Anal. Chem. l S W , 85, 601-605
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Comparison of Covalent and Noncovalent Labeling with Near-Infrared Dyes for the High-Performance Liquid Chromatographic Determination of Human Serum Albumin Richard J. Williams, Malgorzata Lipowska, Gabor Patonay; and Lucjan Strekowski Department of Chemistry, Georgia State University, University Plaza, Atlanta, Georgia 30303
Noncovaknt and covalent methods of Iabellng proteln wlth near-lnfrared polymethlne cyanlne dyes were compared for use In analyzlng human serum albumln (HSA) by hlghperformance llquklchromatography(HPLC) wlth near-lnfrared absorbance detection. Whlle noncovalent Iabellngwas faster than coveknt Iabellng and took place In the phyrlologlcal pH range, covaknt Iabellng was more stable under condltlons encountered In many of the wMely used types of HPLC. Covalently labeled HSA proteln peak8 Indkated unlform Iabdlng of amino groups at both hydrophllk and hydrophobk blndlngsltes, whlle noncovalent Iabellngshowed a preference for hydrophoblc blndlng sltes.
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INTRODUCTION Many polymethine cyanine dyes absorb and fluoresce in the near-infrared region (600-1200nm). They typically have large molar absorptivities and good fluorescent properties. Semiconductor laser absorbance and semiconductor laser fluorimetry have been wed to detect polymethine cyanine dyes at ultratrace levels.'-4 Since interference from solvents and biomoleculesis minimal in the near-infrared (NIR)region, background detection limits are negligible in comparison to instrument detection limits.1.2s4For these reasons, the HPLC determination of protein with NIR polymethinecyaninedyes is characterized by a better detection sensitivity than conventional ultraviolet (UV)detection methods.2 Until now, only noncovalent labeling through electrostatic adsorption forces wing hydrophobic, nonfunctionalized polymethine dyes, such as indocyanine green (Figure 11, has been reportede5J6 While successful labeling and improved detection Author to whom correspondence should be addressed. (1) Imasaka, T.; Yoshitake, A,; Ishibashi, N. Anal. Chem. 1984, 56, 1077. (2) Ishibashi, N.; Imasaka, T.; Sauda, K. Anal. Chem. 1986,58,2649. (3) Imasaka, T;Tsakamoto, A,; Ishibashi, N. Anal. Chem. 1989, 61, 2285. (4) Imasaka, T.;Ishibashi, N. Anal. Chem. 1990, 62, 363A. (5) Ishibashi, N.;Nakagawa, H.; Okazaki,T.; Imasaka, T. Anal. Chem. 1990,62,2404. (6) Karush, F. Adv. Immunol. 1963, 2, 1. (7)Chromatography: Conceptsand Contrasts; Miller,J. M., Ed.;John Wiley and Sons, Inc.: New York, 1988. ( 8 )HPLC in Biochemistry; Henschen, A., Hupe, K., Lottapeich, F. Voelter W., Eds.; VCH Publishers: Weinheim (Federal Republic of Germany), 1985. (9) Strekowski, L.; Lipowska, M.; Patonay, G. J.Org. Chem. 1992,57, 4578.
(10) Strekowski, L.; Lipowska, M.;Patonay, G. Synth. Cornmun. 1992, 22, 2593. (11) Patonay, G.;Antoine, M. D.;Devanathan, S.; Strekowski, L. Appl. Spectrosc. 1991, 45, 451. (12) Antibodies: A Laboratory Manual; Harlow, E., Lane, D., Eds.; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1988. (13) Tsang, V. C. W.; Wilkins,P. J. Immunol.Methods 1991,138,291. (14) HorvBth, C.; Melander, W.; Molnb, I. J. Chromatogr. 1976,125, 129. (15) Brodersen, R. CRC Crit. Rev. Clin. Lab. Sci. 1980,ll 305. (16) Patonay, G.; Antoine, M. D. Anal. Chem. 1991,63, 321A. 0003-2700/93/0365-0601$04.00/0
sensitivity have been accomplished with noncovalent polymethine cyanine labels, the major drawback has been damage to size-exclusion columns caused by free unassociated dye.2 In this study, both noncovalent and covalent labeling methods were investigated in labeling human serum albumin (HSA) for HPLC determination. A polymethine NIR dye 1 (Figure 1) and its isothiocyanato-substitutedanalogues 2-6 were used as noncovalent and covalent labeling agents to label HSA. The isothiocyanato group is known to react selectively with amino groups of protein forming a stable thiourea bond.17.18 Labeling was monitored wing characteristic absorbance ratios at conditions known to affect the electrostatic adsorptionforcea responsible for the noncovalent binding of proteins,6 and the comparisons were reported. These conditions are frequentlyencounteredin different typea of HPLC analyses of various molecule^.^^^ Reversed-phase and size-exclusion HPLC were used to compare covalently and noncovalently labeled HSA determinations. A binding constant was calculated for the association of dye 1 and HSA (17) Jobbagy, A,; Kiraly, K. Biochim. Biophys. Acta 1966,123, 166. (18) Klugerman, M. J. Zmrnunol. 1965,95, 1165. 0 1993 American Chemical Society
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under reversed-phase conditions to emphasize the wide changes in distribution of the relative amounta of the associated and free dye. EXPERIMENTAL SECTION The HSA used in this study was obtained from the Sigma Chemical Co. (St. Louis, MO). The sodium carbonates, phosphates, and chloride were obtained from the Mallinckrodt Chemical Co. (St.Louis, MO). 2-Amino-2-(hydroxymethyl)-l,3propanediol (Tris) and urea were obtained from the Schwartzl Mann Co. (Orangeburg,NY). 2-Morpholinoethanesulfonicacid (MES) and 2- [4-(2-hydroxyethyl)piperazino]ethanesulfonic acid (HEPES),sodium salts, were purchased from the CalbiochemBehring Co. (La Jolla, CA). The taurocholic acid (TCA) was purchased from Eastman Fine Chemicals (Rochester, NY). The methanol was purchased from the Aldrich Chemical Co. (Milwaukee, WI). The synthesis and purity of dyes 1-6 have been reported in previous literature.+ll The HPLC analyses were performed on an SSI 401 system that included an SSI 232 gradient mixer and an SSI 500 variablewavelength UV-vis detector with an SSI 501 fixed wavelength detector connected in sequence for simultaneous detection at different wavelengths. The column system used for reversedphase analysis consisted of a C8 column (5 cm X 4.6 mm) from Rainin packed with Microsorb of 3-pm particle size. The column system used for size-exclusion analysis (25 cm X 4.6 mm) was obtained from Synchrom, Inc. It was packed with GPC300 and preceded by a 5-cm guard column. The size-exclusion short columns used for buffer exchanges were 5-cm PD-10's packed with Sephadex G-25M. They were obtained from Pharmacia LKB BiotechnologyAB (Uppsala, Sweden). UV-vis absorbances and absorbance spectra were obtained on a Beckman scanning UV-vis spectrophotometer. Noncovalent Labeling of HSA. The utility of noncovalent labeling was studied under different conditions. The most efficient noncovalent labeling pH condition was determined to be at pH = 6, in 0.1 M MES buffer. Labeling with dye 1 was complete after 10min. An 80-pL aliquot of 2 mg/mL HSA in 0.1 M carbonate buffer (pH = 9.5) was added to 4-mL samples of different pH buffers that contained 0.025 M citric acid 0.025 M MES, 0.025 M HEPES, 0.025 M TRIS, and 0.5 M NaC1. The buffers had pH's that ranged from 4.5 to 8 in increments of 0.5. An 160-pL aliquot of 1mg/mL noncovalent dye in DMSO was added to each of the buffered HSA solutions to give a 1:100molar ratio of HSA to dye. After 10 min, unbound dye was removed by PD-10 size-exclusion short columns. The molecular size ratio of HSA to NIR dye (600:l) and the strong affinity of unbound dye for the packing of the PD-10 size-exclusion columns suggest only HSA labeled with dye was eluted. Under equilibrium conditions, the noncovalent dye is either associated with HSA or the Sephadex G-25M packing of the PD-10 size-exclusion columns. This was confirmed by UV-vis absorbance analysis of the eluted product. The eluted product contained absorbance maxima at both 275 and 783nm, indicatingthe presence of labeled HSA. UV-vis absorbances characteristicof bound dye (783 nm) and total protein (280 nm, 216 nm) were plotted over the given pH range along with the absorbance ratio of bound dye to total protein, A783/A216* Covalent Labeling of HSA. The covalent labeling of HSA was adapted from the antibody labeling procedure of Riggs et al.12 and involves the formation of a thiourea linkage in the reaction between the isothiocyanate functional group of dyes 2-6 and the amino groups of HSA. Various aliquots of a stock solution of the covalent analogues in DMSO and water (1mg/ mL) were slowly added to a solution of 2 mg of HSA in 1mL of a carbonate buffer, pH = 9.5, and allowed to react in the absence of light for 4 h. The mixtures were quenched by adding 300 pL of 0.1 M ammonium chloride. Under these conditions, the isothiocyanate group of the unbound dye reacts rapidly with ammonia to give a thiourea derivative. The derivatized dye was then removed by PD-10 size-exclusion short columns. The effectsof pH on the hydrolytic degradation of the thiourea linkage over time were studied by equilibrating equal amounts of conjugated HSA into samples of different pH buffers that contained 0.5 M phosphate. A l-mL aliquot of covalentlylabeled
HSA was added to 3.5-mL samples of different pH buffers. The buffers had pHs that ranged from 3.5 to 9 in increment of 0.5. The solutions were passed through PD-10 columns every 15min up to 120 min. The absorbance ratio, A783/A216,was determined after each column separation. Comparison of Noncovalent and Covalent Labeling of HSA. Noncovalent labeling of HSA was compared to covalent labelingunder typical conditions frequently used in HPLC. These conditions were chosen because they demonstrate the effect of three main components of the electrostatic adsorption forces responsible for noncovalent binding.13 Using PD-10 short columns equal ahounts of noncovalently and covalently labeled HSA's were equilibrated into 0.1 M MES buffer, pH = 6, as a control; into 4.0 M MgzCl to study the effects of ionic strength; into 1% Triton-X detergent to study the effects of van der Waals and hydrogen bonding forces; into 8.0 M ureato study chaotropic effects; and into a mixture, 4.0 M MqCl, 8.0 M urea, and 1% Triton-X to study a maximum hydrophobic disruptive effect. Absorbances were measured at the characteristic wavelengthsof bound dye and free protein, and the absorbance ratio of A783/A216 was compared for each condition. Reversed-Phase HPLC Determination of Labeled HSA. Comparable amounts of noncovalently and covalently labeled HSA were detected on a C8 column, simultaneously at 280 and 780 nm. Two 10 mg/mL solutions of HSA in a 0.1 M carbonate buffer, pH = 9.5, were separately labeled with dyes 1 and 3-6 to give a HSA to dye molar ratio of 0.2. A sample containing 100 pL of each type of labeled HSA was injected at an initial mobilephase composition of 100% water, 5 mM TCA. The analysisrun took a total of 50 min with the first 30 rnin as a linear gradient with a final mobile phase composition of 100% methanol, 5 mM TCA. Fractions correspondingto each major chromatogrampeak were collected and analyzed to monitor the binding of dye to protein. A binding constant for the noncovalent association of HSA and dye 1 under these reversed-phase column conditions was determined. Chromatograms were obtained from column injections of 100pL from solutions with HSA to dye molar ratios of 5.0, 0.5, 0.2, 0.05, and 0.02. These chromatograms were integrated, and the peak areaswere used to determine the relative concentrations of complexed and free dye. These concentrations were then substituted into the Scatchard equation to calculate a binding constant for the given column conditions. Peak areas were also used to compare the extent of protein labeling for noncovalent and covalent labeling. Size-Exclusion HPLC Determination of Labeled HSA. Comparable amounts of noncovalently and covalently labeled HSA were detected on a GPC300 size-exclusion column, simultaneously at 254 and 780 nm. Labeled HSA was equilibrated into an 8.0 M urea solution using PD-10 size-exclusion short columns. Chromatograms were compared for column injections of 100pL of covalently and noncovalently labeled HSA at isocratic column conditions of pH = 7.0 in 0.2 M phosphate buffer. The analysis run took a total of 10 min.
RESULTS AND DISCUSSION The spectral absorption properties of the noncovalent dye used inthisstudyarevirtuallyidenticaltothoseofthecovalent dyes. All the dyes have molar absorptivities near 130 OOO and absorb near 780nm. Absorbance near800 nm is preferred in this case because this region corresponds well with the output of readily available semiconductor laser diodes.14 The scanning UV-vis spectrograph (Figure 2) compares the absorption spectra of free dye and dye bound to HSA. Bound dye has more than a 10-nm shift to a longer wavelength of 783 nm. The significant shift makes 783 nm a characteristic wavelength of HSA bound dye. Detection at 216 nm is preferred over detection at the conventional 280 nm because detection at 216 nm is a more sensitive measurement of protein content. The absorbance ratio, A783/A218,is a measure of the relationship between bound dye and total HSA and is used to monitor the degree of dye association with HSA. The dependence of noncovalent dye-HSA association and pH is shown (Figure 3) with a plot of A,83/Az16 vs pH. The
ANALYTICAL CHEMISTRY, VOL. 65, NO. 5, MARCH 1, 1093
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absorbance ratio of bound dye to total protein is a maximum and therefore mostly independent of pH at pH = 6. The most favorablepH for noncovalent association of dye to HSA is at pH = 6. Earlier work by Imasaka et al.l-ssuggests an NIR covalent label might be practical as a label for HPLC protein determination. When noncovalentlabeling of HSA was done with the NIR dye ICG, moderate amounts of unassociated ICG severely damaged the size-exclusion column. This damage is thought to be due to the extremely hydrophobic nature of ICG, along with the resonance stabilized positive charge on the indole nitrogen and the attraction to exposed silanol groups in the column packing. A covalent label should be more stable and specificas well as less damagingto columns with silica and hydrophobic packing. A comparison of noncovalent labeling of HSA to covalent labeling that used A7~3/A2~6 to monitor the degree of binding is shown (Figure 4). The conditions represented are frequently encountered in many types of HPLC analyses (size exclusion,ion exchange, affinity, etc.).788 Noncovalent and covalent labeling of HSA were compared under conditions of ionic strength, van der Waals and
hydrogen bonding, and chaotropic effects. In every case, the NIR label in covalently labeled HSA was found to be more stable than in noncovalently labeled HSA. Also, the use of a nonionic detergent, such as Triton-X, appears to minimize the damaging effects of free polymethine dyes on HPLC columns. The absorbance ratios for NIR dye labeled HSA treated with Triton-X are more than twice those observed for labeled HSA not treated with Triton-X. One explanation for this observation is the hydrophobic effects of the detergent are enough to overcome the polar interaction of the dyes with the column packing, thereby allowing more unbound dye to remain in solution with HSA. Hydrolytic degradation of the thiourea linkage had its maximum effect at pH = 4 over a 2-h period and was minimal at pH above 6 over the same time period (Figure 5). Chromatograms of covalently and noncovalently labeled HSA from reversed-phase HPLC are compared (Figure 6, Table I). When compared to chromatogramsof free dye, the chromatogram of covalently labeled HSA shows almost complete association of dye with HSA. Relative change in peak area percentage for the four covalent labels studied indicates a greater percentageof the covalent labels associates with HSA than in the case of the noncovalent label. One possible explantionfor this observation is that the solvophobic equilibrium forces that are necessary for reversed-phase separation14 produce a competition for the dyes between the hydrophobic packing surface and the hydrophobic binding
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ANALYTICAL CHEMISTRY, VOL. 85, NO. 5, MARCH 1, 1993
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Table I. Comparison of Percent Peak Area Change for Noncovalent and Covalent Labels after Conjugation to
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sites of the HSA molecule. This competition is more pronounced in the case of the noncovalent label because only noncovalent binding forces are involved. This also explains why chromatogrampeaks observed for noncovalently labeled HSA are larger than those for covalently labeled HSA. The nature of hydrophobic forces is such that a substantial amount of free unbound noncovalent dye associates with HSA and travels along with each HSA molecule as it passes through the column. With covalentlylabeled HSA, only dye molecules linked to HSA aminogroupsthrough the thioureabond remain associatedwiththe protein. The average value for the binding
constant determined from the Scatchard equation is 1.7 X lo6. The value of the binding constant demonstrates the preference of the dye for the hydrophobic packing of the column and ita weak affinity for HSA hydrophobic binding sites, as well as the transfer of dye molecules between binding sites. The amount of noncovalently labeled HSA should change as column conditions change. The covalent label is more stable and less affected by the conditions of reversedphase separation because of the highly stable amino moiety formed at the amino sites of HSA. The transfer of covalent dye molecules between the hydrophobic packing surface and the hydrophobicbinding sites of the HSA molecules does not occur as easily as with the noncovalent dye molecules. Earlier work has shown the affinity of noncovalent polymethine cyanine dyes for the hydrophobic cornponenta of human serum.2 Studies suggest HSA is divided into three domains with varying degrees of hydrophobicity, and combinations of these domains are responsible for HSA binding ~ites.~5J+-22 Chromatogramsof NIR-labeled HSA from sizeexclusion HPLC are compared (Figure7). The samples were treated with 8.0 M urea to partially unfold HSA. The chromatogram of noncovalently labeled HSA contains three distinct peaks of unequal peak height. The chromatogram of covalently labeled HSA contains four distinct peaks of virtually equal peak height. This confirms the expectation that the covalent label is specific for all parta of the protein containing exposed primary amine groups. The uneven peak heights of the noncovalently labeled HSA are characteristic of hydrophobic affinity labeling with the largest peak (19) Brown, J. FASEB Fed. R o c . 1976,35, 2141. (20) Jacobsen, J. FEBS Lett. 1969,5, 112. (21) Minshetti,P.;Ruffner, D.; Kuang, W.; Dennison, 0.;Hawkins, J.; Beattie, W.; Dugaiczyk, A. J. Biol. Chem. 1986,261, 6747. (22) Carter, D.; Xiao-Min; Munson, S.;Twias, P.;Gernert, K.; Brown, M.; Miller, T. Science 1989,244, 1195.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 5, MARCH 1, 1993
representing the most hydrophobic component of HSAS2
CONCLUSION The limitations of the conditions for noncovalent HSA labeling were investigated. At those conditions, covalentand noncovalent labels were compared for their specificity and stability. HSA labeled with covalent near-infrared polymethine cyanine dyes were detected at 780 nm with a conventional UV-vis detector. The degree of improvement in the HPLC determination of HSA at 780 nm compared to the the 280-nm determination compared favorably to the results of Imasakaet obtainedusing a noncovalent label, but without the column damage reported. Covalent labeling of HSA is more stable and specific and can be used over a wide range of column conditions. The use of NIR polymethine cyanine dyes as labels offers many advantages over conventional UV-vis detection methods for ultratrace determination of proteins using HPLC. Until now, the major disadvantage was the lack of specificity of the NIR labels. Covalent labeling with functionalizedNIR dyes offers this specificity. The procedure for noncovalent labeling with NIR dyes, while not specific, is faster than covalent labeling, and takes place near physiological pH. Ita main advantage is that it affects the functional activity of
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labeled proteins to a lesser extent. On the other hand, the -NCS moiety of the covalent label might decrease the functional activity of the labeled protein if the functional activity is in close proximity to a primary amine residue. To further develop this NIR labeling technique, other types of functionalizedNIR labels which label in a timely fashion and at nondenaturing conditions are needed. Several functional groups may be used for this purpose, e.g., aldehyde,carboxylic acid, etc. When coupled with spectroscopic techniques that use semiconductor laser diodes as a light source, such as fluorimetry, selective covalent labeling can have wide applications in areas involving trace analyses of biomolecules including immunoassays,nucleic acid sequencing,and protein synthesis.
ACKNOWLEDGMENT This work was supported in part by a grant from the National Science Foundation (CHE-890466) and in part by a grant from the National Institutes of Health (Grant 1RO 1Al 28903-01A2). RECEIVED for review May 20, 1992. Revised manuscript received October 23,1992. Accepted November 19,1992.
