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I t allows a determination over distances of meters and in a rather small sample volume which, in principle, is limited only by the size of the fiber. It therefore provides a suitable means for invasive detection and determination of enzyme activities. Since assays can be performed in vivo, real-time probing becomes possible. The fluorescent dye, which, conceivably, may be physiologically harmful, remains in the immobilized state so that it does not present a risk in in vivo sensing. The acid part, in contrast, is released, but this is the “natural” part of the synthetic substrate, for instance, a fatty acid anion, phosphate, sulfate, or a sugar molecule such as glucose. Because the substrate is irreversibly consumed, the probe has a limited lifetime. Consequently, it does not lend itself to continuous monitoring, which is in contrast to known invasive sensors for pH and oxygen (9). Therefore, the device cannot be called a true sensor which, by definition, is able to indicate continuously and reversibly a chemical or physical parameter. The probe works irreversibly, so that its calibration may be more difficult than in the case of reversible sensors. Since, however, a single membrane can be used for several short-time measurements (with gradual degradation in performance as the background from hydrolyzed HPTS increases), calibration appears feasible by making an initial measurement using an enzyme solution of defined activity. If separate membranes are to be used, one has to make sure that each sensor of a lot responds in the same way. This new principle has been shown to work with carboxylesterases in this paper. Numerous other chromgenic or
fluorogenic substrates are known ( I ) . Once they can be immobilized on a solid support and provided their substrate properties are conserved, they appear to be applicable for in vivo sensing of the respective enzymes via the fiber-optic approach presented here. Registry No. HPTS (acetate),85353-19-1;HPTS (butyrate), 85353-20-4; carboxylesterase, 9016-18-6.
LITERATURE CITED Guilbault G. G. Enzymatic Methods of Analysis ; Pergamon Press: Oxford. 1970. Methods of Enzymatic Analysis; Bergmeyer, H. U., Ed.: Verlag Chemie: Weinheim, Deerfield Beach, 1984. Walter B. Anal. Chern. 1983, 55, 498A-512A. Wolfbeis 0. S.; Koller E. Anal. Blochem. 1983, 129,365-370. Koller E.; Wolfbeis 0. S. Anal. Biochern. 1984, 143, 146-151. Wolfbeis 0. S. Trends Anal. Chem. 1985, 4, 184-188. Peterson J. I.; Goldstein S. R.; Fitzgerald R. V.; Buckhold D. K. Anal. Chern. 1980, 52, 864-869. Peterson J. I.; Fitzgerald R. V.; Buckhold D. K. Anal. Chem. 1984, 56, 62-67. Gehrich J. L.; Lubbers D. W.; Opitz. N.; Hansmann, D. R.; Miller, W. W.; Tusa, J. K.; Yafuso, M. IEEE Trans. Horned. Eng. 1986, 33, 117-132. Arnold, M. A. Anal. Chern. 1985, 57, 565-566. ZhuJun, Z.; Seitr, W. R. Anal. Chirn. Acta 1984, 160, 47-55. Wolfbeis, 0. S.; Furiinger, E.; Kroneis, K.; Marsoner, H. Z. Anal. Ch8m. 1983, 314, 119-124.
Otto S. Wolfbeis Analytical Division Institute of Organic Chemistry Karl Franzens University A-8010 Graz, Austria RECEIVED for review May 6, 1986. Accepted July 7, 1986.
Photothermal Quantitation of Nanogram Quantities of Coomassie Brilliant Blue Stained Proteins in Denaturing Polyacrylamide Gels Sir: Photothermal spectroscopies are a class of ultrasensitive indirect absorption measurements, in which the heat evolution accompanying light absorption is measured. The techniques all measure the local changes in sample refractive index caused by the heat evolution (I-3), as the expansion, deflection, or refraction of a laser beam. Photothermal techniques have been shown to provide superior sensitivity to direct absorption measurements in liquid chromatography (4-6)and thin-layer chromatography (7,8). In the crossed beam thermal lens experiment, a pump/probe configuration is used with the two lasers crossed, rather than collinear. This configuration is simpler to align than the more familiar collinear laser version. In a preliminary report we demonstrated that crossed beam thermal lens spectroscopy can be used to quantitate bovine serum albumin stained by Coomassie Brilliant Blue G250 in nondissociating polyacrylamide gel electrophoresis (9). We obtained detection limits of 1ng using He-Ne laser radiation to generate a thermal lens. The detection limits are about 2 orders of magnitude lower than the visual limit of detection in the same system. For separation purposes, nondissociating electrophoresis is often adequate. However, the characterization of a new protein often begins with an electrophoretic estimate of molecular weight using polyacrylamide gel electrophoresis of a denatured protein. The electrophoretic mobility of a protein depends on its net charge, molecular weight, shape, and the
rigidity of packing of the polypeptide chain. The contribution of these factors varies considerably with experimental conditions. Polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS), the Laemmli procedure (IO), allows elimination of all the factors except molecular weight. In the present communication we apply the photothermal detection technique to the Laemmli SDS-polyacrylamide gel procedure. We describe experimental problems in the use of photothermal detection with SDS-polyacrylamide gel electrophoresis and p r o p e solutions to them. The detergent SDS causes problems in fixation, staining, and quantitation of proteins with dyes (11). Therefore, we compare the performance of Coomassie Brilliant Blue R250 and G250 in the presence of SDS in this application.
EXPERIMENTAL SECTION Polyacrylamide gel electrophoresis was performed by Laemmli’s dissociating discontinuous method (SDS-PAGE). All electrophoresis reagents were obtained from Sigma. Acrylamide and N,”-methylenebis(acry1amide) were recrystallized by Loening’s method (12). Other materials were used without further purification. Low molecular weight protein standards (BioRad), phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, and lysozyme were used to evaluate the procedures. Polyacrylamide gels (10% acrylamide) were cast in 1 cm thick slabs. The protein samples were loaded onto the wells 10 mm
0003-2700/86/0358-2676$01.50/0 0 1986 American Chemical Society
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Flgure 1. Absorption spectra of Coomassie Brilliant Blue R250 and G250 dyes in polyacrylamide gel with and without bound bovine serum albumin: (A) Coomassie Brilliant Blue R250; (B) Coomassie Brilliant Blue R250 bound to albumin; (C)Coomassie Brllllant Blue G250 bound
to albumin. in width. The glass plates used as forms were acid washed and wiped with acetone and methanol before use. All solutions were prepared in deionized water and filtered through Millipore 0.45-pm membrane filters before use. Gels were subjected to preelectrophoresis for 4 h before application of protein standards. Coomassie Brilliant Blue G250 dye was prepared in 3.5% perchloric acid solution (13). Coomassie Brilliant Blue R250 dye was prepared in 12.5% trichloroacetic acid solution (14) or in 50% methanol, 10% acetic acid solution (15). Electrophoresis was carried out at room temperature in a vertical Studier chamber (Markson). Power to the chamber was maintained constant at 8 W by an ISCO Model 494 power supply. A 1-pg protein standard was run in the track adjacent to each sample track. The 1-pg protein standards serve to locate the proteins in the adjacent channel because nanogram quantities used were not visible to the eye even after staining with Coomassie dyes. After electrophoresis the polyacrylamide gels were fixed and stained by gentle rocking overnight in Coomassie Brilliant Blue R250 or G250 solution. The Coomassie Brilliant Blue G250 stained gels were destained for 4 h in 5% acetic acid with several changes of solution. The gels stained by Coomassie Brilliant Blue R250 prepared in trichloroacetic acid were destained in 10% trichloroacetic acid overnight with several changes of solution. The gels stained by Coomassie Brilliant Blue R250 prepared in methanol required at least 24 h of destaining unless the solution was changed frequently. After destaining, gels were cut to size and mounted between 25 X 100 mm quartz plates in a locally constructed holder. Absorption spectra of stained gels were obtained with a Hewlett-Packard 8450A spectrophotometer. Crwed beam thermal lens measurements were carried out with the apparatus previously described (9). For most of these measurements the He-Ne laser was replaced with an argon ion laser (Lexel Model 85-1) operated at 528.7 nm with an output power of 50 mW at the laser head. The pump beam was modulated at 13 Hz. The pump and probe beams were crossed at a 5.7" angle. The gel holder was positioned to give the largest thermal lens signal. A diffusing plate was placed in front of the iris which defines the thermal lens signal to minimize possible interference fringes and scattered light from scratches and dust particles on the elements of the optical train. The lock-in amplifier time constant was maintained at 0.3 s. Band heights were used as measures of amount of proteins in a sample. Base lines were estimated by extrapolation of the background values immediately adjacent to each peak. Base line noise was calculated as the root mean square value of the fluctuations in the base line over 3-5 cm. Calibration curves were prepared by normalizing to the signal from the 1pg quantity in an adjacent track. Measurements were made on data as collected. For data display, noise was removed from data files by the Bussian
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Thermal lens signals from proteins stained by (A) Coomassie Brilliant Blue R250 and (B) Coomassie Brilliant Blue G250. Each graph shows both 70-ng and 10-ng quantities of proteins. Flgure 2.
and Hardle robust smooth (16), implemented on a personal computer. Curved base lines were removed by subtraction of a base line calculated by least-squares fit to a cubic equation using points from regions of the trace containing no major protein bands.
RESULTS AND DISCUSSION To define the most suitable of the easily available argon ion and helium-neon laser lines, we measured the visible absorption spectra of Coomassie Brilliant Blue G250 and R250 and their albumin complexes. Figure 1shows the absorption spectra of Coomassie Brilliant Blue R250 in polyacrylamide gel with and without bound bovine serum albumin. Also shown is the absorption spectrum of Coomassie Brilliant Blue G250 bound to bovine serum albumin. For the albumin complex of Coomassie Brilliant Blue R250 the absorptivity is slightly higher at 528.7 nm than at 632.8 nm. For the G250 albumin complex the absorptivity at 632.8 nm is about twice as large as a t 528.7 nm. The thermal lens signals of the complexes measured at these wavelengths were proportional to absorptivity, as expected. However, the ultimate detection limits (see below) were about the same a t either wavelength and were about the same with both dyes used. These observations suggest that detection limits are not limited by the magnitude of the thermal lens signal itself, but by the presence of some source of background noise, as described later. Figure 2 shows typical thermal lens traces for 70-ng and 10-ng quantities of proteins stained with Coomassie Brilliant Blue R250 and G250. Although the base lines appear noisy, they actually contain many reproducible small peaks caused by the presence of minor impurities or degradation products in the protein mixtures used. Some of the more prominent minor peaks are easily visible in the traces from 70-ng samples. Only four of the six proteins in the commercial preparation
ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1986
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Figure 3. (A) Photothermal signals from 10 ng of proteins. Signals are contaminated by noise from scratches or dust particles. (B) Noise spikes removed from data file by robust smooth method, wlth base line
correction applied. are shown in the figure. The limited scan range (50 mm) of our apparatus precluded measurement of all six proteins in a single experiment. However, the apparatus could easily be modified to include a larger gel holder and a translation stage with a longer travel. The data in Figure 2 have been subjected to software removal of noise spikes and a sloping base line by the procedures described above. Figure 3 shows the thermal lens trace for a 10-ng protein sample before and after software removal of noise spikes and base line correction. The Bussian and Hkdle robust smoothing procedure (16) was found to be an efficient method to remove the spikes and facilitate visual examination of thermal lens traces from samples containing only a few nanograms of protein. The spikes in the data arise from several sources. The staining/destaining and mounting procedures involve extensive manipulation of the gel. Minor damage to the gel occurs in the course of the workup. Small nicks and tears become centers for light scattering. The probe laser intensity is especially susceptible to noise from this source. In addition, the gel contains small quantities of solid impurities from the acrylamide itself, the staining dye, or even from the laboratory air. These particles also serve as scattering centers. After repeated use, the quartz plates used to confine the gel in the densitometer eventually become scratched. Reusable plates were necessary, since the gels are larger than standard microscope slides or any other mass-produced plates of high optical quality. The sloping base line was caused by slight misalignment of the gel holder with respect to the incident laser beams. The signals were optimized a t the second peak (BSA). Because the gel holder was not exactly perpendicular to the line bi-
secting the laser beams, a sloping base line was obtained as the gel position changed relative to the beam foci. This effect demonstrates the importance of positioning the gel holder correctly and reproducibly in the optical path. We examined two different Coomassie Brilliant Blue R250 staining solutions. The same results were obtained with Coomassie Brilliant Blue R250 dissolved in trichloroacetic acid and in methanol solutions. Gels stained with the methanolic solutions required more extensive destaining than gels stained with the trichloroacetic acid. However, gels were more fragile after immersion in the trichloroacetic acid solutions. Protein bands stained with Coomassie Brilliant Blue R250 faded after 2 or 3 days. All measurements were made on freshly stained samples. We found that the Coomassie Brilliant Blue G250 procedure gave results equivalent to those obtained with R250. Destaining in 5% acetic acid was necessary to enhance the sensitivity of G250 stains. To some extent, the great advantage of the Coomassie Brilliant Blue G250 staining method, rapid visualization of protein bands, was lost. Protein band intensities in G250 remained a t least for several weeks. By contrast, R250 stains faded in 2 to 3 days. With the thermal lens densitometer, we are able to detect trace amounts of proteins down to about 1 ng using argon ion 528.7-nm excitation. Both Coomassie Brilliant Blue R250 and G250 yield essentially the same detection limits for various proteins. The detection limits obtained by thermal lens densitometry for proteins stained with Coomassie Brilliant Blue R250 are 0.3 ng for phosphorylase B, 1.3 ng for bovine serum albumin, 0.8 ng for ovalbumin, and 0.5 ng for carbonic anhydrase. For proteins stained by Coomassie Brilliant Blue G250 the detection limits are 0.4 ng for phosphorylase B, 1.1 ng for bovine serum albumin, 1.1ng for ovalbumin, and 0.5 ng for carbonic anhydrase. These resulb confirm and extend our earlier observation of nanogram detection limit for G250 stained bovine serum albumin. The limit of visual sensitivity for bovine serum albumin stained with either Coomassie Brilliant Blue R250 or G250 has been reported by several groups to be around 100 ng (12,131. Our observations confirm these results. The wells in the polyacrylamide gel were cast to be 10 mm wide. But the focused laser beam diameter is 50-100 pm. Only a small fraction of each protein band contributes to the thermal lens signal. The absolute detection limit could be lowered by using narrower wells. It is also quite feasible to apply photothermal densitometry to ultrathin gel electrophoresis. In most ultrathin configurations, proteins are manipulated a t higher molar concentrations, so that smaller absolute amounts would be measured. In addition, looser focusing of the laser beam would average local concentration variations and might lead to improved precision in the measurements. However, this approach is limited by the requirements of the thermal lens effect itself for a sharply defined temperature gradient. Figure 4 shows calibration curves obtained with R250 and G250 stained proteins. The curves are linear over the 10-100 ng region but fall off quite steeply below 10 ng. The most probable explanation of this phenomenon is dissociation of the dye/protein complexes a t these low concentrations. Dissociation, if confirmed by further experiments, may set the ultimate detection limit of Coomassie stains at values not much below those which we have reached. Some improvement might be achieved by optimizing the amount of residual stain left in the gel. The dynamic range of the photothermal densitometer can easily be extended to larger quantities of proteins. Because of the nonlinear responses of the Coomassie Brilliant Blue dye and the saturation of thermal lens signals at high concen-
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power CW lasers. The wavelengths available from heliumcadmium, argon ion, and helium-neon lasers will cover almost all cases. In preliminary measurements we have confirmed that the thermal lens technique can be applied to proteins silverstained by the Merril (17) procedure. With this technique, photothermal densitometry may well be extended to low picogram quantities, but with the usual experimental complications accompanying silver staining. This possibility is under active investigation.
ACKNOWLEDGMENT We thank Robert Broman, ISCO, Inc., and Hugh Montgomery, the Anspec Co., Inc., for the loan of a power supply for this work.
_28
LITERATURE CITED I
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12
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20
40
80
80
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P U A N n N OF PROTEINS (no)
Figure 4. (A) Calibration curves for Coomassle Brilliant Blue R250 stains. (B) Callbration curves for Coomassie Brilliant Blue 0250 stains.
trations, constitutive or marker proteins will be needed as references. Photothermal densitometry is not limited to Coomassie Brilliant Blue stained proteins. Other protein stains are used in special cases and staining is used with nucleic acids (16). For example, Stains-All is often used for phosphoproteins, as well as RNA. The procedures described here should be equally applicable to any stain whose absorption spectrum includes the wavelengths of any of the commercially available low-
(1) Fang, H. L.; Swofford, R. L I n Ufirasensfilve Laser Spectroscopy; Kiiger, D. S., Eds.; Academic Press: New York, 1982; pp 175-232. (2) Harris, J. M.; Dovichi, N. J. Anal. Chem. 1980, 52, 695A-706A. (3) Morris, M. D.; Peck, K. Anal. Chem. 1986, 58, 811A-822A. (4) Sepaniak, M. J.; Vargo, J. D.; Kettier, C. N.; Maskarinec, M. P. Anal. Chem. 1984, 56, 1252-1257. (5) Leach, R. A.; Harris, J. M. J . Chromatogr. 1981, 278, 15-19. (6) Pang, T.-K. J.; Morris, M. D. Anal. Chem. 1984, 56, 1457-1459. (7) Masujlma, T.; Sharda, A. N.; Liyod, L. B.; Harris, J. M.; Eyring, E. M. Anal. Chem. 1984, 5 6 , 2977-2979. (8) Peck, K.; Fotiou, K. F.; Morris, M. D. Anal. Chem. 1885, 5 7 , 1359-1362. (9) Peck, K.; Morris, M. D. Anel. Chem. 1988, 5 8 , 506-507. (10) Laemmli, U. K. Nature (London) 1970, 227, 680-685. (11) Sedmak, J. J.; Grossberg, S. E. Anal. Biochem. 1977, 79, 544-552. (12) Loening, U. E. Blochem. J. 1987, 702,251-256. (13) Reisner, A. H.; Nemes, P.; Bucholtz, C. Anal. Blochem. 1975, 6 4 , 509-516. (14) Chrambach, A., Reisfeld, R. A., Wyckoff, M., Zaccari, J. Anal. 810chem. 1967, 20, 150-154. (15) Hames, B. D. I n GelNectrophoresis of Proteins; Hames, B. D., Rickwood, D., Eds.; IRL Press: Washington DC, 1981; pp 44-45. (16) Bussian, B.4.; Hardie, W. Appl. Spectrosc. 1984, 38, 309-313. (17) Switzer, R. C., 111; Merril, C. R.; Shifrin, S.Anal. Blochem. 1979, 98, 231-237.
Konan Peck Michael D. Morris* Department of Chemistry University of Michigan Ann Arbor, Michigan 48109 RECEIVED for review May 8, 1986. Accepted July 24, 1986. We thank the National Science Foundation for financial support through Grant CHE-8317861.
AIDS FOR ANALYTICAL CHEMISTS Simultaneous Determination of Free Homovanillic Acid, (3-Methoxy-4-hydroxypheny1)ethylene Glycol, and Vanllmandellc Acid in Human Plasma by High-Performance Liquid Chromatography Coupled with Dual-Electrode Coulometric Electrochemical Detection Greg A. Gerhardt,* Carla J. Drebing, and Robert Freedman' Department of Psychiatry, University of Colorado Health Sciences Center, Denver, Colorado 80262 The methoxylated metabolites of the catecholamine neurotransmitters in human plasma have received considerable Also D e p a r t m e n t of Pharmacology, U n i v e r s i t y of Colorado H e a l t h Sciences Center, Denver, CO 80262, and M e d i c a l Research, Veterans A d m i n i s t r a t i o n M e d i c a l Center, Denver, CO 80220.
attention as possible indexes of central nervous system catecholamine activity in recent studies of schizophrenia and affective disorders. The measurement of the dopamine metabolite homovanillic acid (HVA) has clearly shown some utility as a therapeutic marker for changes in dopaminergic activity with neuroleptic drug treatment (1-4). The use of
0003-2700/86/0358-2879$01.50/00 1986 American Chemical Society
