Anal. Chem. 1883, 65, 2029-2035
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Native Protein Separations and Enzyme Microassays by Capillary Zone and Gel Electrophoresis Dan Wu and Fred E. Rsgnier’ Purdue University, Department of Chemistry, West Lafayette, Indiana 47097-1393
Native protein separations by capillary gel electrophoresis are achieved using linear acrylamide gel matrices. Polyacrylamide gels with a concentration range of 3.5-5% did not exhibit size separations for native proteins with molecular weights from 20 000 to 47 000. The separation of native proteins in gel-filled capillaries is based solely on the charge of the protein as in normal zonal electrophoresis. Retention of protein activity in the acrylamide matrix was demonstrated by performingenzymaticassays in the gel matrix. Alkaline phosphatase (ALP) and 8-galactosidase assays were conducted in bothC18-PFlO8-modified and polyacrylamide gel-filled capillaries. Enzyme assays were achieved by filling the capillary with an appropriate substrate dissolved in the electrophoresis buffer. The product formed by the reaction of enzyme with substrate was monitored using a standard UV-visible detector. Both constant potentialand zero potential modes of analysis were demonstrated. The polyacrylamide gel columns provide the advantages of minimized diffusion and limited band spreading due to the high viscosity of the gel matrix. The lowest detection mol (7.6 X 10-l2 M limit achieved was 5.2 X sample injected) of ALP. The dual enzyme assay of ALP and 8-galactosidase was achieved in gelfilled capillaries simultaneously.
INTRODUCTION Enzyme assays have been widely used in traditional chromatographic and electrophoretic systems to (i) identify enzymes among the separated components, (ii) establish that the three-dimensional structure of an enzyme is intact after fractionation, (iii) quantify recovery and purification, (iv) assess the possibility of enzymatic isotypes in the sample, and (v) obtain diagnostic evidence from the sample as in clinical and forensic analyses. These applications are equally important in capillary electrophoretic systems. Recent reports indicate that enzyme activity may be determined either in postcolumn assays’ or by electrophoresis of the substrate into capillaries.213 Postcolumn assays have been achieved through the use of pressure to mix substrate and the effluent from a capillary and force the mixture into a second capillary where incubation and detection occur.1 An alternative is to continuously transport substrate into the capillary by a combination of electroendosmosis and electrophoresis where it is electrophoretically mixed with enzyme.2 Although the enzyme catalyzes product formation as it moves electrophoretically through the capillary, product does not (1) Roeraade, J.; et al., poster from HPCE ’93. (2) Bao, J. B.; Regnier, F. E. J. Chromatogr. 1992,608, 217. (3) Yao, X.W.; Wu,D.;Regnier, F. E. J. Chromatogr. 1993,636, 21. 00092700/93/0365-2029$04.00/0
accumulate. Under constant potential the enzyme and product are continuously separated. It is only when the operating potential of the system is interrupted that separation of product and enzyme ceases and product accumulates. When a detectable amount of product has accumulated, potential is again applied to the system and product is transported to the detector. This mode of enzyme assay in which electrophoretic mobility is used to separate protein analytes, mediate the mixing of assay reagents, and transport products to a detector will be referred to below as electrophoretically mediated microassay (EMMA). The work of Bao and Regnier? in which electrophoretically mediated enzyme assays were carried out in open tubular capillaries, suggests that product diffusion during lengthy zero potential assays will seriously diminish resolution. Theory would predict* that this problem could be overcome through the use of gel-filled capillaries in which the internal viscosity is sufficiently high to restrict product and analyte diffusion. The ability to conduct enzyme assays in open tubular and polyacrylamide gel-filled capillaries depends on the separation of proteins in their native state. Most of the previous work on capillary gel electrophoresis of proteins was accomplished in the presence of sodium dodecyl sulfate (SDS). Because SDSdenatures proteins,c7 the columns used in these studies were incompatible with EMMA. The objective of the work presented here was to determine the highest sensitivity that could be obtained in enzymebased EMMA with UV absorbance detection. In a comparison of open tubular systems modified with a nonionic, nondenaturing surfactant, and acrylamide gel-filled capillaries, electrophoretically mediated enzyme assays of alkaline phosphatase and &galactosidase were used to examine sensitivity. These two enzymes were chosen because they are of high turnover number and both cleave nitrophenyl-containing substrates. Nitrophenolate anion is produced, which may be detected at 405 nm.MO
EXPERIMENTAL SECTION Materials. All proteins, p-nitrophenyl phosphate, and o-nitrophenyl 8-D-gdactopyranoside were purchased from Sigma ChemicalCo. (St.Louis,MO). Acrylamide,ammonium persulfate (APS),NJVJV’JV’-te~amethylethylenediamie (TEMED),NJV’methylenebis(acry1amide) (bis), and trizma (Tris) base were purchased from Bio-Rad (Richmond, CA). Glycine and direct yellow 50 were purchase from Aldrich Chemical Co. (Milwaukee, WI).Pluronic F-108nonionic surfactant (PF-108)was supplied by BASF Co. (Parsippany, NJ). Buffer solutions were prepared by using double-distilled water that was passed through a 0.45(4) Ganzler, K.; Grew, K. S.; Cohen, A. 5.;Karger, B. L.; Guttman, A.; Cwke, N. C. Anal. Chem. 1992,64,2665. ( 5 ) Dolnik, V.; Cobb, K. A.; Novitny, M. J.Microcol. Sep. 1991,3,165. (6) Tsuji, K. J. Chromatogr. 1991, 550, 823. (7) Cohen, A. S.; Karger, B. L. J. Chromatogr. 1987,397,409. (8) Tietz, N. W., Ed. Fundamentals of Clinical Chemietry; W. B. Saunders Co.: Phhdelphia, PA, 1976. (9) McComb, R. B.; Bower, G. N., Jr. Clin. Chem. 1972,18, 97. (10) Wallen Fels, K. In Methods in Enzymology; Colowick, S . P., Kaplan, N. O., Eds.; Academic Press Inc: New York and London, 1961; VOl. 5. Q 1993 American Chemlcal Soclety
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pm nylon filter. Protein sampleswere prepared in Tris (25mM)glycine (125mM) buffer (pH 8.3) and stored below 0 O C between analysis. Instrumentation. An electrophoreticsystem was built with the buffer vials, fused-silica capillary, and electrodes contained in a Lucite cabinet fitted with a safety interlock that interrupted power to the instrument when the cabinet door was opened. High voltage was supplied by a Glassman PS/EL30P01.5 (Glassman high voltage,Whitehouae Station, NJ) power supply. On-column detection was achieved with an Isco CV4 (Lincoln,NE) variablewavelength UV-VIS absorbance detector. Polyacrylamide Gel-Filled Capillaries. Fused-silica capillaries of 375-pm o.d and 75-pm i.d were purchased from Polymicro Technologies (Phoenix, AZ). The polyimide coating was removed 20 cm from the inlet of 45-cm lengths of tubing to provide a detector window. These capillaries were washed with distilled water for 30 min to wet the surface. Gel-filed capillaries were prepared by a modification of the procedure described by Wu and Regnier.11 A 5-mL aliquot of 2 % acrylamide solution was vacuum degassed for 30 min followed by the addition of 50 pLofAPSsolution (10%)and 20pLofTEMED solution (10%). After the reaction had proceeded for 20 min, 2%by weight solid acrylamide,with 0.5% bis (w/w),was added to the solution with stirring. Another addition of 50 pL of APS (10%)and 20 p L of TEMED (10%) was made, and stirring continued for an additional 1min. A reservoir of this solution was then connected to a capillary and the solution forced into the column at 120 psi pressure. Columns filled in this manner were allowed to sit for 1h before use. C18-PFlOS-Modified Capillaries, A modification of the procedure by Towns12 in which toluene was substituted for methylene chloride was used to prepare octadecykilane-derivatized capillaries. After silylation,the capillary was washed with THF for 10 min followed by a 10-min wash with methanol and with double-deionizedwater. The surfactant solution was prepared by dissolving 1% (w/v) PF-108in buffer. This solution was pulled through the capillary for aminimum of 1.5 hto ensurecompletecoating of the capillary. The capillary was then washed with a 0.5% (w/v)solution of the surfactant. Runningbuffers contained 0.1 % (w/v)surfactant to replace surfactant that leached during operationof the capillary.
RESULTS AND DISCUSSION Enzymes can convert lO"lO6 molecules of substrate to product in 18. This is a type of chemical amplification that has been widely exploited in enzyme assays and enzymelinked immdosorbent assays for high-sensitivity detection. Sensitivity is directly proportional to the turnover number of the enzyme and to incubation time. Reaction times of several hours are used when a high degree of amplification is required. On the basis of the Einstein equation, o = (2Dt)lI2, it is anticipated that lengthy reactions may be a problem in electrophoretically mediated enzyme assays. Band spreading may become so large in extended zero potential assays that both sensitivity and resolution will be compromised. This issue was examined using gel-filled capillaries to diminish diffusion. The utility of these gel-filled capillaries was compared to open tubular capillaries of similar electroendosmotic flow. Because suitable capillaries were not commercially available, it was necessary to synthesize and characterize the capillaries used in this work. Preparation and Characterization of Polyacrylamide Gel Columns. Previous studies from this laboratory and otheraon the preparation and use of gel-filled capillaries were directed at the use of sodium dodecyl sufate (SDS)-polyacrylamide gel electrophoresis (PAGE) for separating proteine.4-'Js In SDS-PAGE, proteins take up 1.4times their (11)Wu,D.; Regnier, F. E.J. Chromatogr. 1992,608, 349. (12) Towns, J. K.; Regnier, F. E.Anal. Chem. 1991, 63, 1126. (13) Guttman, A.; Cooke, N. Anal. Chem. 1991,63, 2038.
Table I. Molecular Weight and p I of the Standard Proteins proteins re1 mol mase PI trypsin inhibitor from soy bean 20 OOO 4.6 ovalbumin 47 OOO 4.7 a-amylase from Bacillus 45 OOO 6.3 carbonic anhydrase from bovine 29 OOO 6.2 weight of SDSand are denatured. Because it was the objective in these studies to carry out enzyme assays in the capillaries, denaturing solvents could not be used. PAGE columns were prepared without SDS. These columns will be referred to as 'native PAGE" columns. SDS-PAGE has been carried out with both cross-linked and linear polyacrylamide.14J5 However, it was observed in this work that column lifetime decreased when the 4% linear gels were used without SDS. New techniques for preparing gel-filled capillaries using a two-stage polymerization method were found to be the most effective in circumventing this problem. In the first stage of this process, linear polyacrylamide was formed outside the capillary. Acrylamide monomer, cross-linker, and catalysts were then added in a second step, and the mixture was pumped into the capillary where it polymerized. A polymeric matrix of high viscosity and a low degree of cross-linking was formed by this process. The viscosity and degree of cross-linking were manipulated by varying monomer concentration in the two stages. Columns prepared in this way were more stable due to the small amount of cross-linking and higher viscosity. The average column lifetime was approximately 10 analyses. This is comparable to reported lifetimes of SDS-PAGE columns. It is expected in polyacrylamide gels that separations of native protein will be based on both size and charge. When this is true, the slope of Ferguson plots will be proportional to protein molecular weight.16 These new PAGE columns were characterized by separating four model proteins with isoelectric points (pl) of less than 6.2 and two different molecular weight ranges (Table I). Separation of these proteins on a 4% acrylamide-O).5%bis gel-filled capillary (Figure 1)indicates that the migration order followed the p1 of the proteins. Trypsin inhibitor (PI=4.6) eluted first and carbonic anhydrase (PI= 6.2) last. Each peak was identified by electrophoresing individual protein. The unlabeled peaks are impurities from trypsin inhibitor and ovalbumin. Ferguson plots (Figure 2) derived by using gels ranging from 3.5 to 5% acrylamide appear to give parallel lines of the same slope for the four proteins. Statistical analysis indicated that the slopes of these lines are not different. From these results it may be concluded that (i) these columns do not discriminate on the basis of size in the molecular weight range examined and (ii) the separation is based on net charge. Stated another way, normal zonal electrophoretic separations were obtained in these gel-filled capillaries. The electropherogram of an Escherichia coli protein extract in Figure 3 illustrates the high resolving power of native PAGE columns. Separations were also conducted at a highgel concentration (12%) in an effort to fiid a concentration range in which sieving became an issue. At this concentration the migration order of the model proteins was according to pl; however, sizing effects began to be observed (Figure 4). Data obtained at 12% polyacrylamide do not fit the extrapolation of the Ferguson plot at lower gel concentrations. In addition, the resolution between a-amylase and carbonic anhydrase is less '
(14) Widhalm, A.; Schwer, C.; Blaas, D.; Kenndler, E. J. Chromotogr. 1991, 549, 446. (15) Heiger, D. N.; Cohen, A. S.; Karger, B. L. J. Chromatogr. 1990, 516, 33. (16) Ham-, B. D.; Rickwood, D., Eds. In Gel Electrophoresis of Proteins, a Practical Approach; IRL Press: Washington DC, 1983.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 15, AUGUST 1, 1993
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with 4 % acryiamlde and 0.5% bis: (1)trypsln Inhibitor, (2) ovalbumin, (3)a-amylase,and (4) carbonicanhydrase. Conditions: capilary length, 45 cm; separation length, 25 cm; 75-fim 1.d.; separation potential, 15 kV, 333 V/cm; Trls-glycine buffer, pH 8.3.
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Figure 9. Electrophoresis separatlon of E. coli protein extract in an acryiamlde gel capillary. Conditions are given in Figure 1. than expected, which suggests the separation is not solely based on charge at this gel concentration. It is seen that, in comparison to free solution, electroosmotic flow and analyte
Figure 4. Separation of native proteins on 12 % linear acrylamlde gel columns: (1) trypsin inhibitor, (2) ovaibumln, (3)a-amylase, and (4) carbonic anhydrase. Conditions are given In Figure 1. diffusion are greatly reduced when zonal electrophoresis is carried out in gels. Preparation and Characterization of Open Tubular Capillaries. It has been shown in traditional electrophoretic systemsthat nonionic surfactants do not denature proteins." In this study, protein separations were performed in C18PF108 capillaries for comparison with polyacrylamide gel systems. These capillarieswere prepared by first derivatizing the fused silica with C18 to limit negative charge on the capillary surface and provide an anchor group for the surfactant. Then a nonionic surfactant, PF108, was adsorbed to the octadecylsilane (C18)layer in a dynamic coating process. A 1.0% solution of PF-108 was forced through columns for 2.5 h to assure that the surface of the capillaries was coated with surfactant. Electroendosmotic flow in the C18-PF108 capillaries was reduced 35-fold relative to that of uncoated columns. A flow of 7.1 X 10-8 m2/v.s was measured in native fused-silicacapillaries compared to 2.0 X 10-9 m2/v.s with the surface-modified capillaries. Protein adsorption was ala0 drastically reduced by this adsorbed coating.12 Separation of the model protein mixture is illustrated in Figure 5. Because electroendosmosis approaches zero in the coated capillaries separation is based on electrophoretic mobility alone. Migration velocity generally follows the PIof the proteins as in the acrylamide gel columns. It was shown above that electroendosmosis is insignificant in octadecylsilanesurfactant ((318-PF108) coated capillaries. In this regard, acrylamide gel-filled capillaries and the surfactant-coated capillariesare the same. Protein migration in these two types of capillaries will be on the basis of electrophoretic mobility alone. The difference in the two systems is that the viscosity in the gel matrix is much higher than in the C18-PF108 system. These systems were used to test the conceptthat high-viscositysystemsminimize analyte and product diffusion. Enzyme Assays in Capillary Electrophoresis. According to theory: electrophoretically mediated enzyme assays are based on the use of differencesin the electrophoretic mobility of the enzyme, substrate, enzymesubstrate complex, and product to cause electrophoretic mixing. Assays may be run in two ways: at zero or constant potential. At zero potential, product is allowed to accumulate and is then transported to the detector. The zero potential assays reported here were carried out in three steps: protein separation, incubation, and product transport. In the sep(17) Stryer, L. Biochemistry; W. H. Freeman and Co.: New York, 1988; Chapter 12.
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carbonic anhydrase. Conditions are ghren In Flgure 1.
aration phase, sample was introduced into capillaries filled with buffered substrate and potential applied for a sufficieht period of time to effect a protein separation. Although product was formed during this phase, migration of the enzyme away from the product did not allow product accumulation. Power was interrupted and product allowed to accumulate for a fixed time in the second phase of the assay. Products formed during this incubation phase were then transported to the detector by reapplying potential to the capillary in the third phase. The profile in this mode is a peak on top of a plateau, with the peak typically being dominant (Figure 6). The enzyme reactions in this study were always conducted under saturating substrate conditions. According to the Michaelis-Menten equation, under these conditionsthe reaction rate approachesthe maximum velocity and is dependent on the enzyme activity and concentration.* The product is accumulated during a fixed incubation time under the zero potential mode. The area of the product peak is therefore determined by enzyme activity, enzyme concentration, and incubation time. The origin and height of the plateau will be explained below in the discussion of constant potential assays. Constant potential assays, in contrast, were achieved in a single step. After the sample was applied to the capillary, potential was maintained at a constant level until both the enzyme and product passed the detector window. Although product is continuously swept away from the enzyme, the rate at which product is produced is sufficiently high relative to the separation time that a small amount of product accumulates.12 When the electrophoretic mobility of the enzyme is negative and less than that of the product, a plateau of product will lead the enzyme through the column. The last product to be formed when the enzymepasses the detector window is the last to be detected. In contrast, product formed when the enzyme was at the capillary inlet will be the first to pass the detector.
Figure 6. Electropherogram of ALP assay In Cl&PF 108 columns under zero potential mode. The flrst peak results from the product generated during the incubation time. The second peak Is the internal standard dye peak. Concentrationof ALP, 0.105 mg/ml, concentration of Dye, 1.0 mg/ml. Initial evaluations of the sensitivity of electrophoretically mediated assayswere done using alkaline phosphatase (ALP). ALP catalyzes reactions involving the hydrolysis of phosphate ester bonds.18 When p-nitrophenyl phosphate (PNPP) is the substrate, ALP cleaves the phosphate group from PNPP to form p-nitrophenyl (PNP) and inorganic phosphate. Ionization of PNP at basic pH produces the nitrophenolate anion, which is a quinonoid structure of intense yellow color, absorbing at 405 nm. Because ALP and PNPP have absorbance maxima of 280 and 340 nm, respectively, accumulated PNP may be measured at 405 nm without interference. The ALP assay was conducted by filling the capillary and the buffer vials with 1mg/mL PNPP in Tris-glycine buffer solution (pH 8.4). Preparation of the Tris-glycine buffer is described in the Experimental Section. The assay was conducted in both the constant and zero potential modes. Assays for 8-galactosidase were carried out in a similar manner. This enzyme cleaves o-nitrophenyl 8-galactopyranoside (ONPG) to produce o-nitrophenyl (ONP) and galactose. Again, nitrophenyl may be detected at 405 nm. Because the electrophoretic mobilities of ALP and 8-galactosidase are different, both of these enzymes may be detected in the same electrophoretically mediated assay. Assays in the Open Tubular Capillary. Electropherograms of ALP and @-galactosidaseassays under constant potential (Figure 7a,b) typically show a plateau. The difference in time between the leading and trailing edges of the elution profile is determined by the difference in electrophoretic mobility of the enzyme-substrate complex and product. The width of the plateau in the ALP assay was 12 min as compared to 2.5 min with &galactosidase at 200 V/cm with a 25-cm separation length. Product mobility is greater than that of either of these enzymes. In the case of ALP, the mobility of product is much greater than the mobility of the (18)Thompson, R. Q.;Barone, G. C.; Haleall, H. B.; Heineman, W. R. Anal. Biochem. 1991,192,W.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 15, AUGUST 1, 1993
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Time ( m i d Flgure 8. Relationship between the applied potenthl and the ALP assay proflle under constant potentlal mode. The experimental conditlons are glven in Flgure 7a.
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enzyme-substrate complex. The product formed migrates towards the detector, leading the plateau. However, the mobilities of the product and the enzyme-substrate complex are relatively close in the case of &galactosidase. This results in a narrow plateau. For singleenzymeassaysunder constant potential the width and the height of the plateau is also controlled by the applied voltage. The amount of product generated by the enzyme reaction is determined by the time period between injection and when the enzyme passes the detector window. At higher voltages,enzyme is transported to the detector window within a shorter period of time. This allows less product to form. The enzyme assay has a plateau of lower height and narrower width. In contrast, at lower potentials more product will form and give a higher and wider plateau (Figure 8). The areas under the curves in Figure 8 are 954,1399,and 3204 for 12,9,and 6 kV, respectively. One will not see a simple linear relationship between the voltage and product area in a constant potential assay where the voltage was varied to control the incubation time. This is because the lowering of the voltage not only increases the incubation time of the enzymereaction but also changesthe electrophoreticmobility of the product. At decreased voltages, the product will pass the detector window at a lower velocity, resulting in an increased peak area. When enzyme concentration is low, it would be expected that the lower potentialmode, approaching zero applied potential, is more beneficial. An ALP assay in the zero potential mode is illustrated in Figure 6. The first peak is product which accumulated at zero potential and the second peak is a dye (direct yellow 50, e400nm) used as an internal standard. Becausethe enzyme was manually introduced into the capillaryhydrodynamically,
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it is difficult to inject with precision or accuracy. The internal standard was mixed with the sampleto overcomethis problem. On the basis of experience gained during these studies, it was concluded that an internal standard improves quantitative information measurements. A calibration curve for the ALP assay in the zero potential mode is shown in Figure 9. Each data point in the figure represents the average of the three injections. Injection errors decreased from 10-15 76 without internal standard to 3-676 with the internal standard. It is worth noting again that samples were introduced manually in these studies. Errors could probably be reduced to less than 3% using a commercial instrument with automated injection. The calibration curve for ALP was linear from 0.005to 0.105 mg/mL. Because product concentration varies with time while the concentration of internal standard is constant, an isoform of the enzyme being assayed would be a better internal standard. An isoform internal standard would better compensate for errors in both sampling and incubation time. Assays in Polyacrylamide Gel-Filled Capillaries. An ALP assay at constant potential in a polyacrylamide-filled
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ANALYTICAL CHEMISTRY, VOL. 85, NO. 15, AUGUST 1, 1993
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capillary is presented in Figure 10. Although the concentration of enzyme in the sample and the running conditions used are identical to those used with the open tubular column in Figure 7a, two differences were observed between the open tubular and gel-filled capillaries. First, it is seen that the electrophoretic mobility is lower in the gel-filled capillaries. The product plateau passed the detector 11 min after injection in the native gel columns compared to 8 min in C18-PF108 columns. This is attributed to the high viscosity of the gel medium restricting electrophoretic migration. Second, the plateau is only l/g as high with the gel-filled capillaries. This is caused by differences in the method of injection. Whereas both electrokinetic and hydrodynamic injection may be used with open tubular capillaries, only electrokinetic injection may be used with the gel-filled capillaries. Restriction of electrophoretic mobility causes less enzyme to be introduced into the gel-filled capillary than with hydrodynamicinjection in the open tubular system. Comparison of Enzyme Assays in C18-PF108and GelFilled Capillaries, The zero potential mode of assay is inherently more sensitive because it allows for greater amplification. The problem with long incubation times in open tubular capillaries is that products are generally low molecular weight and diffuse away from the enzyme during incubation. Based on the Einstein equation, u = (Wt)1/2, the diffusion of particles in the solution is proportional to the squareroot of the time. When the incubationtime is increased from 2 to 8 min, the band spreading will increase 2-fold. This was observed in the ALP enzyme assay in open tubular columns. Diffusion coefficients are also related to the viscosity (7) of the solution and Stokes radius of paticles (a) as D = kT/ 67rqa. From previous studies,11 the viscosity measured in a buffer solution and a 4% linear polyacrylamide gel solution are 1.0 and 100.0cP, respectively. It can be calculated that band spreading of the product in an open tubular capillary will be 10 times greater than in a gel-filled column. If a 2-h incubation is carried out in an open tubular capillary, the product diffuses sufficiently that detection is difficult. This occurs because the area under the peak remains constant as time elapses. The peak becomes short and wider to the point that it cannot be distinguished from the baseline. In gelfilled capillaries, diffusion is limited by the high viscosity of the medium. This allows for much longer incubation times and lower detection limits. Figure 11 shows the electropherogramfor a zero potential ALP w a y in a gel-filled capillary with an incubation time of 2 h. The peak at 13 min is from the PNP product. The concentration of the enzyme solution was 7.6 X lo-'* M, and approximately 5 X 10-20 mol of enzyme was injected. This
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Time ( m i n ) Figure 11. ALP assay in an acrylamlde gel-filled capillary under zero potential. Peak 1 1s the product of ALP enzyme reaction. ALP sample concentration, 7.8 X lo-'* M; injection, 3 kV, 30 8; incubation, 2 h. Other conditions are the same as given in Figure 8.
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Time (min) Dual enzyme assay of ALP and @glactosk!ase in an acrylatmide gel-filled capillary under zero potential. ALP concentration, 0.008 mg/mL; &galactoSklase concentration, 2.3 X mg/mL. Incubation time, 10 min; Injection, 3 kV, 30 s. The two substrate concentrations are 1 mg/mL. Figure 12.
detection limit exceeds that obtainable by direct UV detection by 4 orders of magnitude and is comparable to detection with laser-induced fluorescence.mP21
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Because the ALP and &galactosidase both produce nitrophenyl, it is possible to detect the two enzymes simultaneously. A dual-enzyme assay was conducted by filling the capillary with buffer containing substrates for both enzymes and injecting the mixture in the same way as with a single enzyme assay. The first peak to pass the window in a zero potential assay (Figure 12) was from &galactosidase and the second from ALP. These peakswere identifiedby conducting ALP and 8-galactosidase assays independently.
CONCLUSIONS Native protein separationswere achieved with open tubular and polyacrylamide gel-filled capillaries. Polyacrylamide gels of 4-12 7% did not show sieving effects on native proteins with molecular weights from 20 OOO to 45 OOO. The separation appeared to be based on charge. Enzyme microassays of ALP and &galactosidase have been conducted in both surfactant- (C18-PFlOS) coated open tubular and gel-fied (19) Jorgeneon, J. W.; Lukacs, K. D. Anal. Chem. 1981,53,1298. (20) Che Y. F.; Dovichi, N. J. Science 1988,242,562. (21) wu,% L.; Dovich, N. J. J. Chromutogr. 1989,480, 141.
NO. 15, AUGUST 1, 1993 2OSS
capillaries under constant and zero potential modes. The zero potential mode has the advantages of being easier to quantitate and of high sensitivity. Zero potential assays are beet with low enzyme concentrations. Diffusionof the product zone during the incubation was diminished in polyacrylamide gel-filled capillaries due to the high viscosity of the gel matrices. Gel-filled columns allow for enzymes to be incubated up to 2 h in the capillary without significant band spreading. The detection limit for ALP in a gel-filled system was 5.2 X lem mol with an initial enzyme concentration of 7.6X M. Dual enzyme assaysof ALP and &galactosidase were alsopossible. Multiple enzyme assays are best achieved in a high-voltage separation with zero potential incubation.
ACKNOWLEDGMENT This work was supported by NIH Grant GM-25431.
RECEIVEDfor review March 9, 1993. Accepted April 1993.
