Anal. Chem. 1984, 56,305-307
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AIDS FOR ANALYTICAL CHEMISTS Densitometer with Tapered Optical Fibers D. Wayne Hughes’ and A. M. Harper*2
Department of Chemistry, University of Georgia, Athens, Georgia 30602 The use in electrophoresis of poly(acry1amide) and agarose gels for the separation of proteins has greatly facilitated the analysis of complex biological systems and has enormously advanced the knowledge of protein structure and chemical behavior ( I ) . Outgrowths of the basic electrophoretic method include isoelectric focusing, discontinuous electrophoresis, and two-dimensional electrophoresis (2); these modifications have vastly improved the resolution and scope of the basic separation. A large number of visualization methods are used for detecting separated proteins. Silver staining (3) for general protein analysis and activity staining (4)for specific enzyme visualization are comparably sensitive methods. Other general protein stains, such as Coomassie Brilliant Blue and Amido Black T (5) are less sensitive. Quantitation of protein bands after staining is a much less developed technology. Densitometry, the measurement of the absorption of radiation of a stained band, is a potentially feasible direct method of quantitation (6). Historically the use of densitometry has been almost exclusively limited to the scanning of gel slabs, cylinders, autoradiograms, and photographic negatives for the sole purpose of recording band positions. Development of instrumentation has therefore been geared toward increasing resolution, with little or no regard for quantitation. One approach for improving resolution has been to focus the source to an increasingly tiny pinpoint of light at the plane of the gel being scanned (7).An equally small pinpoint source would be imaged for this purpose. Another approach has been to enlarge the image of the scanned gel by means of a zoom lens; the projected image is then sampled by a narrow slit (8). Both of these approaches demand an intense light source to compensate for the reduction in source intensity imposed by the design. The use of filters of monochromators to select narrow wavelength bands is prohibited for reasons of sensitivity. The resultant dependence on intense white light has made quantitation unattainable. Calibration curves obtained with instruments of these design types are generally nonlinear over even small ranges of protein concentration (9); usually the results are explained by changes in protein-dye binding with respect to protein concentration. An alternative explanation proposed by our laboratory is poor adherence to Beer’s law due to the use of white light. More recently a demand for quantitative capability has stimulated instrumental development in other directions. A densitometer design incorporating a HeNe laser (LKB, Rockville, MD) with a narrow beam width and an output wavelength of 633 nm resolves test lines drawn 100 pm apart and gives linear response over 2 orders of magnitude of protein concentration. The single output wavelength feature, while a positive approach in one respect, restricts the system to analysis of protein stained with a blue dye; the brown silver ‘Present address: Department of Botany, University of Georgia, Athens, GA 30502. Present address: Departments of Pathology and Chemistry and Biomaterials Profiling Center, 391 S. Chipeta Way, Suite F, Research Park, Salt Lake City, UT 84108.
strain would be poorly measured at 633 nm. Optical fibers have been employed in another commercial instrument (Kontes, Vineland, NJ). Interchangeable fiber optic heads give a variety of beam widths. Phosphor-coated disks excited by a low-pressure Hg lamp provide a selection of ten peak wavelengths of excitation radiation with bandwidths ranging from 10 nm to 300 nm. While these approaches have considerably enhanced the feasibility of quantitation of protein bands in gel slabs and cylinders, problems still remain. The laser approach is relatively expensive and inflexible in terms of its output wavelength. The use of phosphor-coated disks as a radiation source improves the range of selectable wavelengths, but narrow bandwidths are not always attainable. While for some stains a narrow bandwidth may not be necessary, the absorption of a stain with a broad absorption wavelength range (e.g., the increasingly popular silver stain, which results in brown stains) must be measured by passing light of a narrow bandwidth. We report that the use of tapered optical fibers circumvents many of these problems in a simple prototype instrument. A normal tungsten source can be used in combination with a monochromator. Sensitivity does not suffer with wavelength selection, since the large end of the fiber optic samples a large source image 2 mm in diameter and effectively concentrates the light at the other end. The optical fiber tapers to a small end and the viniaturized source image is focused onto the gel, with a minimal loss in light intensity.
EXPERIMENTAL SECTION Materials. Creatine kinase (CK), the MM isoenzyme form, was purchased from Sigma Chemical Co. (St. Louis, MO) as Type I (C3755). Acrylamide, N,N-methylenebis (acrylamide) (bis), N,N,N’,N’-tetramethylenediamine(TEMED), and ammonium persulfate were purchased from BioRad Laboratories (Richmond, CA), as were the Teflon spacers and combs required for poly(acrylamide) gel construction. All other chemicals were Baker Reagent grade. The vertical electrophoresis rig was constructed by the machine shop (University of Georgia, Athens, GA), as was the densitometer module. The spectrophotometer was a GCA/McPherson Model 707 spectrophotometer system (Acton, MA). The tapered fiber optic assembly was constructed by Galileo Electro-Optics Corp. (Sturbridge, MA). Electrophoresis. Separation of CK isoenzymes was done by the method of Smith (IO) except that 0.1 M borate/boric acid buffer, 1 mM Na2EDTA, and 1mM dithiothreitol were used as the running buffer. Water at 4 OC was circulated in the electrophoresis rig during application of 13 V/cm. Volumes of 3.0 wL sample containing CK at concentrations ranging from 0.01 to 1.0 mg/mL were applied to sample wells formed during the polymerization. Proteins migrated toward the anode under these conditions, and separation was achieved in 2 h. Gels were 0.75 mm thick. Staining. Two staining methods were used. For general protein staining, silver staining was done by the method of Oakley ( I I ) , except 0.5% potassium ferricyanidein distilled H20was used to soak the gels for 5 min after fixation. The brownish stained bands, with essentially no background, were scanned at 505 nm, as determined by adsorption spectra. For specific CK activity staining a method modified from Smith (10) and Yue (12) was used. All materials were from Sigma Chemical Co. Glucose
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6-phosphate dehydrogenase (G7878) was added to the acrylamide monomer solution a t a concentration of 0.1 mg/mL just prior to polymerization. Polymerization causes covalent immobilization of the enzyme (13) onto the polymer. Hexokinase (H1131) was introduced into the gel by inclusion in the cathodic running buffer at a concentration of 13 mg/L and preelectrophoresis for at least 30 min at 13V/cm. After electrophoresis of samples, dithiothreitol was removed from the gel by soaking twice in pH 6.8, 0.1 M magnesium imidazole acetate buffer for 10 min each a t room temperature. The gel was then soaked in activity stain consisting of 35 mM creatine phosphate (P6502), 2 mM adenosine diphosphate (ADP; A6521), 2 mM nicotinamideadenine dinucleotide phosphate (NADP; N0505), 20 mM glucose (G5000), 2 mM phenazine methosulfate (PMS; P9625), and 2 mM 3-(4,5-dimethylthiazoI-2-yl)-2,5-diphenyltetrazolium bromide (MTT; M2128), all in pH 6.8,O.l M magnesium imidazole acetate buffer. The blue bands reached suitable intensity in 2-5 min, and the reaction was stopped by several soaks in 5% acetic acid. Gel scanning was done a t 570 nm, as determined by the adsorption spectrum. The Instrument. The densitometer was designed to focus a spot of monochromatic light delivered by a bundle of 20 tapered optical fibers onto the plane occupied by the slab gel. The gel was affixed in a frame which was moved on a lead screw in a direction perpendicular to the beam. The entire assembly replaced the double beam sample module in the GC/McPherson spectrophotometer system. The scanner proper was enclosed in a machined aluminum box (Figure 1). Entrance and exit port dimensions allowed lightight fit with the rest of the spectrophotometer. A removable top allowed external manipulation of the interior and gave a lighttight seal when in place. A long lead screw turned by a stepper motor (K82501-P2,North American Philips Control Corp., Cheshire, CT) driven through
a interface controlled by the GCA/McPherson controller (Model EU-700-32) provided precise linear motion. The gel was clamped between two glass plates and set in a frame which rested atop the lead screw via Thompson ball bushings. Two fixed rods running parallel to the lead screw provided support for and restricted nonlinear motion of the frame. The frame was adjustable in that the gel/frame assembly could be moved vertically over a distance of 15 cm. Additionally, the gel within the frame could be rotated in-plane over a limited range. The optical assembly is shown in Figure 2. The fiber optic large end sampled a 2.0-mm light spot exiting from the monochromator. This spot was reduced to a 50 wm diameter spot a t the small end of the fiber optic. The light emerging from the fiber optic was focused by lens L1 onto the gel. Lens L2 focused the diverging beam through the exit port and onto the photomultiplier. The electronic interface was constructed according to specifications included with the driver control chip (SAA-1027, N.A. Philips). The DVM trigger output at pin 4 of connector J8 of the controller was sampled by a N3904 transistor, which switched the trigger input to the driver chip. The driver chip operated the motor. The frequency of pulses from the controller was selectable by the signal averaging control on the spectrophotometer from 50 ms to 5 s per pulse. This allowed a linear rate of 0.012 to 0.2 cm/min, in a reverse or forward direction. The scanner was usually operated a t a 0.2-s step-integration time. Corresponding rate of linear motion was 0.30 cm/min. Thus a 10 cm long gel could be scanned in 30 min. For a 2-mm spot of radiation of 570 nm wavelength and 2 nm bandwidth sampled by the fiber optic, a 5.00-V signal corresponding to 100% transmittance could be output with 800 V applied to the photomultiplier. T r e a t m e n t of Data. Electropherograms obtained by scanning were machine-copied, cut, and weighed to the nearest 0.1 mg. These raw weights were then normalized to a common band length. Band lengths were measured to within f2% while the gel was clamped between glass plates. Normalized data were then suitable for subsequent data reduction.
RESULTS AND DISCUSSION The resolution of the densitometer is demonstrated in Figure 3. Parallel lines were drawn on transparent tape a t the distances indicated. The tape was sandwiched between glass plates and scanned t o give the output as shown. Resolution was complete when the lines were less than 1 m m apart. Figure 4 shows a comparison of the scans obtained with this densitometer and the Ortec 4310 densitometer. Scans were made on a n autoradiogram negative lane containing multiple bands (not shown). The two scans demonstrate comparable degrees of resolution obtained by the two instruments. In contrast t o the fiber optic densitometer, the ORTEC 4310 utilizes unfiltered white light reduced to a small
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beam diameter by a mask. While this does not necessarily impair resolution, linearity of measured absorbance with respect to intensity of a stained band would be poor over a large range of stain intensities. The linearity of the measurements obtained by the densitometer was excellent for both the silver stain and the revised activity stain. Identical volumes (3.0 yL) of serially diluted samples of CK-MM were loaded with a 5-pL Hamilton syringe, subjected to electrophoresis, strained as described, and scanned. Both stains gave linear response over nearly 2 orders of magnitude of concentration (30-900 ng loaded). This is an achievement not obtained by previous work with the silver stain (91,which attributed nonlinearity to concentration dependence on relative stain response. The linear response for the activity stain was obtained only after modifying substrate constituent concentrations from those recommended in the original references (10,12). The limit of detection, expecially for the activity stain, is primarily a function of the stain development time. Devel-
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opment for long periods of time in activity stain generally led to greater intensification of the protein bands, along with a background coloration which was removed without loss of protein band intensity upon soaking in 5% acetic acid. The activity stain was unstable, and development eventually slowed and stopped after 15 min at room temperature. For the silver stain, further development led to an increase in background staining, which was irreversible without attenuation of the stained protein. Perhaps the major problem encountered appeared to be that of band shape heterogeneity. Dumbbell-shaped and wavy bands were not uncommon, especially for slowly migrating proteins. This heterogeneity in band shape gave rise to unreliable results when a pinpoint of light was used for scanning. We compensated when necessary by making multiple scans across different cross sections of the bands and averaged the peak weights obtained. A more direct method for eliminating this problem would consist of replacing the fiber optic cluster with a fiber optic ribbon, such that the small end would consist of a single file of fibers. Thus the entire band would be scanned by a knife edge, rather than a pinpoint. This would allow a single absorption measurement over the entire band length, obviating the necessity for making multiple scans over several cross sections of a stained band. This approach would represent a simple and effective solution to elimination of the heterogeneous band shape problem. This instrument can only be considered a pcototype at this stage. Technical and mechanical difficulties with the vertical frame arrangement suggest that a horizontal frame upon which the gel rests may be a less tedious design to work with. Some unnecessary dispersion of the light beam appears to be a result of the sandwiching of the gel between glass plates and multiple reflection and refraction from the interfaces. Again, a horizontal arrangement would minimize this problem and improve resolution. Despite these drawbacks to the design presented here, the results we have obtained regarding linear stain response and moderately high resolution demand further investigation of this approach to densitometric analysis.
ACKNOWLEDGMENT The authors wish to thank the University of Georgia instrument shop for their construction of the densitometer and Geoffery N. Coleman for advice on the optical system. Registry No. Creatine kinase, 9001-15-4. LITERATURE CITED (1) Chrarnback,',A.; Rodbard, D. Science 1971, 172, 440-451. (2) Gaal, 0. Electrophoresis in the Separation of Biological Macromolecules"; Wiley: New York, 1980. (3) Merril, C. R.; Goldrnan, D.; Sechman, S. A.; Ebert, M. H. Science 1981, 211, 1437-1438. (4) Wlerne, R. J. I n "Methods of Enzymatic Analysis", 2nd ed.; Bergrneyer, H. U., Ed.; Academic Press: New York, 1974; Vol. 1, pp 261-282. (5) Gabrlel, 0. Methods Enzymol. 1971, 22, 578-604. (6) Wierne, R. J. J . Chromafogr. 1958, I , 166-171. (7) Taber, H. W.; Sherman, F. Ann. N . Y . Acad. Sci. 1964, 121, 600-6 15. (8) Petrakls, P. L. Anal. Biochem. 1969, 28, 416-427. (9) Poehling, H. M.; Neuhoff, V. I n "Electrophoresis '81"; Allen, R. C., Arnaud, P. A., Eds.; Walter de Gruyter: New York, 1981; pp 133-147. (10) Smith, A. F. Clin. Chlm. Acta 1972, 39, 351-359. (11) Oakiey, 6. R.; Kirsch, D. R.; Morrls, N. R. Anal. Biochem. 1980, 705, 361-363. (12) Yue, R, H.; Jacobs, H. K . ; Okabe, K.; Keutel, H. J.; Kuby, S. A. Biochemtsfry 1968, 7, 4291-4298. (13) Harrison, R. A. P. Anal. Biochem. 1974, 6 1 , 500-507.
RECEIVED for review June 24, 1983. Accepted October 17, 1983. The Petroleum Resource Foundation (#12082-G5,4)is gratefully acknowledged for support of this work.
