvent (85% aqueous methanol). The organic solvent separated the soluble, unbound 99mT~ (which migrated with the bound ~ to the protein (which solvent front) from g g m T remained at the origin). The method of use and subsequent processing of data were similar to that described elsewhere (Rhodes et al., 1986). Both cysteine and histidine, as used in the competition studies, are soluble in alcohols, and theirmTc complexeswere found in control studies to migrate with the solvent front. The percentage of protein-bound 99mT~ was the ratio of the cpm in the origin half of the strip minus the background divided by the total cpm. The total cpm was the cpm in the origin half of the strip minus the background plus the cpm in the solvent front half of the strip minus the background cpm. Competition for g 9 m Tby ~ Free Amino Acids. Individual lyophilized kits of IgG or reduced IgG were hydrated with 40 mM phthalate/lO mM tartrate buffer, pH 5.6, containing various concentrations of free amino acids. Histidine was used at concentrations of 50,5,0.5, 0.05, 0.005, 0.0005, and 0.0 mM (control). Cysteine was use at concentrations of 5,0.5,0.05,0.005,0.0005,and 0.0 mM (control). The labeling reaction was initiated by adding 0.5-2.0 mCi of 9 9 m T(sodium ~ pertechnetate in saline) to the vial. Radiochemical analysis was begun 30 min after the introduction of the pertechnetate. Modification of Imidazole Groups. Commercially available DEPC (diethyl pyrocarbonate, Aldrich Chemical Co., Milwaukee, WI) was used to modify the imidazole groups on histidine, and presumptively block potential binding. DEPC, a liquid (6.9 M), was diluted with anhydrous acetonitrile. DEPC has a half-life of 24 min at room temperature pH 6.0 (Miles, 19791, and was prepared immediately prior to use. The final concentration of DEPC used in this study was 7 mM. The working dilution was prepared from a 0.69 M working stock solution. An aliquot of 3 mL of immunoglobulin solution (50 mg/mL) was placed in an amber vial and 60 pL of stock solution of DEPC was added with mixing. The mixture was incubated either at room temperature for 1h or at 4 "C overnight, and the volume was then adjusted to lOmL with 0.9% NaC1. The mixture was chromatographed through Sephadex G-25 to remove unreacted components. The void volume, containing the immunoglobulin, was collected and filtered through a 0.22pm filter. This preparation was analyzed by HPLC and found to be chromatographically uniform, with a single peak which eluted at a time corresponding to the molecular weight of standard IgG. The sample was then divided into two aliquots and further processed into IgG and IgG-r labeling kits as described above, except that the kita were frozen at -70 "C until use, rather than lyophilized. Modification of Thiolate Groups. N-Ethylmaleimide (NEM) was used as a thiolate blocking agent. It was prepared fresh in an amber vial as a 100 mM stock solution in 95% ethanol. Actual modification mixtures contained either 60 mg of IgG or IgG-r in 0.9% NaCl prepared by buffer exchange over Sephadex G-25. The pH was adjusted to 8.1 by the addition of 1:lO volume of 0.1 M phosphate buffer, pH 8.1. The reaction was started by adding NEM stock solution to a final concentration of 1mM NEM. The reaction was allowed to proceed for 30 min at room temperature and was terminated by precipitation of the antibody in approximately 20 % polyethylene glycol (MW 8000, 52% PEG stock solution). The precipitated antibody was collected by centrifugation (lOOOg

gemTcBlndlng to HlstMlne and Thlolates

for 15min) and the supernatant discarded. The precipitate containing the antibody was dissolved in 0.9 % NaCl and chromatographed over Sephadex G-25 in degassed 0.9% NaC1. The eluate was collected and filtered through a 0.22-pm filter. The IgG and IgG-r preparations were then prepared for labeling as described above, except that the final kits were stored frozen rather than lyophilized. Modification of Both Thiolate and Imidazole Groups. Immunoglobulin (300mg) was reacted with 7 mM DEPC for 1 h a t room temperature. After incubation, the solution was chromatographed over Sephadex G-25 (0.9% NaCl equilibration and elution buffer) and the void volume containing the antibody was collected. The DEPCtreated immunoglobulin was then split into two aliquots. One aliquot was treated with NEM and used to prepare IgG labeling kits. The other aliquot was reduced with stannous ions, treated with NEM, and used to prepare IgG-r labeling kits. For treatment with NEM, one aliquot of DEPC-treated IgG was incubated in 10 mM NEM in 0.01 M phosphate buffer, pH 8.0, for 30 min. Unreacted NEM was separated from the antibody by chromatography over Sephadex G-25 (0.9% NaCl equilibration and elution). The antibody was then subjected to buffer exchange by chromatography over Sephadex G-25 (equilibrated and eluted with 40 mM phthalate/lO mM tartrate, pH 5.6). The immunoglobulin was made into IgG labeling kits as described above, and frozen until used. The other aliquot of DEPC-treated immunoglobulin was first reduced with stannous ions and then treated with NEM. The DEPC-treated immunoglobulin was reduced in phthalatehartrate buffer, pH 5.6, containing 2 mM stannous tartrate for 21 h. The stannous ions were removed by chromatography over Sephadex G-25 (0.9% NaCl equilibration and elution). The IgG-r was then incubated in 10 mM NEM in 0.01 M phosphate buffer, pH 8.0, for 30 min. The unreacted NEM was separated from the antibody by chromatography over Sephadex G-25 (0.9% NaCl equilibration and elution). The IgG-r was subjected to buffer exchange by chromatography over Sephadex G-25 (equilibrated and eluted with 40 mM phthalatel10 mM tartrate, pH 5.6). The immunoglobulin was made into IgG-r labeling kits as described above, and frozen until used. Sham DEPC- and NEM-modified kits were also made, in which immunoglobulin was subjected to the pH shifts, buffer exchanges, and column chromatography steps, without addition of either DEPC or NEM. These IgG and IgG-r kits were used as controls in DEPC- and NEMmodified kit experiments, to insure that the reaction conditions did not affect ggmTcbinding. Preparation of Peptides for Labeling. Synthesized peptides were obtained commercially as lyophilized preparations. Each peptide was dissolved directly in P / T buffer, resulting in a peptide concentration of 1.4mg/mL. This solution was mixed (7:3)with P / T buffer containing 1.25 mM stannous tartrate. Aliquots of 0.5 mL were dispensed into individual vials and stored frozen until used. Each kit contained 0.5 mg of peptide, 40 mM phthalate, 10 mM tartrate, and 22 p g of stannous tartrate. All preparations were labeled with 99”Tcusing methods similar to those described above. RESULTS

The IgG labeling kits were found to have 0.6 thiolatesl molecule as determined by use of Ellman’s reagent (Jocelyn, 1987). The IgG-r labeling kits, treated with stannous ions to reduce disulfide bonds and increase

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f

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8 E

8 IgG PLUS HISTIDINE

ELUTION TIME

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Figure 1. Radiochemical analysis by high-pressure liquid chromatography of IgG and IgG-r after labeling with mTc in the absence (controls) or presence of 0.5 mM histidine. Similar amounts of protein and &Tc were used, and the incubation and analytical conditions were also similar. The dark arrows indicate protein-boundhTc (elution time = 9.3 min). The outlinedarrow indicates *Tc presumably bound to histidine (elution time = 14.2min).

thiolate concentration, were found to have 6-8 thiolatesl molecule. The IgG kits consistently bound some, but not all, of the 9 9 m T(34.0 ~ f 6.0% total semTc, n = 10) as determined by quantitative radio-HPLC. The IgG-r kits (96.5 ~ f 2.0% total consistently bound nearly all the 9 9 m T 99mT~, n = 40). Competition for 9 9 m Twith ~ Free Histidine and Cysteine. To evaluate the hypothesis that both imidazoles in histidine and thiolates in cysteine bind 99mTc, competition studies were performed in which either histidine or cysteine was added during hydration of IgG and IgG-r labeling kits. Histidine, in concentrations ranging from 0.005 to 50 mM, was able to compete for 9 9 m Twith ~ the immunoglobulin in IgG labeling kits, as is illustrated in Figures 1 and 2. At a concentration of 0.05 mM histidine, approximately 50% of the gQmTcwas removed from the protein. However, the immunoglobulin in IgG-r labeling kits was able to bind approximately 90% of the 99mT~, even in the presence of 50 mM histidine. IgG labeling kits were very sensitive to histidine, while IgG-r kits were relatively insensitive. In those cases where histidine was , able to compete with the antibody protein for m T ~HPLC profiles revealed a second, low molecular weight radioactive peak which was consistent with the known elution time of histidine. As an aside, in control experiments where free histidine, but no protein, was used, a discrete peak of radioactivity was associated with the histidine. Cysteine, in concentrations ranging from 0.005 to 5 mM, was able to compete successfully for 99mT~ in both IgG and IgG-r labeling kits (Figure 2). The competition curves demonstrate that IgG kits were more sensitive to competition with free cysteine than were IgG-r kits. In studies where glycine was used as a competitor, no effect on the binding of 99mTc with either IgG or IgG-r kits

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5 m M HISTIDINE (LCG,J

5 m M CYSTEINE (LOG,J

Figure 2. Comparative effect of adding histidine or cysteine to IgG and IgG-r prior to radiolabeling with 99mT~. All experimental values are expressed as a percentage of the appropriate control value, e.g. the percentage of 99mTcbound to either IgG or IgG-r containing 0 mM free amino acid. Note the dissimilarity in the shape of the curves obtained with histidine and the overall similarity of the curves obtained with cysteine.

Table 1. Comparison of the Relative Percentage of 9 9 m T ~ Bound in IgG and IgG-r Kits, Both with and without Chemical Modification of Amino Acid Side Chains. % of total ~

IgC-r

T protein-bound C IgG-r

group modified IgG 30.2 100.0 none histidine only 5.7 85.0 thiolate only 38.8 60.1 histidine and thiolate 0.5 11.5 Histidine was modified by the use of diethyl pyrocarbonate. Thiolates were modified by use of N-ethylmaleimide. All values are compared relative to IgG-r labeling. See methods section for details on modification conditions. All evaluations were performed using an equivalent amount of protein and radionuclide,and similar labeling conditions.

was detected at any concentration of glycine tested (up to 20 mM). Similarly, essentially no effect was noted when 5 mM of either lysine or arginine was used as competitors. Amino Acid Side Chain Modification. In both IgG and IgG-r labeling kits, the relative percentages of as determined by quantiimmunoglobulin-bound 99mT~, tative HPLC, changed depending on whether (a)histidines were modified with diethyl pyrocarbonate (DEPC), (b) thiolates were modified with N-ethylmaleimide (NEM), or (c) both histidines and thiolates were modified. A summary of the results of these experiments is shown in Table I. When histidine groups of IgG were modified with DEPC, the subsequent labeling of the IgG kits with g g m labeling T~ was greatly inhibited. This is illustrated by the HPLC profiles shown in Figure 3. However, when DEPCmodified IgG-r kits were labeled with 99mT~, the amount of 99”Tc bound to immunoglobulin was only slightly reduced compared to that in unmodified, control kits (IgGr). Some dimerization or aggregation was noted with the DEPC-modified IgG-r kits, but an evaluation of the absorbance profile a t 280 nm suggested that little or no immunoglobulin was lost during the evaluation. With NEM-modified IgG kits, in which thiolate groups were presumptively blocked, 99mT~ labeling was slightly higher than in control IgG kits (Table I). This higher value is believed to be within experimental error. However, NEM-modified IgG-r kits showed a sharp decrease in labeling efficiency. When both imidazole and thiolate groups in the imlabeling ~ universally munoglobulin were modified, g g m T decreased. With DEPC- and NEM-modified IgG kits,

L DEPC. TREATED IgC-r

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DEPC.TREATED IgC

TIME OF ELUTION

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Figure 3. The effect of diethyl pyrocarbonate (DEPC), a

chemical modifier of histidine, on the subsequent “Tc labeling of IgG as determined by radio-HPLC. The profiles in the top panels are paired controls for the DEPC-treated preparations in the bottom panels. In the left panels are results obtained with IgG. In the right panels are results obtained with IgG-r.

99mT labeling ~ was negligible, approximately 0.5 9%. With DEPC- and NEM-modified IgG-r kits, labeling efficiency was substantially reduced, but was not eliminated. In studies with preparations receiving sham treatments, in which the steps for NEM and DEPC modification were followed, but without addition of either NEM or DEPC, IgG kits were determined to bind approximately 30% total 99mT~, and IgG-r kits essentially 100%total SmTc (Table I). Results with sham treatments were thus similar to results with regular IgG and IgG-r kits.

ODmfc Binding to Histldine and Thloiates

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1 It should be noted that selection of NEM as a thiolate modifying agent was based on a series of preliminary IgG-r I studies evaluating a number of thiolate blocking agents (POSITIVE CONTROL) 1 I (iodoacetamide, dibromobimane, monobromobimane, N(iodoethy1)trifluoroacetimide)(Dailey, 1984). NEM was found to decrease the number of thiolates in IgG-r from 6-8 thiolates/molecule to approximately 1thiolate/moli ecule, and was the most effectivethiolate-modifying agent evaluated. These preliminary studies showed a direct POLY-TYROSINE (NEGATIVE CONTROL) relationship between the number of thiolates/molecule and %Tc labeling efficiency in IgG-r kits. IgG-r kits with 6 or more thiolates/molecule labeled with essentially 100% efficiency, with the labeling efficiency decreasing approximately 20% for every decrease of 2 thiolates/molecule. Labeling Synthesized Peptides Containing Histidine or Cysteine. To further evaluate the potential & c binding of m T ~to histidine as well as cysteine, we CYSTEINYL-PEPTDE E ~ in three peptides with known evaluated 9 9 m Tbinding e amino acid sequences. These peptides were specifically I1 selected to be unrelated to IgG. One peptide, with the I I amino acid sequence HzN-Cys-Asp-Pro-Gly-Tyr-Ile-GlySer-Arg, contained a single cysteine residue and no histidines. Another peptide, with the sequence Ac-AspHISTIDWYL-PEPTIDE Arg-Val-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Asp, contained histidine residues but no cysteines or cystine. The control, polytyrosine, contained neither histidine nor cysteine. The histidine-containing peptide bound some but not all of the added %Tc (Figure 4). The cysteine-containing peptide bound essentially all of the added %Tc (Figure 4). Polytyrosine, the negative control material, did not label. These results were confirmed by conventional thinELUTION TIME d layer chromatography. To further confirm that 99mT~ Figure 4. The binding of 99mT~ to synthesized peptides as could bind to histidine-containing peptides, a number of determined by radio-HPLC. Polytyrosine (negativecontrol) was other peptides were examined and found to bind 99mT~. used as an example of a peptide containing neither histidine nor cysteine. IgG-r was used as a positive control material. One These peptides include the following: N-acetylrenin substrate tetradecapeptide (Ac-Asp-Arg-Val-Ile-His-Pro- peptide, Ac-Asp-Arg-Val-Ile-His-Pro-Phe-His-Leu-Val-Ile-HisAsp (elution time = 13.8min), contained histidine but no cysteine Phe-His-Leu-Val-Ile-His-Asp), renin inhibitor (Pro-Hisor cystine. Another peptide, HZN-Cys-Asp-Pro-Gly-Tyr-IleHis-Pro-Phe-His-Phe-PheLeu-Val-His), angiotensin (AspGly-Sel-Arg (elution time = 13.9min), contained a single cysteine Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu), hypercalcemia of but not histidine. malignancy factor (PTH-like) 1-16 (Ala-Val-Glu-His-GlnLeu-Leu-His-Asp-Lys-Gly-Lys-Gly-Ser-ne-Gln), and polyresidues (Rhodes, 19911, a number of other amino acids histidine. Polyhistidine was found to have very limited may be involved in the direct binding of wmTcto proteins, solubility under the conditions of the experiment, but analogous to the binding that takes place with other nonetheless, evidenced 99mTcbinding. 99mTc binding was transition metals (Henkin, 1975). Histidine, for example, and to also observed in poly(His,Glu)-poly-Ala-poly-Lys, is well-known for its ability to coordinate metals, and as a much larger extent than poly(Tyr,Glu)-poly-Ala-poly- a free amino acid binds w m T(Seifert ~ et al., 1983). Lys. The results of Steigman et al. (1975) and Lanteigne and Hnatowich (1984) clearly indicate that proteins contain DISCUSSION thiolate binding sites as well as other Tc-binding sites. Lanteigne and Hnatowich (1984), for example, were able Rhodes et al. (1980)were the first to develop an “instant” to Tc label myoglobin, a molecule which contains neither labeling kit for immunoglobulins in which stannous ions cystine nor cysteine. Immunoglobulins (the constituent were used to reduce proteins, thereby generating reactive component of IgG kits of this study) contain histidines in This thiolates, for subsequent direct labeling with 99mT~. both the light and heavy chains (Edelman et al., 1969), strategy, applied primarily to monoclonal antibodies, has and while they do not normally contain reactive thiolates, been widely emulated using a number of reducing agents they do contain significant numbers of disulfide bridges. (for reviews see Eckelman & Steigman, 1991;Rhodes, 1991; Reduced immunoglobulin (the constituent component in also Schwarz & Steinstrasser, 1987,and Mather & Ellison, the IgG-r kits used in this study) contains 6-8 reactive 1990). Thakur et al. (1991) has directly related the type thiolates per molecule. of reducing agent to the number of thiolate groups produced in antibodies and the resulting radiochemical In the study reported here, when histidine was added incorporation. Thakur also suggested treating reduced immediately prior to radiolabeling, it was able to sucantibody with iodoacetate to validate the hypothesis that cessfully compete for “Tc with immunoglobulin,but not thiolate groups provide the binding sites for wmTc. reduced immunoglobulin. This suggeststhat the primary binding site in unreduced immunoglobulin is related to Thiolate groups in cysteine have long been implicated histidine groups. This observation explains reports of in the direct labeling of proteins with Tc (Steigman et al., 1975). In spite of the implication that WmTcis bound to direct labeling of unmodified immunoglobulins,albeit with low radiochemical incorporations (Pettit et al., 1980).The reduced immunoglobulin via the thiolates of cysteine

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inability of histidine to compete with reduced immunoglobulin for "Tc does not necessarily preclude Tchistidinyl-protein bond formation. The results do suggest that a different site, presumably involving thiolates, is predominant in reduced immunoglobulin. When cysteine was added immediately prior to radio~ both labeling, it successfully competed for 9 9 m Twith immunoglobulin and reduced immunoglobulin. Since immunoglobulin contains virtually no reactive thiolates, these observations suggest that cysteine may be involved in binding to reduced immunoglobulin. However, since cysteine is a reducing agent known to bind 99mT~, an alternative explanation is that the cysteine reduced Tcprotein bonds and subsequently competed for the released Tc. To further explore the relative involvement of imidazoles and thiolates in 9gmTcbinding, we monitored the labeling of IgG and IgG-r labeling kits with amino acid side chain modification (Means and Feeney, 1990). The results T~ site in unreduced indicate that the primary 9 9 m binding immunoglobulin is related to imidazole groups, and not to thiolates. In IgG-r kits, containing reduced immunoglobulin, both imidazole and histidine groups are involved I t is clear that reduction of IgG to ~ in 9 9 m Tbinding. generate thiolates is required to obtain radiochemical incorporations of 99mT~ which approach 100%. In the immunoglobulin preparations tested, residual stannous ions used to reduce pertechnetate could potentially reduce disulfide bonds, and complicate the interpretation of the results. We thus evaluated the binding of 99mT~ to peptides with defined amino acid sequences. One peptide contained a terminal cysteine residue, but no histidines, while the other peptide contained histidine residues, but no cysteine or cystine. Both peptides were found to label with 99mT~, while in control studies, a peptide free of cysteine and histidine did not label. The utility of histidine group binding in peptides and proteins lacking cysteine remains to be fully developed, but suggests that such a labeling strategy deserves further study. In conclusion, both histidine and cysteine groups of proteins can bind 99mT~. When cysteine is not available binds to histidine groups, and the binding in IgG, 99mT~ efficiency is only moderate. When cysteine is available, as in reduced IgG, 9 9 m Tbinds ~ to the protein almost quantitatively, and the binding is essentially to cysteinyl groups. ACKNOWLEDGMENT

This study was funded by Small Business Innovative Research Grant 2 R44 CA50877-02 from the National Cancer Institute, Department of Health and Human Services. The editorial assistance of T. Coons and S. Slusher is gratefully acknowledged, as is the technical assistance of P. Budd, C. Lambert, and K. Sass. The professional expertise of M. Marek in preparation of some of the immunoglobulinkits is also gratefully acknowledged. LITERATURE CITED Arnold, F. H., and Haymore, B. L. (1991) Engineered metalbinding proteins: Purification to protein folding. Science 252, 1796-1797. Dailey, H. A. (1984) Effect of sulfhydryl group modification on the activity of bovine ferrochelatase. J. Biol. Chem.259,27112715.

Zamora and Rhodes

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