Potential of albumin labeled with nitroxides as a contrast agent for

32

Bioconjugate Chem. 1990, 1, 32-36

Potential of Albumin Labeled with Nitroxides as a Contrast Agent for Magnetic Resonance Imaging and Spectroscopy Hsiao-Chang Chan,t>fKeqin Sun,$ Richard L. Magin,$ and Harold M. Swartz*St Bioacoustic Research Laboratory, Department of Electrical and Computer Engineering, Department of Biochemistry, and Departments of Medicine and Biophysics, University of Illinois, Champaign-Urbana, Illinois 61801. Received June 17, 1989

Urbana,

The biological and physical properties of albumin and nitroxides make them attractive candidates as special purpose MRI contrast agents which could be used to study the intravascular compartment or specific targets in tissues. In this study, albumin-nitroxide complexes were prepared by reduction and alkylation of the disulfide bonds of the protein and characterized by electron spin resonance and ultraviolet absorption spectroscopy. An average of six nitroxides were bound covalently to each molecule of human serum albumin. The water proton relaxivity of the protein-bound nitroxide (at 20 MHz and 37 "C) was 4-fold greater than that of the free nitroxide. The digestion of the nitroxidealbumin complexes by cells or by trypsin decreased the relaxivity of the nitroxide-protein complex. The rate of reduction of albumin-bound nitroxide by cells was much slower than that of the free nitroxide but still was oxygen-sensitive (2-3-fold increase in the rate of reduction in the absence of oxygen).

The attachment of paramagnetic nitroxide free radicals to biomolecules has been an important biophysical tool for structural and functional studies in biological systems for more than 2 decades (1). The effect of the attachm e n t of nitroxides t o macromolecules on waterrelaxation properties has been noted (2-51, and recently, nitroxides and their protein complexes have been suggested as contrast agents for magnetic resonance imaging and spectroscopy (MRI and MRS) (3-7).l This approach parallels the use of conjugates of albumin and paramagnetic metal ions as MRI contrast agents, which also has been investigated recently (11, 12). Although there are concerns regarding the antigenicity, toxicity, and slow excretion clearance of albumin, albumin with a paramagnetic species attached has several useful features that include provision of more optimum correlation times for relaxivity, a biodistribution primarily confined to the vascular space after intravenous administration (13),the ability to bind a wide variety of substances including monoclonal antibodies or specific receptors (14), and a rate of biodegradation of the complex which could be used as a potential additional parameter to measure metabolic function. Although the relaxivity induced by nitroxides is usually less than that of many paramagnetic metals or metal chelates @-IO), nitroxides have some special features which make them attractive potential contrast agents. Nitroxides can be reduced by cells to their nonparamagnetic state a t rates that reflect the metabolic condition of the cells (15);in particular, hypoxia increases the rate of reduc-

tion of nitroxides, and the rate of oxidation back to the nitroxides can be proportional to the concentration of oxygen (16). This metabolic responsiveness may enable nitroxides to provide contrast in MRI or MRS for various pathophysiological states characterized by hypoxia, e.g., cancer, ischemia, and inflammation ( I 7 ) . Nitroxides can be attached to albumin covalently with a maleimide nitroxide which interacts principally with the single reactive sulfhydryl group of a native albumin molecule (I, 18-20). This method is easy, but the amount of nitroxide bound to each molecule of albumin is small, with a typical ratio of nitroxide to albumin of about 0.6 to 1 (2, 20). Chemical modifications of albumin with nitroxides have been reported which result in a higher ratio of nitroxides to albumin (3,21);however, these preparations involve some special reagents and a certain degree of difficulty. In this study, we have used a relatively easy preparation which involves simple reduction of the disulfide bonds in albumin and the alkylation of the disulfide bonds with maleimide nitroxides; this allows the binding of several nitroxides to albumin. We report here on the relaxivity of the nitroxide-albumin complex and its interaction with two cell lines.

EXPERIMENTAL PROCEDURES Materials. Human serum albumin (HSA, essentially fatty acid free), urea, chloroquine, and the nitroxide 3maleimido-2,2,5,5-tetramethylpyrrolidine-N-oxyl (Mal3) were purchased from Sigma Chemical Co. (St. Louis, MO). Cell culture media, serum, and trypsin were purchased from Gibco Laboratories (Grand Island, NY). Preparation of Nitroxide-Labeled Albumin. The * Author to whom correspondence should be addressed: procedure involved reduction and alkylation of the disDr. Harold M. Swartz, ESR Center at the University of ulfide bonds in albumin as described by Hunter and Illinois, 190 Medical Sciences Building, 506 S. Mathews, McDuffie (22) with some modification. The denaturing Urbana, IL 61801. agent urea was added to an albumin solution (17.5 mg/ College of Medicine. mL) to a final concentration of 8 M, and the pH was Bioacoustic Research Laboratory. adjusted to 8.2 with sodium carbonate. A solution of the Department of Biochemistry. reducing agent sodium thioglycolate (pH 8.2) then was Abbreviations: MRI and MRS, magnetic resonance imagadded to a final concentration of 0.3 M, and the flask ing and spectroscopy; ESR, electron spin resonance; HSA, human serum albumin; Mal-3,3-maleimido-2,2,5,5-tetramethylpyrroli- was immediately evacuated and filled with nitrogen. The mixture was left in the dark for 20 h at room temperadine-N-oxyl;TB cells, mixed cultures of thymus and bone marture. The mixture then was put in dialysis tubing and row; CHO, Chinese hamster ovary. 1043-1802/90/2901-0032$02.50/0 0 1990 American Chemical Society

Bioconjugate Chem., Vol. 1. No. 1, 1990

Nitroxide-Labeled Albumin

dialyzed against 1 L of phosphate-buffered saline (PBS, pH 8.4) for 4 h a t 4 "C with two changes of the buffer to eliminate the reducing agent. After dialysis the albumin solution was mixed with 10 mg of Mal-3, and the mixture was stirred for 24 h at 4 "C in a sealed vial flushed with nitrogen. The mixture was then dialyzed against PBS (pH 7.4) to remove unbound nitroxide. Magnetic Resonance Measurements. All electron spin resonance (ESR) measurements were obtained a t 9 GHz with a Varian E 109-E spectrometer equipped with a Varian gas flow temperature controller. The concentration of Mal-3 was determined by the double integration of ESR spectra with a Zenith 2-100 computer. The rate of reduction of nitroxide was calculated from the recorded initial linear decay of ESR signal intensity as a function of time. The spin-lattice relaxation time (TI) of the water protons were measured a t 20 MHz on a Bruker PC/20 Minispec operating at 37 "C, using an inversion recovery pulse sequence. UV Spectrophotometry. The concentration of HSA was measured on a 8415A diode-array spectrophotometer with the value of 6.0 for the specific absorption of a 1% solution through a path length of 1 cm (peak a t 280 nm). Spectra were taken a t wavelengths from 240 to 300 nm. Cell Cultures. T B cells were established from mixed cultures of thymus and bone marrow from CFW/D mice (23). They were grown in McCoy's medium with 10% serum in a 150-cm3flask. Chinese hamster ovary (CHO) cells were grown in suspension in a spinner flask (10' cells/mL) with the same medium. T o prepare samples for ESR measurements, IO' cells were transferred to 100mm petri dishes to grow as monolayers for 8 h, an aliquot of 5 mL of serum-free medium was substituted for the old medium a t the end of the 8 h, and 0.25 mL of the nitroxide-albumin complex (1 mM Mal-3) was then added, and the samples were incubated for various periods of time a t 37 "C. The treatment of cells with chloroquine was carried out by adding the agent (0.5 mM final concentration) 30 min prior to the addition of the nitroxide-albumin complex. After incubation, the cells were washed three times with PBS (pH 7.4). The cells were then exposed to 1 mL of 0.25% trypsin for 1 min. A 5-mL volume of medium with 10% serum was added to the trypsinized cells and the cell suspension was centrifuged a t 500g for 5 min. The pelleted cells were transferred into gas-permeable Teflon tubing for ESR measurements. For measurements of relaxation times, 1.5 X lo8 CHO cells per sample were taken from the spinner flask, and the pelleted cells were packed in a 7-mm NMR tube. A 0.2-mL sample of the nitroxide-albumin complex was added to make up the total volume of 0.6 mL. Protein Digestion by Trypsin. The NMR sample was prepared by adding 0.2 mL of 0.25% trypsin to 0.4 mL of the nitroxide-albumin complex (0.17 mM of albumin) in a 7-mm NMR tube. A 70 pL aliquot of the mixture was drawn into a glass capillary tube for study by ESR. The process of digestion a t 37 "C was monitored by observing changes in the line widths of the spectra as a function of time. RESULTS ESR Characterizationof the Complex. After exhaustive dialysis, the nitroxide-albumin complex had a broader and more complex ESR spectrum than the isotropic narrow spectrum of the free nitroxide (Figure l),indicating that most of the nitroxides were motionally restricted due to binding to albumin. The concentration of bound

33

Figure 1. Binding of Mal-3 to albumin. The ESR spectrum

of maleimide nitroxide (for structure see insert) in aqueous solution (A) and the ESR spectrum of Mal-3 covalently bound to albumin (B) are shown.

-.

I

\

Wavelength (nm)

Figure 2. UV absorption of native albumin (-) and the albumin-nitroxide complex (- -). The UV spectra indicate that there were structural changes in the albumin after reduction and alkylation of the disulfide bonds. The protein concentration for the two samples was indentical.

nitroxide was 1.1 mM as measured by double integration of the ESR spectrum. In a control experiment, albumin was mixed with Mal-3 without reduction; these samples had ESR spectra indicating that a much lower proportion of Mal-3 was bound to albumin. UV Characterization of the Complex. The protein concentration of the complex was 0.17 mM as determined by UV absorbance at 280 nm. The combined results of UV and ESR indicated that an average of six molecules of nitroxide were bound to each molecule of albumin. The altered UV spectrum of the complex (Figure 2) probably results from the unfolding of the polypeptide chains and the addition of nitroxides to albumin. TIMeasurements. The spin-lattice times (T,)of Mal3 and albumin-bound Mal-3 were measured and compared. The relaxivity (mM-' 9-l) of the albumin-bound Mal-3, calculated by subtracting out the diamagnetic contribution of the protein and dividing by the concentration of the bound Mal-3 (over a range of concentrations, 0.1-5 mM), is 1.38 mM-' s-'. This value is about 4 times higher than the relaxivity of Mal-3 alone, 0.38 mM-' s-'.

34

Chan et al.

Bioconjugate Chem., Vol. 1, No. 1, 1990

Figure 3. Interaction of the albumin-nitroxide complex with cells. The ESR spectrum of the albumin-nitroxide complex after incubation for more than 4 h with CHO cells (A) shows more isotropic motion than the ESR spectrum of the same nitroxide after incorporation into cells to which chloroquine has been introduced (B).

Interaction with Cells. The interaction of the nitroxide-albumin complex with cells was followed by both ESR spectroscopy and NMR relaxation measurements. The line width of the ESR spectrum of the nitroxide-albumin complex after incubation (2 h or longer) with either T B or CHO cell became narrower (Figure 3A), indicating greater motion of the nitroxide. This did not occur if the cells were treated with chloroquine (Figure 3B), an agent that inhibits proteolysis by raising the pH of lysosomes (24,30,31),indicating that the change in the spectrum of the nitroxide-albumin observed in untreated cells (Figure 3A) was due to digestion of the protein by the cells. The relaxation measurements were consistent with the interpretation, showing a 40% decrease in the relaxivity of the nitroxide-albumin complex after a 3-h incubation with CHO cells. Digestion by Trypsin. In order to confirm that proteolysis could account for the changes described above, we studied the effect of the proteolytic enzyme trypsin (25). After the digestion of the nitroxide-albumin complex by trypsin, the ESR spectrum (Figure 4C) was similar to that of free Mal-3 (Figure 1A). The relaxivity of the digested complex was 0.58 mM-' s-l, compared to 1.38 mM-' s-l for the undigested complex. Reduction of Mal-3 by TB and CHO Cells. The reduction of albumin-bound Mal-3 by T B and CHO cells was studied in parallel to the study of the reduction of the free nitroxide (Table I). In both types of cells, free Mal-3 was reduced more rapidly than the albuminbound nitroxide, and the rate of reduction of both free and bound Mal-3 increased under hypoxic conditions. DISCUSSION

The relaxivity observed for the nitroxidealbumin complex is a factor of 4 greater than that for free Mal-3. Enhancement of relaxation due to binding of the paramagnetic species to albumin has been observed previously and the physical basis for such enhancement has been described ( 2 , 4 ,10, 26). The effect is attributed to

Figure 4. Effect of proteolysis (by trypsin) on ESR spectra of the albumin-nitroxide complex. The process is indicated by changes (decreases) in line width as a function of time of incubation: 1 min (A), 5 min (B), and 25 min (C). The receiver gains for each of the three spectra are indicated in the figure.

Table I. Reduction of Free and Albumin-Bound Mal-3 by TB and CHO Cellss HSA-Mal-3

free

TB CHO

air 23.0f 2.3 6.0f 2.0

N,

air

N,

35.3 f 0.4 11.7 f 3.4

0.5 f 0.4 0.4 f 0.2

1.5 f 0.5 0.7 f 0.1

Rates are initial rates in units of lo6 molecules/min per cell. The results are given as mean f SD from four independent measurements

the changes in correlation times, especially the rotational correlation time of the nitroxide. Binding free nitroxides to macromolecules such as albumin results in restricted motion or slower tumbling rate of the nitroxides. It has been demonstrated that the inner-sphere component, which is negligible for the rapidly tumbling, uncomplexed nitroxides, dominates the relaxivity of macromolecule-bound nitroxides ( 4 ) . In this case, the overall correlation time (t,) of the alumin-bound Mal-3 more closely approximates the time scale of the resonant (Larmor) frequency of water to give rise to more efficient proton relaxation. The relaxivity of 1.38 mM-' s-l for the nitroxide-albumin complex from the current study is fairly consistent with reported values (2, 4 ) . Water-soluble nitroxides previously have been shown to increase contrast in MRI and have been used to identify breaks in the blood-brain barrier (27) and to delineate renal structures ( 7 ) and tumors (28) in experimental animals. The albumin-nitroxide complex used in the current study has an enhanced relaxivity compared to free nitroxides and thus should be able to provide equivalent MRI contrast with a lower injected dose. Toxicity studies of nitroxides indicate relatively high LD,, doses in rats (15-25 mmol/kg) and an apparent lack of induc-

Bioconjugate Chem., Vol. 1, No. 1, 1990 35

Nitroxide-Labeled Albumin tion of mutations in CHO cells (29). However, the toxicity of this albumin-nitroxide complex has not yet been evaluated. The changes in ESR spectra and relaxation measurements over time in cell suspensions incubated with the albumin-nitroxide complex indicate that the complex can be digested, resulting in decreased relaxivity. This phenomenon might be exploited to extend MRS techniques to follow metabolic processes associated with cellular uptake and digestion of proteins. Similarly, the observation that the relaxivity of the albumin-nitroxide complex changes during the course of protein digestion raises the possibility of using such complexes as metabolically responsive, contrast-enhancing agents for detecting various pathological conditions, such as disorders of renal function, under which the rate of protein turn over is affected (32, 33). Rapid metabolic conversion in vivo of nitroxides to nonparamagnetic states has been considered a disadvantage of nitroxides as MRI contrast agents. However, the finding that the rate of reduction of nitroxides responds to the metabolic state of the cells has led to the notion of using nitroxides as metabolically responsive MRI contrast agents (15, 16). The results from this study also indicate that the oxygen-dependent cellular reduction of the albumin-nitroxide complex could be exploited to provide contrast for pathophysiological hypoxic areas such as ischemic and neoplastic regions. Biodistribution studies in rats have shown that albumin-metal chelate complexes remain largely in the intravascular space after iv injection (12, 13). Similar studies of t h e albumin-nitroxide complex must be performed. The potential antigenicity of this complex (an altered form of albumin) could be a drawback to in vivo use of the conjugate, and therefore the antigenicity as well as the toxicity and excretion rate of the complex should be carefully assessed. If these studies show that the albumin-nitroxide complex is safe and remains in the blood pool, it could be used in MR assessments of blood volume a n d tissue perfusion and diagnosis of ischemic and neoplastic disorders. In addition, the ability of albumin to bind antibodies and specific receptors (14) could be exploited to make such complexes targetspecific contrast agents. Aggregation of albumin molecules into entities such as microspheres drastically changes their biodistribution; they are cleared rapidly from blood after iv injection and concentrated in the reticuloendothelial system (liver, spleen, and bone marrow) (34),and thus they also could be used to study the reticuloendothelial system. ACKNOWLEDGMENT We thank Maoxin Wu for preparing the cell cultures. This work was supported by NIH Grants CA 42388 and CA 40665. Hsiao Chang Chan received support from NIH Training Grant CA 09067. The ESR data were obtained at the University of Illinois ESR Research Center, supported by NIH Grant RR 01811. LITERATURE CITED (1) Stone, T. J., Buckman, T., Nordio, P. L., and McConnell, H. M. (1965) Spin-labeled biomolecules. Proc. Natl. Acad. Sci. U.S.A. 54, 1010-1017. (2) Polnaszek, C. F., and Bryant, R. G. (1984) Nitroxide rad-

ical induced solvent proton relaxation: measurement of localized translational diffusion. J. Chem. Phys. 81, 4038-4045. (3) Sosnovsky, G., Rao, N. U. M., Lukszo, J., and Brasch, R. C. (1986) Spin labeled bovine serum albumin, spin labeled

bovine serum albumin chelating agents and their gadolinium complexes. Z. Naturforsch 41b, 1170-1177. (4) Bennett, H. F., Brown, R. D., 111, Koenig, S. H., and Swartz, H. M. (1987) Effects of nitroxides on the magnetic field and temperature dependence of 1/T, of solvent water protons. Magn. Reson. Med. 4, 93-111. (5) Slane, J. M. K., Lai, C. S., and Hyde, J. S. (1986) A proton relaxation enhancement investigation of the binding of fatty acid spin labels to human serum albumin. Magn. Reson. Med. 3,699-706. (6) Brasch, R. C. (1983) Work in progress: methods of contrast enhancement for NMR imaging and potential applications. Radiology 147, 781-788. (7) Brasch, R. C., London, D. A,, Wesbey, G. E., Tozer, T., Nitecki, D., Williams, R., Doemeny, J., Tuck, L., and Lallemand, D. (1983) Work in progress: nuclear magnetic resonance study of a paramagnetic nitroxide contrast agent for enhancement of renal structures in experimental animals. Radiology 147, 773-779. (8) Wolf, G. L., Burnett, K. R., Goldstein, E. J., and Joseph, P. M. (1985) Contrst Agents for Magnetic Resonance Imaging. In Magn. Resonance Annual pp 231-266, Raven Press, New York. (9) Kang, Y. S., Gore, J. C., and Armitage, I. M. (1984) Studies of factors affecting the design of NMR contrast agents: manganese in blood as a model system. Magn. Reson. Med. 1,396-409. (10) Koenig, S. H., and Brown, R. D., I11 (1984) Relaxation of

solvent protons by paramagnetic ions and its dependence on magnetic field and chemical environment: implication for NMR imaging. Magn. Reson. Med. 1, 478-495. (11) Lauffer, R. B., and Brady, T. (1985) Preparation and water relaxation properties of proteins labeled with paramagnetic metal chelates. Magn. Reson. Zmaging 3, 11-16. (12) Ogan, M. D., Schmiedl, U., Moseley, M. E., Grodd, W., Paajanen, H., and Brasch, R. C. (1987) Albumin labeled with Gd-DTPA, an intravascularcontrast-enhancing agent for magnetic resonance blood pool imaging: preparation and characterization. Invest. Radiol. 22, 665-671. 3) Schmiedl, U., Ogan, M., Paajanen, H., Marotti, M., Crook, L. E., Brito, A. C., and Brasch, R. C. (1987) Albumin labeled with Gd-DTPA as an intravascular,blood pool-enhancingagent for NMRimaging: biodistribution and imagingstudies. Radiology 162, 205-210. 4) Peter, T. (1975) Serum Albumin. In The Plasma Proteins (F. w. Putnam, Ed.) pp 133-181, Academic Press, New

York.

(15) Swartz, H. M., Chen, K., Pals, M., Sentjurc, M., and Morse, P. D., I1 (1986) Hypoxia sensitive NMR contrast agents. Magn. Reson. Med. 3,169-174. (16) Chen, K., and Swartz, H. M. (1988) Oxidation of hydrox-

ylamines to nitroxides spin labels in living cells. Biochim. Biophys. Acta 970,270-277. (17) Swartz, H. M. (1989) Metabolically Responsive Contrast Agents. In Advances in Magnetic Resonance Imaging (E. Feig, Ed.) pp 49-71, Ablex Publishing Company, New Jersey. (18) Griffith, 0. H., and McConnell, H. M. (1966) A nitroxidemaleimide spin label. Proc. Natl. Acad. Sci. U.S.A. 55, 8-

11. (19) Benga, G., and Strach, S. J. (1975) Interaction of the elec-

tron spin resonance spectra of nitroxide-maleimide-labeled proteins and the use of this technique in the study of albumin and biomembranes. Biochim. Biophys. Acta 400, 6979.

(20) Cornell, C. N., and Kaplan, L. J. (1978) Spin-labeled stud-

ies of the sulfhydryl environment in bovine plasma albumin. 1. The N-F transition and acid expansion. Biochemistry

17,1750-1754. (21) Berliner, L. J., Ed. (1976) Spin Labeling. Theory and

Applications Academic Press, New York. (22) Hunter, M. J., and McDuffie,F. C. (1959) Molecular weight

studies on human serum albumin after reduction and alky81, 1400-1406. lation of disulfide bonds. J. Am. Chem. SOC.

Bioconjugate Chem. 1990, 7, 36-50

36

(23) Ball, J. K., Huh, T. Y., and McCarter, J. A. (1959) On the

distribution of epidermal papillomata in mice. Br. J. Cancer 18, 120-123. (24) Ohkuma, S.,and Poole, B. (1978)Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc. Natl. Acad. Sci. U.S.A. 75, 3327-3331. (25) Blackburn, S. (1976)Enzyme Structure and Function pp 105-141, Marcel Dekker, New York. (26) Wien, R. W., Morrisett, J. D., and McConnell, H. M. (1972) Spin-labeled-inducednuclear relaxation. Distances between bound saccharides, histidine-15, and tryptophan-123 on lysozyme in solution. Biochemistry 11, 3707-3716. (27) Brasch, R. C., Nitecki, D. E., Brant-Zawzdzki,M., Enzamann, D. R., Wesby, G. E., Tozer, T. N., Tuck, L. D., Cann, C. E., Fike, J. R., and Sheldon, P. (1983) Brain nuclear magnetic resonance imaging enhanced by a paramagnetic nitroxide contrast agent: preliminary report. Am. J . Radiol. 141, 1019-1023. (28) Ehman, R. L., Wesbey, G. E., Moon, K. L., Williams, R. D., McNamara, M. T., Couet, W. R., Tozer, T. N., and Brasch, R. C. (1985) Enhanced MRI of tumors utilizing a new

nitroxyl spin label contrast agent. Magn. Reson. Imaging 3, 89-97. (29) Afzal, V., Brasch, R. C., Nitecki, D. E., and Wolff, S. (1984) Nitroxyl spin label contrast enhancers for magnetic resonance imaging: studies of acute toxicity and mutagenesis. Znuest. Radiol. 19, 549-552. (30) Segal, H. L., and Doyle, D. J., Eds. (1978) Protein Turn-

over and Lysosome Function Academic Press, New York.

(31) Hershko, A., and Tomkins, G. (1971) Studies on the deg radation of tyrosine aminotransferase in hepatoma cells in culture. J . Biol. Chem. 246, 710-714.

(32) Schimke, R. T., and Doyle, D. (1970) Control of enzyme levels in animal tissues. Annu. Reu. Biochem. 39, 929-976. (33) Strober, W., Mogielnicki,R. P., and Waldmann, T. A. (1972) The Role of the Kidney in the Metabolism of Serum Proteins. Protein Turnover pp 25-41, Elsevier, Amsterdam. (34) Widder, D. J., Greif, W. L., Widder, K. J., Edelman, R. R., and Brady, T. J. (1987) Magnetite albumin microspheres: a new MR contrast material. Am. J. Radiol. 148, 339-404. Registry No. Trypsin, 9002-07-7.

Site-Directed Chemical Modification and Cross-Linking of a Monoclonal Antibody Using Equilibrium Transfer Alkylating Cross-Link Reagents Frederick A. Liberatore,'vt Robert D. Comeau,t James M. McKearin,t Daniel A. Pearson,t Benjamin Q. Belonfa III,t Stephen J. Brocchini,* John Kath,$ Terri Phillips,$ Kira Oswell,§ and Richard G. Lawton A Medical Products Department, E. I. du Pont de Nemours and Company, 331 Treble Cove Road, North Billerica, Massachusetts 01862, and Department of Chemistry, University of Michigan, 2525 Chemistry Building, Ann Arbor, Michigan 48109. Received July 21, 1989

A new, more reactive group of protein cross-linkers in the class of equilibrium transfer alkylating cross-link (ETAC) reagents has been synthesized. These compounds include a,a-bis[ (p-chlorophenyl)methyl]- and a,a-bis[ (p-tolylsulfonyl)methyl]acetophenonessubstituted in the acetophenone ring with chloro, nitro, amino, and carboxyl groups and derivatives. Included are an '251-labeled ETAC reagent and a "'In-labeled DTPA (diethylenetriaminepentaaceticacid) ETAC for site direction and biodistribution studies. These ETAC compounds were reacted with unreduced and partially reduced antibody under mild pH (pH 4-8) and room temperature conditions to give cross-linked structures. Examination of resultant cross-linked antibody via size-exclusion HPLC, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, and an enzyme linked immunosorbent assay revealed that (1) both interantibody as well as intraantibody cross-linking had occurred; (2) the level of inter- and intraantibody cross-linking varied with the substituent on the ETAC; (3) the stability of the crosslinks on the reducing SDS gels varied with substituents on the ETAC; (4) little if any immunoreactivity was lost after reaction with one of the more effective ETAC cross-linking compounds; (5) the '251-labeled ETAC sulfhydryl cross-linking in partially reduced antibody increased with p H whereas amine cross-linking with the unreduced antibody decreased with pH; (6) the optimum pH for sulfhydryl site direction was p H 5.0; (7) the "'In DTPA ETAC labeled antibody had a biodistribution in CD1 mice similar to that of the "'In bis cyclic anhydride DTPA labeled antibody.

The only chemical method that allows the identification of the tertiary and quaternary structure of proteins involves the use of organic hetero- and homobifunctional cross-linking reagents (1-4). These same reagents can be used to join similar or dissimilar proteins together ~

~

~~~~~~

~~

~

E. I. du Pont de Nemours and Co. University of Michigan. University of Michigan Undergraduate Research Participants.

*

or to cross-link smaller molecules to a protein (5-8). Virtually all bifunctional cross-links described are believed to covalently couple to reactive functions through links that are nonreversible. However, one class of crosslinkers has been described whose members have the potential ability to transfer from the initial site of protein attachment to another reactive site to form a (relatively) more stable bond. These types of compounds have been given the acronym ETAC for equilibrium transfer alkylating cross-link (9).The original ETAC reagents consisted of

1043-1 802/90/2901-0036$02.50/0 0 1990 American Chemical Society