Use of Heavy-Metal Clusters in the Design of N-Succinimidyl Ester

Domenico Osella,*,† Paola Pollone,† Mauro Ravera,† Miche`le Salmain,‡ and Gérard ... Corso T. Borsalino, 54, I-15100 Alessandria, Italy, and ...
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Bioconjugate Chem. 1999, 10, 607−612

607

Use of Heavy-Metal Clusters in the Design of N-Succinimidyl Ester Acylation Reagents for Side-Chain-Specific Labeling of Proteins Domenico Osella,*,† Paola Pollone,† Mauro Ravera,† Miche`le Salmain,‡ and Ge´rard Jaouen*,‡ Dipartimento di Scienze e Tecnologie Avanzate, Universita` del Piemonte Orientale “Amedeo Avogadro”, Corso T. Borsalino, 54, I-15100 Alessandria, Italy, and Ecole Nationale Supe´rieure de Chimie de Paris, Laboratoire de Chimie Organome´tallique, UMR CNRS 7576, 11, rue Pierre et Marie Curie, F-75231 Paris Cedex 05, France . Received November 19, 1998; Revised Manuscript Received March 10, 1999

New heavy transition metal carbonyl markers for protein labeling, containing an "Mn(CO)11" (M ) Ru, Os, n ) 3; M ) Ir, n ) 4) moiety, were prepared by reaction of “lightly stabilized” clusters with an N-succinimidyl ester functionalized phosphine, namely N-succinimidyl 3-diphenylphosphinepropionate (DPPS). The reaction of Os3(CO)11(DPPS) with the model amino acid β-alanine was performed and led to the expected amide. From the reaction of Mn(CO)11(DPPS) with bovine serum albumin (BSA) in mixed organic/aqueous medium, conjugates bearing a fairly high number of metal carbonyl fragments were obtained, thus demonstrating the usefulness of this class of reagents for the selective and covalent graft of heavy metal clusters to side chain of proteins.

INTRODUCTION

Amine-reactive compounds are widely used in protein chemistry for probing protein structure and/or function or to introduce radioactive or cold reporter groups (Wong, 1991). Some examples of application of the resulting bioconjugates are immunoanalysis and immunohistochemistry. In these applications, the stability of the chemical bond between the marker and the biomolecule is particularly important. The most widely used amine-reactive probes are reagents that form carboxamide, sulfonamide, urea, or thiourea bonds upon reaction with proteins amino groups (Means and Feeney, 1990). In particular, N-succinimidyl esters are able to acylate amines and lead to the formation of a peptide bond (Anderson et al., 1964). This family of reagents displays a high reactivity with protein amino groups (Brinkley, 1992). This is particularly interesting because virtually all proteins have lysine residues and, owing to their hydrophilic nature, most of them are located at the surface of proteins, readily accessible to reagents. Moreover, they have a very low reactivity toward tyrosine, serine, histidine, and cysteine residues (Anjaneyulu and Staros, 1987). Water-soluble N-sulfosuccinimidyl esters have also been described (Staros, 1982). The prototypical compound of this category is the Bolton-Hunter reagent, N-succinimidyl 3-(4-hydroxy-5iodophenyl)propionate (Bolton and Hunter, 1973), commercially available as labeled with 125I. Proteins and peptides radiolabeled via the Bolton-Hunter reagent often exhibit a lower degree of deiodination compared with directly radioiodinated proteins and higher conjugation efficiencies are reached (Bolton et al., 1976; Vaidyanathan and Zalutsky, 1990). Although radioisotopic labeling is a very sensitive method, owing to legal and technical problems associated * To whom correspondence should be addressed. † Universita ` del Piemonte Orientale “Amedeo Avogadro”. ‡ Ecole Nationale Supe ´ rieure de Chimie.

with radioactivity, the implementation of “cold” labeling procedures has been an important goal (Gosling, 1990). Application of organotransition metal complexes containing biologically relevant ligands have attracted considerable attention in this field in recent years (bioorganometallic chemistry) (Severin et al., 1998). In this area, Jaouen et al. (1993) developed the so-called carbonylmetallo-immunoassay (CMIA),1 where biomolecules are labeled with metal carbonyl fragments and conjugates detected by FT-IR spectroscopy in the carbonyl ligands stretching vibration modes region (2150-1800 cm-1), where proteins do not absorb. Transition metal carbonyl complexes containing an N-succinimidyl ester function (Varenne et al., 1993; Salmain et al., 1994; Gorfti et al., 1996), a pyrylium group (Salmain et al., 1994; Malisza et al., 1995), or an isothiocyanate (Kazimierczak et al., 1997), all reacting at the N-terminal position and/or the lysine residues of proteins, have been developed for this purpose. In the same context, we covalently linked a third-row transition metal cluster fragment, namely "Os3(CO)10", to a model amine (i.e., benzylamine), to model amino acids (i.e., β-alanine and NR-protected lysine), and finally to bovine serum albumin (BSA) by using N-succinimidyl4-pentynoate as the linker. The average coupling ratio (CR) between the Os3-marker and BSA was fairly high, i.e., around 20 (Osella et al., 1996). This was the first example of a marker containing a metal carbonyl cluster of predefinite nuclearity. To make the Bolton-Hunter-like procedure more widely applicable to polymetallic carbonyl compounds, we have designed a new phosphine linker functionalized by an N-succinimidyl ester, namely N-succinimidyl 3-diphe1 Abbreviations: BSA, bovine serum albumin; CMIA, carbonylmetallo-immunoassay; CR, coupling ratio; DCI-MS, desorption chemical ionization mass spectrometry; DPPS, N-succinimidyl 3-diphenylphosphinepropionate; FT-IR, Fourier transform infrared; RP-HPLC, reversed-phase high-performance liquid chromatography; STEM, scanning transmission electron microscopy; TFA, trifluoroacetic acid; THF, tetrahydrofuran.

10.1021/bc980139n CCC: $18.00 © 1999 American Chemical Society Published on Web 06/19/1999

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nylphosphine propionate (DPPS), 1. In this paper, we report the synthesis of three polymetallic carbonyl complexes containing the DPPS ligand, namely Ru3(CO)11(DPPS), 5, Os3(CO)11(DPPS), 6, and Ir4(CO)11(DPPS), 7, and their reactivity toward β-alanine and BSA. Our goal is to label proteins with metal carbonyl clusters that we expect to be useful in scanning transmission electron microscopy (STEM) studies, owing to their electron-dense cores. In contrast to colloidal gold, derivatives 5-7 are not adsorbed but covalently linked to the target biomolecules, avoiding nonspecific interactions. An interesting precedent is the use of the rigid dimer [Ir4(CO)11][Ph2POC6H4-C6H4OPPh2], which showed paired spots (ca. 6 Å in diameter) in STEM micrograph. The average separation between paired spots allowed the dimensions of molecular electronics structures to be established (Furuya et al., 1988). Moreover, compounds 5-7 could be useful for the production of isomorphous heavy metal derivatives for protein X-ray crystallographic structure determination (Weinstein et al., 1989). Derivatization with heavy atom compounds at a unique lattice site is frequently used to obtain phase information and enable X-ray structure resolution of proteins (Branden and Tooze, 1991). This project may substantially contribute to the better understanding of the structure and role of proteins and other biomolecules. EXPERIMENTAL PROCEDURES

Materials and Methods. Reactions were carried out under nitrogen using dried and prepurified solvents. Chromatographic separations were performed using microcrystalline cellulose (Merck Avicel). Routine FT-IR and FT-NMR spectra were recorded on a Bruker Equinox 55 and on a JEOL-EX 400 spectrometer, respectively. NMR chemical shifts are referenced to the internal standard tetramethylsilane (TMS) and 85% H3PO4 for 1H and 31P spectra, respectively. UV-vis spectra were recorded on a Safas UV/mc2 spectrometer. DCI-MS spectra were recorded on a Finnigan-MAT 95Q instrument with magnetic and electrostatic analyzers. Isobutane was used as the reagent gas at 0.5 mbar pressure. The ion source temperature was kept at 50 °C, the electron emission current at 0.2 mA, and the electron energy at 200 eV. Positive ion spectra were collected. A computer program provided a digital readout of peak intensities and their comparison with the simulated isotopic pattern. The fit was satisfactory for all compounds under investigation. Quantitative FT-IR analyses were performed by depositing 10 mL samples on 9 mm diameter nitrocellulose membranes (Alltech), and recording spectra on a Bomem MB100 FT-spectrometer equipped with a liquid nitrogencooled MCT detector as described (Salmain et al., 1993). N-Succinimidyl 3-diphenylphosphinepropionate, 1, (DPPS) (Santimaria et al., 1995), lightly activated clusters 2 and 3 (Drake and Khattar, 1988), and 4 (Chini et al., 1978) were synthesized according to the literature methods and their purity checked by spectroscopic techniques. Synthesis of M3(CO)11(DPPS) (M ) Ru, 5, and Os, 6). Solid M3(CO)11(NCCH3) (M ) Ru, 2, and Os, 3) (2 200 mg, 0.31 mmol; 3 100 mg, ca. 0.11 mmol) was added to a freshly distilled THF solution (ca. 10 mL) of DPPS (1:1 ratio) at 0 °C in a Schlenk tube equipped with a magnetic stirring bar and an inlet arm for purging the solution with nitrogen. The color turned from yellow to orange. The mixture was allowed to reach room temperature and chromatographed on a cellulose column. Petroleum ether

Osella et al.

first eluted adventitious M3(CO)12, then mixtures containing petroleum ether and increasing amounts of CH2Cl2 eluted the monosubstituted compound (5 or 6) and then the higher substituted compounds. The polysubstituted compounds M3(CO)12-n(DPPS)n (n ) 2, 3) were identified by comparison of their IR spectra with those of original samples (Bruce, 1987). The desired compounds were crystallized in petroleum ether/CH2Cl2 (1:1 v/v) at -10 °C, giving yellow or orange microcrystals. Analytical Data for 5. IR (CH2Cl2): νCO 2097m, 2047vs, 2027sh, 2014vs, 1990sh, 1817w, 1790w, 1745ms cm-1. UV-vis (absolute ethanol): λmax 419 nm ( ) 5100). 1H NMR (400 MHz, CDCl ): δ 7.51 (m, 10H, Ph), ca. 2.9 3 (partially overlapped to the following signal, 2H, P-CH2CH2), 2.83 (s, 4H, N-succinimidyl cycle), 2.50 (q, 2H, P-CH2-CH2-). 31P NMR (161.9 MHz, CDCl3): δ 27.6. MS (DCI, isobutane): [M + H]+ ) 969 m/z. Anal. Calcd. for C30H18NO15PRu3: C, 37.28; H, 1.88. Found: C, 37.06; H, 1.98. Analytical Data for 6. IR (CH2Cl2): νCO 2108m, 2055s, 2033ms, 2018vs, 2000ms, 1989ms, 1818w, 1789w, 1745ms cm-1. UV-vis (absolute ethanol): λmax 407 ( ) 4600), 345 ( ) 12000) nm. 1H NMR (400 MHz, CDCl3): δ 7.51 (m, 10H, Ph), 3.03 (q, 2H, P-CH2-CH2-), 2.83 (d?, 4H, N-succinimidyl cycle), 2.47 (q, 2H, P-CH2-CH2-). 31P NMR (161.9 MHz, CDCl3): δ -9.21. MS (DCI, isobutane): [M + H]+ ) 1236 m/z. Anal. Calcd for C30H18NO15POs3: C, 29.20; H, 1.47. Found: C, 29.01; H, 1.62. Synthesis of Ir4(CO)11(DPPS), 7. Ir4(CO)12 (100 mg, 0.09 mmol) was added to a freshly distilled THF solution (ca. 20 mL) of (C2H5)4NBr (25 mg, 0.12 mmol) in a threenecked flask equipped with a magnetic stirring bar and an inlet arm for purging the solution with nitrogen. The solution was refluxed for 4 h, then DPPS (25 mg, 0.12 mmol) was added and refluxed for 1 h. The mixture was chromatographed on a cellulose column. Mixtures of petroleum ether and 10% CH2Cl2 first eluted 7, then pure CH2Cl2 eluted the disubstituted compound Ir4(CO)10(DPPS)2 in trace amounts. 7 was crystallized in dichloromethane/petroleum ether (1:1 v/v) at -10 °C, giving yellow microcrystals. The disubstituted compound Ir4(CO)10(DPPS)2 was identified by comparison of its IR spectrum with that of an original sample of Ir4(CO)10(PPh3)2 (Darensbourg and Baldwin-Zuschke, 1982). Analytical Data for 7. IR (CH2Cl2): νCO 2089m, 2057vs, 2020s, 1843m, 1819m, 1791w, 1745s cm-1. 1H NMR (400 MHz, CDCl3): δ 7.50 (m, 10H, Ph), 2.83 (s, 4H, N-succinimidyl cycle), 2.73 (q, 2H, P-CH2-CH2-), 2.60 (q, 2H, P-CH2-CH2-). 31P NMR (161.9 MHz, CDCl3): δ -15.5. MS (DCI, isobutane): [M + H]+ ) 1434 m/z. Anal. Calcd. for C30H18NO15PIr4: C, 25.16; H, 1.27. Found: C, 24.80; H, 1.52. Reaction of 6 with β-Alanine Ethyl Ester. β-Alanine ethyl ester hydrochloride, dissolved in CH2Cl2, was neutralized with a slight excess of triethylamine, then solid 6 was added in a 1:1 molecular ratio. The mixture was stirred overnight at room temperature, and chromatographed on a silica gel column. CH2Cl2 removed side-products, then acetone eluted the desired product Os3(CO)11(Ph2P(CH2)2C(O)NH(CH2)2COOEt), 8, in 80% yield. IR (CH2Cl2): νCO 2107m, 2054s, 2033sh, 2017vs, 1999ms, 1990sh, 1730w, 1679w, 1607m, 1518w cm-1. 1H NMR (400 MHz, CD2Cl2): δ 7.61 (m, 10H, Ph), 6.07 (t, br, 1H, NH), 4.27 (m, 2H, -CH2CH3), 3.57 (m, 2H, -NHCH2CH2-), 3.18 [m, 2H, -P-(CH2)2-], 2.62 (t, 2H, -NH-CH2CH2-), 2.17 [m, 2H, -P-(CH2)2-], 1.41 (t, 3H, -COOCH2CH3). 31P NMR (161.9 MHz, CDCl3): δ -9.01.

Use of Metal Clusters in the Design of Acylation Reagents

Bioconjugate Chem., Vol. 10, No. 4, 1999 609

Scheme 1. Synthetic Steps for Preparing Clusters 2-3 and 5-6

Scheme 2. Synthetic Steps for Preparing Clusters 4 and 7

MS (DCI, isobutane): [M + H]+ ) 1238 m/z. Anal. Calcd for C31H24NO14POs3: C, 30.12; H, 1.96. Found: C, 29.98; H, 2.01. Labeling of BSA with 5-7. Two stock solutions were prepared, the first being 10 µM in BSA (crystallized, Serva), dissolved in borate buffer (0.1 M pH 9.0 prepared from demineralized water), the second being 4 mM in 5-7 dissolved in DMF (5 and 6), or 2-propanol (7). To 0.45 mL of protein solution was added 0.05 mL of marker solution (the resulting solutions have a 10% v/v organic solvent content and a 45:1 marker-protein molar ratio). The solutions were stirred 5 h at room temperature, filtered on 0.2 µm porosity PVDF membranes, and chromatographed on a prepacked gel filtration column (Kwiksep 5 mL, Pierce) using NH4HCO3 10 mM as eluent. Twelve 0.5 mL fractions were collected and analyzed as described below. Analysis of the Conjugates. Protein concentration [P] was determined by the Coomassie blue method (Bradford, 1976) while marker concentration [M] was measured by UV-vis spectroscopy for 5 and 6 (5 at λ ) 419 nm,  ) 5100; 6 at λ ) 407 nm,  ) 4600) or by FTIR spectroscopy of the νCO band at 2058 cm-1 (k ) 85) for 7, following a technique previously described (Salmain et al., 1993). The combination of these data provided the final ratio between marker and protein concentrations, namely the so-called coupling ratio CR ) [M]/[P], and the coupling yield CY defined as {([M]/[P])final/([M]/ [P])initial} × 100. Reversed-phase HPLC of the conjugates was perfomed on a 4.6 × 150 mm Si-C4 column (Vydac 214 TP). Species were eluted by applying a linear gradient from 20 to 60% of 0.1% TFA-CH3CN in 0.1% TFA-H2O in 30 min (flow rate ) 1 mL min-1) and were detected at 280 nm. RESULTS AND DISCUSSION

Synthesis and Characterization of Markers. Since the parent clusters Ru3(CO)12, Os3(CO)12, and Ir4(CO)12

are quite inert toward CO substitution, activated derivatives should be used. The “activated” M3(CO)11(NCMe) (M ) Ru, 2, and Os, 3) and Ir4(CO)11Br, 4, have been prepared from the reaction of the parent clusters with trimethylamine-N-oxyde, (CH3)3NO (Drake and Khattar, 1988) (Scheme 1), and tetraethylammonium bromide, (C2H5)4NBr (Chini et al., 1978) (Scheme 2), respectively. These intermediates are very convenient precursors in the preparation of the corresponding derivatives with a variety of phosphines in mild experimental conditions (Foulds et al., 1985; Ros et al. 1986). Hence, compounds 2-4 underwent fast reaction with 1 in THF giving M3(CO)11(DPPS) (M ) Ru, 5, and Os, 6) and Ir4(CO)11(DPPS) (7) in excellent yields for Os and Ir (ca. 80%) (Schemes 1 and 2). Unfortunately, 5 was always obtained in low yield (ca. 15%) due to further substitution of CO ligands with DPPS, yielding the higher substituted Ru3(CO)10(DPPS)2, and Ru3(CO)9(DPPS)3, even when a low stoichiometric ratio between ligand and activated cluster was employed. Thermal reaction of Ru3(CO)12 with a wide variety of phosphines (L) led directly to the formation of trisubstituted Ru3(CO)9(L)3, suggesting that replacement of carbonyl ligands by phosphorus ligands accelerates the rate of further substitution. This rate enhancement has been confirmed by a number of kinetic investigations (Muetterties et al., 1982). Alternative synthetic strategies have been attempted employing benzophenone ketyl (BPK) (Bruce, 1987) or [PPN+] salts (PPN+ ) bis(triphenylphosphine)iminium) as catalysts (Lavigne and Kaesz, 1984). In both cases, the major product was again the disubstituted derivative. Compounds 5-7 were characterized by elemental analysis, IR, 1H and 13C NMR spectroscopy, and DCIMS (see Experimental Procedures). Noteworthy, compounds 5-7, which have fairly high molecular weights and moderate to low polarity, were much more effectively characterized by the DCI technique than both electron impact (EI, ionizing energy ) 70 eV) or fast-atom

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Scheme 3. Coupling Reaction of 6 with β-Alanine Ethyl Ester

Figure 1. Experimental and simulated DCI mass spectra of 7 in the region corresponding to the [M + H]+ peak. The two isotopic patterns have been compared by setting at 100% intensity the most abundant peaks at m/z 1434 in each spectrum.

bombardment (FAB, glycerol matrix) ionization methods. Among these techniques, only DCI-MS provided clean spectra, good sensitivity and well-defined isotopic patterns for the parent ions [M + H]+ (Figure 1). Coupling Reaction of 6 with β-Alanine Ethyl Ester. Once the synthesis and characterization of derivatives 5-7 was completed, it was still necessary to check that the coordination by metal fragments had not altered the acylating property of 1. It was done by testing its reactivity with a model amino acid in dichloromethane. The reaction of 6 with β-alanine ethyl ester proceeded smoothly and completely within 24 h at ambient temperature giving the expected amide Os3(CO)11[Ph2P(CH2)2C(O)N(H)(CH2)2C(O)OEt], 8, in good yield (Scheme 3). As already reported (Osella et al., 1996), the IR, in the νCO region, and UV-vis spectra of 6 and 8 are quite similar, representing good fingerprints for the presence of metallic markers in the conjugate. Labeling Tests of BSA. Bovine serum albumin (BSA) is a very convenient and inexpensive model to test the binding capacity of newly developed amine reactive compounds. In particular, its contents in lysines is of 59 (i.e., a total of 60 amino groups), but in nondenaturating conditions (low temperature and low amount of organic cosolvent), 30-40 lysines are believed to be accessible to reagents (Erlanger, 1980). Solutions of BSA at pH 9.0 were allowed to react with compounds 5-7 dissolved in DMF (5 and 6) or 2-propanol (7), so that the percentage of organic cosolvent was 10% and the initial reagent to protein molar ratio was 45. Reaction proceeded at room temperature for 5 h, and samples were submitted to gel filtration chromatography after filtration. The protein-containing fractions were analyzed by several spectroscopic methods in order to calculate the coupling ratio CR, corresponding to the final number of organometallic clusters bound per protein molecule. Results are reported in Table 1.

Figure 2. RP-HPLC of BSA (A) and BSA-6 conjugate (B). For elution conditions, see the Experimental Procedures. Table 1. Labeling of BSA with Compounds 5-7 (10% organic solvent in borate buffer pH 9.0, initial reagent to protein molar ratio ) 45) reagent

[M]final (M)

[P]final (M)

coupling ratio CRa

coupling yield CYb

5 6 7

48 × 10-6 c 88 × 10-6 d 59 × 10-6 e

4.2 × 10-6 6.5 × 10-6 6.1 × 10-6

11.4 13.2 9.7

25% 30% 22%

a Coupling ratio CR ) [M] b final/[P]final. Coupling yield CY ) [([M]/[P])final/([M]/[P])initial] × 100 ) CR/45. c Measured by UVvis spectroscopy at 419 nm. d Measured by UV-vis spectroscopy at 407 nm. e Measured by FT-IR spectroscopy at 2058 cm-1.

Because of the high molecular weight of the reagents and their hydrophobic nature, we encountered some hurdles during the purification of the conjugates. Dialysis was unable to remove excess reagents (some part precipitated). Gel filtration chromatography of the reagents incubated in the same conditions without protein showed that some of them eluted in the same tubes as the protein while the rest precipitated in the column. We conclude that this means of purification is likely to lead to conjugates contaminated with noncovalently bound cluster.

Use of Metal Clusters in the Design of Acylation Reagents

The ultimate evidence of covalent binding of clusters 5-7 to BSA was brought by RP-HPLC. Chromatograms of BSA and BSA-6 conjugates are displayed Figure 2. In the elution conditions chosen, BSA had a retention time of 28.3 min while BSA-6 gave 4 major peaks at higher retention times (43 < tr < 47.3 min), in agreement with the conjugation of several hydrophobic clusters to the protein. The presence of unbound cluster was detected by elution with 100% CH3CN. In summary, we observed a similar behavior of compounds 5-7 toward BSA; in particular, compound 6, namely Os3(CO)11(DPPS), represents the best compromise between the moderate propensity toward hydrolysis showed by the isostructural compound 5 and the bulky hydrophobic character exhibited by the tetranuclear cluster 7, so giving the highest CR and CY values. CONCLUSIONS

Coordination of the ligand N-succinimidyl 3-diphenylphosphine propionate to activated triruthenium, triosmium, and tetrairidium clusters was achieved and the resulting complexes were shown to label BSA with yields in the range 22-30%. Within the Bolton-Hunter-like protocol for obtaining metallic-labeled acylating reagents for proteins, this phosphine ligand appears as a more widely applicable linker than the previously employed N-succinimidyl 4-pentynoate, since almost all metal carbonyl clusters react with phosphorus ligands and maintain their geometry. On the contrary, the coordination of the triple bond to clusters is not straightforward and often causes structural rearrangements (Osella and Raithby, 1989). By a similar strategy, it should also be possible to prepare derivatives targetted toward thiols (by introducing a maleimide function in place of the N-hydroxysuccinimide ester) or water-soluble compounds by exchanging some of the remaining CO ligands with water-soluble phosphines. ACKNOWLEDGMENT

We thank Johnson Matthey for a generous loan of precious metals, European Union (EU) and MURST (COFIN 98) for financial support, and Dr. M. Vincenti (Department of Analytical Chemistry, University of Torino) for recording DCI-M.S. spectra. We are indebted with the two reviewers for valuable and constructive suggestions. LITERATURE CITED (1) Anderson, G. W., Zimmerman, J. E., and Callahan F. M. (1964) The use of esters of N-hydroxysuccinimide in peptide synthesis. J. Am. Chem. Soc. 86, 1839-1842. (2) Anjaneyulu, P. S. R., and Staros, J. (1987) Reactions of N-hydroxysulfo-succinimide active esters. Int. J. Pept. Protein Res. 30, 117-124. (3) Bolton, A. E., and Hunter, W. M. (1973) The labeling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem. J. 133, 529-539. (4) Bolton, A. E., Bennie, J. G., and Hunter, W. M. (1976) Innovations in labeling techniques for radiommunoassays. Protides Biol. Fluids 24, 687-693. (5) Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248254. (6) Branden, C., and Tooze, J. (1991) Introduction to protein structure, Garland Publishing Inc., New York and London. (7) Brinkley, M. (1992) A brief survey of methods for preparing protein conjugates with dyes, haptens and cross-linking reagents. Bioconjugate Chem. 3, 2-13.

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