Characterization of Reaction Dynamics in a Trypsin-Modified Capillary

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Anal. Chem. 1998, 70, 1902-1908

Characterization of Reaction Dynamics in a Trypsin-Modified Capillary Microreactor Larry Licklider and Werner G. Kuhr*

Department of Chemistry, University of California, Riverside, California 92521

Application of mild vibration to an immobilized trypsin capillary microreactor can enhance digestion rates for many globular and glycosylated proteins (12-70-kDa range) without additional sample handling. A sinusoid wave form generator and a simple piezoelectric transducer were used to apply vibration in a wide frequency range to the 50-µm-i.d. enzyme microreactor over its entire length. The mass transport properties of the microreactor were quantitatively examined for protein digestions through the use of an artificial globular protein. This was synthesized by covering the surface of 35-nm-diameter latex beads with a peptide (Leu-Arg-Leu). Capillary electrophoresis analysis of the microreactor products showed there were no mass transport-related effects for vibration of the capillary. Digestions of a range of globular protein structures were performed and the products analyzed by capillary electrophoresis. The rate enhancements were found to be related to the stability of the protein tertiary and secondary structure. Cytochrome c showed a dramatic acceleration in rate of digestion as the vibration frequency increased over a range of 200 Hz to 7.1 kHz. The ability to enhance reaction rates for very stable proteins can be gained by additional means of destabilizing the protein, as shown by removal of calcium from r-lactalbumin. Vibration of the enzyme capillary will have the greatest utility for extremely limited protein samples since chemical modification to completely denature proteins usually requires considerable sample handling. Immobilized-enzyme fused-silica capillary microreactors have been developed for peptide mapping of picomole or smaller protein samples using on-line capillary electrophoresis (CE) analyses or off-line mass spectrometry.1-3 After a few nanoliters of sample migrated into a 50-µm-i.d. enzyme capillary (which has a volume of 20 nL/cm of length), the protein digest could be generated in less than 30 min, and several nanoliters of the digest could be sampled, separated by CE, and detected in less than 20 min. Denatured proteins are normally employed in peptide mapping procedures to facilitate the digestion, since the enzyme then has greater access to internal peptide bonds in the protein structure.4 In contrast, we have found that digestion rates in microreactors (1) Amankwa, L. A.; Kuhr, W. G. Anal. Chem. 1993, 65, 2693-7. (2) Licklider, L. J.; Kuhr, W. G. Anal. Chem. 1994, 66, 4400-7. (3) Licklider, L. Kuhr, W. G.; Lacey, M. P.; Keough, T.; Purdon, M. P.; Takigiku, R. Anal. Chem. 1995, 67, 4170-7. (4) Ripley, J. A. Methods Enzymol. 1967, 11, 905-17.

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can be increased for many globular or glycosylated proteins without exposure of the sample to denaturing conditions. This was accomplished though application of mild vibration at low frequency (120 Hz) to the trypsin microreactor in order to obtain digestions which otherwise were very slow or impossible to obtain.2,3 Several mechanisms might be proposed to explain this phenomenon. Rate enhancements could be related to increased mass transport derived from acoustic microstreaming, generated either at the inner surface of the capillary or through volume oscillations of vapor pockets or bubbles interacting with the sound wave.5,6 There have been a number of recent reports of immobilized enzyme rate enhancements by ultrasound (>18 kHz) applied to packed-column enzyme reactors of relatively large volumes (>1 mL).7-9 Alternatively, the incident acoustic vibration might influence the stability of the higher ordered structure of the intact protein sample. Low-power vibration in the acoustic range (1-20 000 Hz) could partially denature the sample, exposing internal peptide bonds, thereby increasing the rate of proteolysis. In this report, the effect of vibration of the microreactor on the protein digestion rate was characterized by varying the protein structure and conformation, the sample residence time, and the frequency of vibration over a wide range. The relevance of mass transport effects to the enhancement of digestion rate in the trypsin microreactor was examined with an artificial globular protein, prepared by covalently binding a tripeptide (LRL) to the surface of 34-nm-diameter latex spheres (beads). In effect, the peptide-labeled beads could serve as a model globular protein with a diffusion rate nearly 3 orders of magnitude less than the diffusion rate of the free peptide. The effect of varying vibration frequency was examined at a single concentration of the beads by measuring the microreactor product (phenylthiocarbamyl-LR) using CE. Similarly, the effect of vibration was examined for digestion of the free peptide, which undergoes a much higher rate of diffusion. Finally, a range of globular protein conformations was utilized to examine the stability in vibrated trypsin capillaries. The native conformations of horse cytochrome c (MW 12 634), bovine R-lactalbumin (MW 14 200), and calcium-depleted bovine (5) Nyborg, W. L. In Physical Acoustics; Mason, W. P., Ed.; Academic Press: New York, 1965; Vol. 2B, pp 265-331. (6) Atchley, A. A.; Crum, L. A. In Ultrasound: Its Chemical, Physical, and Biological Effects; Suslick, K. S., Ed.; VCH: New York, 1988; pp 1-64. (7) Schmidt, P.; Rosenfeld, E.; Millner, R.; Schellenberger, A. Ultrasonics 1987, 25, 295-9. (8) Ishimori, Y.; Karube, I.; Suzuki, S. J. Mol. Catal. 1981, 12, 253-9. (9) Sinisterra, J. V. Ultrasonics 1992, 30, 182-5. S0003-2700(97)00852-4 CCC: $15.00

© 1998 American Chemical Society Published on Web 03/27/1998

R-lactalbumin were utilized, as well as fully denatured reduced R-lactalbumin. EXPERIMENTAL SECTION Chemicals and Reagents. Poly(oxyethylene)diamine 2000 MWav, (Jeffamine ED-2003, Huntsman Chemical, Houston, TX), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC; ICN Biomedicals, Aurora, OH), N-hydroxysulfosuccinimide (sulfo-NHS), sulfosuccinimidyl-6-(biotinamido)hexanoate (s-NHSLC-biotin), Sepharose-immobilized trypsin TPCK-treated (Pierce Chemicals, Rockford, IL), H-Leu-Arg-Leu-OH (LRL; Bachem, Torrance, CA), acetone, heptane, triethylamine, ammonium hydroxide, sodium hydroxide, calcium chloride, ethyl acetate, ethanol, dimethyl sulfoxide, methanol, sodium acetate, phosphoric acid, disodium phosphate (Fisher Scientific, Fairlawn, NJ), sodium azide, benzamidine, 2-(morpholino)ethanesulfonic acid (MES), Sepharose-immobilized p-aminobenzamidine, phenylisothiocyanate (PITC), triethanolamine, sorbital monolaurate (Tween 20), hexadimethrine bromide (Polybrene), histidine, bovine R-lactalbumin (calcium-depleted), bovine carboxymethyl-R-lactalbumin, horse heart cytochrome c, ExtrAvidin (avidin) (Sigma Chemical, St. Louis, MO), 3-(aminopropyl)triethoxysilane (APTES), tris(hydroxymethyl)aminomethane (TRIS), and poly(ethylene oxide) (PEO) 8 million MWav (Aldrich, Milwaukee, WI) were used as received. Supplies and Equipment. Carboxylate-modified latex beads (34-nm diameter ( 15%) were supplied by Interfacial Dynamics, Corp. (Portland, OR) as a surfactant-free 4.3% aqueous suspension. The specified density of surface carboxylate groups corresponded to an effective concentration of 20 mM. Regenerated cellulose dialysis tubing of 100 000 MWCO (Spectrum Microgon, Laguna Hills, CA) and fused-silica capillaries (360 µm o.d. × 50 µm i.d., 184 µm i.d. × 50 µm i.d., and 150 µm o.d. × 75 µm i.d.) were obtained from Polymicro Technologies (Phoenix, AZ). A commercial CE instrument (ABI 270A, Perkin-Elmer/Applied Biosystems, Inc.) and a home-built CE instrument, which has been described previously, were utilized for CE separations.10 Piezoelectric speaker elements (part no. 273-091, Radio Shack, Ft. Worth, TX) were purchased locally and powered with an audiooscillator model 200AB (Hewlett Packard, Palo Alto, CA). All DI water was type 1 reagent grade (Barnstead E-Pure, Dubuque, IA). Microreactor Preparation. A procedure reported previously for derivatization of the capillary wall with APTES was modified to improve the stability of microreactors during vibration.11 Briefly, bare fused-silica capillaries were filled with 6 M HCl for 6 h at 24 °C and then rinsed with DI water, methanol, and acetone for 30 min each. APTES (2%) in acetone was rinsed through for 20 min, followed by a flush using nitrogen (filtered in-line). The capillaries were then placed in an oven at 110 °C for 6 h with the capillary ends covered. Afterward, the capillaries were rinsed with 10 mM HC1 for 3 h and rinsed thoroughly with DI water. The remainder of the procedure to immobilize trypsin is similar that reported earlier. Immobilization of Peptide on Carboxyl-Modified Beads. The bead suspension was placed in dialysis tubing and dialyzed (10) Amankwa, L. N.; Scholl, J.; Kuhr, W. G. Anal. Chem. 1990, 62, 2189-93. (11) Amankwa, L. N.; Kuhr, W. G. Anal. Chem. 1992, 64, 1610-3.

overnight in 2 L of Na4HPO4 buffer, 15 mM, pH 6.8, which contained 10 mM poly(oxyethylene)diamine (2000 MWav). With care to avoid foaming, the dialysate was transferred into a 1.5-mL polypropylene vial and was mixed with EDC added in ∼10-fold molar excess of surface carboxylate groups. The mixture was placed on a rocker and allowed to react for 12 h at 4 °C. Afterward, extensive dialysis was performed. The phenylthiocarbamyl (PTC) derivative of LRL (HCl salt) was formed by a microscale procedure adapted from Kuhn and Crabb.12 After performing solvent extractions and filtering of the aqueous phase, PTC-LRL was redissolved at a concentration of ∼20 mM in 0.5 mL of 10 mM NaHPO4 buffer (pH 7.2) containing 100 mM EDC and 20 mM sulfo-N-hydroxysuccinimide. An equal volume of the amino-modified beads was carefully added, and the reaction mixture was gently rocked overnight at 4 °C. Extensive dialysis was performed afterward to remove free peptide and raise the pH by increments to pH 8.1, in 50 mM Tris-HCl, 0.1% sodium azide. The final 0.2% suspension of bead-immobilized peptide was stored at 0-4 °C and used over a period of 8 weeks. Protein Digestions. Digestion buffer (50 mM, pH 8.1 or pH 8.3, Tris HCl) was siphoned into 90 cm length, 190 µm × 50 µm microreactors by connecting one end to an in-house vacuum line. Cytochrome c, calcium-depleted R-lactalbumin, and reduced carboxymethyl-R-lactalbumin were used as supplied. CaCl2 (10 mM) was added to the digestion buffer before preparing samples of calcium-bound R-lactalbumin, which were allowed to equilibrate for 1 h. One end of the capillary was placed in ∼100 µL of a protein sample (1 or 2 mg/mL) at 25 °C and then rinsed with the sample solution for 2-3 min. The capillary ends were then placed in septa and each was tightly coiled before placing over a 2-cmdiameter piezoelectric surface. The vibration was driven by a sinusoid waveform (∼0.75 W) which was delivered by the audio oscillator at a frequency of 200 Hz or at another frequency characteristic of the piezoelectric element (i.e., 750 Hz, 2.3 kHz, 3.3 kHz, and 7.1 kHz). Microreactor digestions of R-lactalbumin were also attempted without applying vibration. After a specified digestion time, the microreactors were either immersed in an ice bath or immediately coupled to a 1-mL syringe to transfer the digestion products into vials. Injection of a sample for a CE analysis was done immediately, and the vials were stored frozen for repeated analyses. Digestion of Bead-Immobilized PTC-LRL and PTC-LRL. Several 90 cm length, 190 µm × 50 µm microreactors were equilibrated at 25 °C with digestion buffer as described above for protein digestions. Afterward, the microreactors were rinsed with several column volumes of a 0.1% suspension of bead-immobilized PTC-LRL which also contained 140 µM PTC-H as internal standard. Other microreactors were used to digest 740 µM PTCLRL and 140 µM PTC-H. In each case, the capillary ends were sealed in septa and each capillary was individually secured to a piezoelectric surface. The microreactor vibration was produced as described for protein digestions, and the vibration was applied in an insulated box (to minimize sound) maintained at 25 °C. After 18 h, the capillaries were removed and immersed in a ice bath before attaching each, in turn, to a syringe and transferring the (12) Kuhn, C. C.; Crabb, J. W. In Advanced Methods in Protein Microsequence Analysis; Wittmann-Liebold, B., Salnikow, J., Erdmann, V. A., Ed.; Springer-Verlag: Berlin, 1986; p. 63.

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contents into a vial. The vials were sealed and briefly stored at -5 °C until CE analysis. CE Separations of Cytochrome c Digests. The CE capillary (50 µm i.d. x 360 µm o.d., 32 cm length to the detector) was prepared as reported before,2 except that the CE buffer contained 50 mM formate instead of citrate for the analyses of digests generated over a range of vibration frequencies. Samples were injected by raising the inlet end 15 cm for 20 s. Separations were accomplished with a field strength of -290 V cm-1. Absorbance data at a wavelength of either 200 or 214 nm were acquired at 2.3 Hz by an 18-bit A/D converter interfaced to a PC computer, as well as by strip-chart recordings of the detector output. CE Separations of Digests of PTC-LRL, Bead-Immobilized PTC-LRL and r-Lactalbumin. The CE capillary (75 µm i.d. x 150 µm o.d., 21 cm length to the detector) was rinsed with 1 M HC1, followed by a 0.2% solution of poly(ethylene oxide) (PEO), 8 million MWav, in 0.1% HCl for 30 min. Next, 0.15 M Na2HPO4 buffer (pH 2.1) was introduced for 5-10 min. This was followed by a sieving medium, 0.7% PEO, in pH 2.1, 0.05 M Na2HPO4, which only filled the separation length before the detection window. All solutions were introduced with a pressure of 20 mmHg. Digest samples were pressure injected using 5 mmHg for 40 s, except for R-lactalbumin samples, which were injected using 5 mmHg for 20 s. Following injection of a sample, the inlet end of the capillary was switched to a vial containing the same buffer as the outlet reservoir (0.15 M, pH 2.1 Na2HPO4). The separations were accomplished by applying 8 kV (52 µA) at the inlet end with the outlet reservoir grounded. Data acquisition was as described for analysis of cytochrome c digest samples. RESULTS AND DISCUSSION Effect of Diffusion Rate on Trypsin Microreactor Rate. There have been a number of recent reports of immobilized enzyme rate enhancements by ultrasound (>18 kHz) applied to packed-column enzyme reactors of relatively large volumes (>1 mL).7-9 To determine whether the vibration-derived enhancement in the rate of proteolysis was due to increased mass transport derived from acoustic microstreaming, generated either at the inner surface of the capillary or through volume oscillations of vapor pockets or bubbles interacting with the sound wave,5,6 we examined the effect of diffusion rate of the sample molecule on the digestion kinetics. Since the average radial diffusion time (t ) 2D/L2) in a 50-µm-i.d. capillary is only on the order of several seconds for the proteins we have examined, any enhancements to diffusion-based transport should become most apparent at the largest protein radius (based on the Stokes-Einstein diffusion constant, D ) kT/6πηr, where kT is the Boltzmann factor and r the radius of a protein in a solution of viscosity η). An artificial globular protein was synthesized to examine the effect of diffusion rate on the apparent rate of proteolysis at maximum protein radius. This was accomplished by covering the surface of 34-nm-diameter carboxyl-modified latex beads with an arginine-containing tripeptide (LRL) coupled through a long hydrophilic tether (∼10 nm). Since this peptide-coated bead has no secondary or higher ordered structure, any changes in the rate of proteolysis during vibration should solely be due to changes in mass transfer. Figure 1 shows the scheme for immobilization of the PTC derivative of the peptide. The release of a single tryptic product (PTC-LR) from this artificial globular 1904 Analytical Chemistry, Vol. 70, No. 9, May 1, 1998

Figure 1. Scheme for modification of beads with phenylthiocarbamyl-labeled peptide.

protein greatly simplified the task of quantifying and interpreting the digestion products. If diffusional limitations to the microreactor rate could be overcome by vibrating the microreactor, then the rate should increase over a large frequency range (0 Hz - 10 kHz). Since only the vibration frequency was varied, the measured rate should be independent of any steric or electrostatic factors. The microreactor rate for free PTC-LRL (a small peptide) was measured over the same frequency range in order to examine the effect with the substrate diffusion rate being nearly 3 orders of magnitude greater. The residence time for both the beads and free peptide was 18 h to generate sufficient product for detection and to minimize product generation during the brief introduction and removal of the samples. There was no significant effect of vibration on the reaction rate for either the peptide-coated beads (0.1% w/v), or free LRL (0.04% w/v; equivalent to 0.37 mg/mL) over a frequency range over 3 orders of magnitude. Table 1 shows the reaction rates measured for vibration frequencies from 0 to 7100 Hz. Thus, it would appear that increased mass transport is an unlikely explanation for the enhancements in the rate of proteolysis which had been evident for many proteins, even though the baseline microreactor rate for the beads was >10% of the microreactor rate for the free peptide. The difference in reaction rates between the gel and free solution rates emphasizes the difference between the peptide and the bead’s diffusion rates

Table 1. Trypsin Microreactor Digestion Rate at Different Vibration Frequencies with PTC-LRL and Bead-Immobilized PTC-LRL as Substratesa microreactor rate PTC-LR (µM h-1) frequency (Hz)

peptide

beads-peptide

0 750 2300 7100

5.0 (0.6) 4.8 (0.6) nab 4.6 (0.6)

0.72 (0.09) 0.78 (0.11) 0.67 (0.06) 0.67 (0.08)

a The rate was determined by CE analyses of digests of PTC-LRL and bead-immobilized PTC-LRL using PTH-H as an internal standard. Errors reported as standard deviations (n ) 3-5). CE conditions are given in the text. b Not available.

and shows that there is no effect of frequency on the rate of digestion. Effect of Residence Time and Vibration Frequency on Cytochrome c Digestions. Proteolysis is a relatively slow process for globular proteins that have substantial tertiary and secondary structure. As would be expected, the time required to generate a complete digestion should depend on the structure of the protein. Linear peptides and amorphous (random coil) proteins, e.g., insulin β-chain and β-casein, were found to undergo rapid digestions (less than 30 min) without applying vibration.1,2 However, the efficient digestion of a variety of globular proteins including a heavily glycosylated protein (R-acid glycoprotein) required mild vibration of the capillary, since no digestion was observed without vibration.2,3 In this work, the effect of vibration on globular protein structure was examined with horse heart cytochrome c as a representative globular protein. The electropherograms shown in Figure 2 were obtained by off-line CE analyses of trypsin microreactor digestions of cytochrome c in the same capillary (1-m length, 50-µm i.d.) with 200-Hz vibration. A very broad latemigrating peak represented the intact protein in the electropherograms produced after 60- and 90-min periods of digestion. It was of particular interest to examine the effect of vibration when the digestion primarily involved the intact protein. Therefore, in subsequent experiments, a 30-min digestion period was used to compare digestions performed with increasing vibration frequency applied to the capillary. Effect of Vibration Frequency of Microreactor on Digestion of Horse Cytochrome c. The electropherograms shown in Figure 3 demonstrate a dramatic enhancement in the digestion rate of cytochrome c in a representative trypsin microreactor by increasing the vibration frequency. Each electropherogram was produced after a 30-min digestion at a single frequency: 750 Hz, 2.3 kHz, 7.1 kHz, and 9.8 kHz. A larger yield of digest peptides was apparent after 750-Hz vibration for a 30-min period than would be produced with 200-Hz vibration (Figure 2). The largest increase in rate for digestion of the intact protein (the slowest migrating component in the peptide map) corresponded to an increase in the frequency from 750 Hz to 2.3 kHz. Analyses of digest samples after 30 min of vibration with frequencies as high as 13.1 kHz gave results qualitatively similar to the 9.8-kHz vibration, indicating that a complete digestion had been obtained. It should be noted that the frequency of vibration and the time of digestion are interrelated, such that analysis of a digest sample

Figure 2. Effect of digestion time on horse heart cytochrome c in a single trypsin microreactor with 200-Hz vibration, from bottom, 60 min, 90 min, and 3 h. CE conditions were similar to those reported previously.2

with less than 30 min of reaction may show rate enhancements for the frequencies above 2.3 kHz. The effect of temperature on the reaction rate was eliminated by performing the vibration at all frequencies at constant power. By securing a small thermocouple to the surface (as done with the capillary), no temperature change was observed for the 750Hz vibration. Slight warming after 30 min was measured at 2.3 and 7.1 kHz (between 1 and 4 °C), but this small increase in temperature should be unimportant when compared to the Analytical Chemistry, Vol. 70, No. 9, May 1, 1998

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Figure 3. Effect of frequency on 30-min digestions of horse heart cytochrome c in a single trypsin microreactor: 750 Hz, 2.3 kHz, 7.1 kHz and 9.8 kHz. CE conditions were identical to Figure 2 except use of 50 mM ammonium formate (pH 3.1) separation buffer.

dramatic change in digestion rate. Finally, the effect of vibration on the rate of proteolysis was also examined in homogeneous solution. A 7.1-kHz vibration of a 150-µm-i.d. bare fused-silica capillaries containing the digestion buffer and a 20:1 excess of cytochrome c to trypsin showed no effect of vibration frequency on the apparent rate of digestion, indicating that the enhancement requires an interfacial process. Effect of Vibration on r-lactalbumin Conformation. Since trypsin’s ability to bind specific residues depends on the flexibility of the peptide chain in the sample molecule, the digestion of proteins having a globular conformation is extremely restricted unless they are first unfolded by exposing them to denaturing conditions.4 Denaturation of single-domain globular proteins usually involves a direct transition to a fully unfolded conformation.13,14 Cytochrome c and R-lactalbumin are among a small number of globular proteins with a stable unfolded intermediate, the molten globular state.15-17 Additionally, R-lactalbumin has four disulfide bonds and thus has less flexibility than cytochrome c (13) Privalov, P. L. Adv. Protein Chem. 1979, 33, 167. (14) Creighton, T. E. Biochem. J. 1990, 270, 1-16. (15) Kuwajima, K. Proteins Struct. Funct. Genet. 1989, 6, 87-103. (16) Baum, J.; Dobson, C. M.; Evans, P. A.; Hanley, C. Biochemistry 1989, 28, 7-13.

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(which lacks disulfide bonds). Thus, R-lactalbumin is particularly well-suited for the examination of the effect of microreactor vibration on protein structure since simple chemical procedures can be used to change its tertiary structure from the calciumbound “native” configuration, to the calcium-free molten globular state, to a completely denatured conformation obtained by reduction of all four disulfide bonds.18 The reaction dynamics induced by vibration of the microreactor can then be compared between these states to examine the effect of protein tertiary structure on the changes in digestion rate. Figure 4 shows CE peptide maps for the molten globular (calcium-depleted) form of R-lactalbumin, which was digested with the trypsin microreactor for 100 min with 7.1 kHz vibration (right) and with no vibration (left). These digests show a significant effect of vibration after 100 min of residence time with 7.1-kHz vibration compared to that observed without vibration, although there is considerable product formation even in the latter case. This indicates that the molten globular form of R-lactalbumin has appropriate peptide bonds accessible for reaction with trypsin and that vibration of the microreactor increases the availability of these (17) Kuroda, Y.; Endo, S.; Nagayama, K.; Wada, A. J. Mol. Biol. 1995, 247, 6828. (18) Ewbank, J. J.; Creighton, T. E. Biochemistry 1993, 32, 3694-3707.

Figure 4. Effect of vibration on molten globular R-lactalbumin after digestion with (right) and without (left) vibration. No vibration was applied during a 100-min digestion shown by peptide map on the left, while 7.1-kHz vibration was applied on the right. Both digestions were done in the same microreactor. CE conditions are given in the text.

Figure 6. No effect of vibration on digestion of reduced carboxymethyl-R-lactalbumin (denatured state): (bottom) no vibration applied during a 30-min digestion; (top) 60-min digestion with 7.1-kHz vibration applied. Both digestions in the same microreactor. CE conditions are given in the text.

Figure 5. No effect of vibration on native R-lactalbumin after a 100min digestion time. CE peptide map for digestion after application of 7.1-kHz vibration for 100 min. CE conditions are given in the text.

sites for reaction. Digestions under a variety of conditions in five different microreactors for periods lasting 1-2.5 h showed that there was a maximum effect of vibration on the rate of digestion of the molten globular form of the protein over periods of 90100 min. The effect of vibration on the rate of reaction could be minimized either by stabilizing the native globular conformation of the protein or by removing any semblence of tertiary structure by denaturing the protein and reducing all its disulfide bonds. The native form of the protein, which is a tight globular protein, is generated by adding calcium to the molten globular form. The native globular protein was introduced to the same microreactor used to digest the molten globular form, and the result after 100

min of reaction with vibration is shown in Figure 5. As shown, the native form of R-lactalbumin proved much less susceptible to the effects of vibration due to its greater conformational stability (especially when compared to cytochrome c, which has no disulfide bonds). Alternatively, reduction of all disulfide bonds in the protein produces the fully unfolded conformation of the protein. This can be rapidly digested without vibration of the microreactor, as shown by the peptide maps in Figure 6, which compare vibration (7.1 kHz) in a representative microreactor during a 60-min digestion (at top) with the unvibrated digestion over a 30-min period (at bottom). The peptide maps obtained after even longer digestions showed no further increase in the rate of digestion (data not shown). CONCLUSIONS The rate of protein digestion was quantitatively examined with respect to several properties of the microreactor and the sample protein in a trypsin-immobilized microreactor that was vibrated over a wide range of frequencies. Mass transport-related effects were ruled out as the cause of a dramatic acceleration of the reaction rate because of the similarity of behavior of large and small substrates. The effectiveness of the applied vibration was examined over a range of globular protein structures. The digestion of cytochrome c dramatically increased in rate as the Analytical Chemistry, Vol. 70, No. 9, May 1, 1998

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vibration frequency increased over a range from 0 Hz to 7.1 kHz. Digestions of R-lactalbumin, which contains four disulfide bonds, offered convincing evidence that the rate enhancements with vibration were related to the stability of the protein tertiary structure. The ability to enhance reaction rates even for very stable proteins, e.g., those having numerous disulfide bonds, can be gained by additional means of destabilizing the native conformation. This was shown by removal of calcium from R-lactalbumin. Vibration of the enzyme capillary will have the greatest utility for extremely limited protein samples since chemical steps to completely denature proteins usually require considerable sample handling.

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ACKNOWLEDGMENT The generous donation of surfactant-free latex nanospheres by Interfacial Dynamics, Inc., and the loan of a CE instrument and a UV spectrophotometer by Applied Biosystems, Perkin-Elmer Inc., are appreciated. This work was supported by the NSF (Grant CHE-897394) and a grant from Procter and Gamble Co.

Received for review August 8, 1997. Accepted January 23, 1998. AC970852Q