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Anal. Chem. 1991, 63,1346-1350
Influence of Column Temperature on the Electrophoretic Behavior of Myoglobin and a-Lactalbumin in High-Performance Capillary Electrophoresis Robert S. Rush, Aharon S. Cohen, and Barry L. Karger* Barnett Institute, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115 The Influence of column temperature on the electrophoretlc behavlor of myoglobin and a-lactalbumin in hlgh-perfonnance caplilary electrophoresls (HPCE) Is presented. The major effect of temperature Is to shorten the analysls time by decreasing the vlscoslty, but speclfk temperature effects on the proteln migratlon behavlor were also observed. Myoglobin, under high fleld (350 V/cm), was essentlaily temperature stable from 20 to 45 O C , but at constant current, a second form of myoglobin could be detected at both 214 and 410 nm. The lnitlal form appeared to correspond to the Fe3+ and the second to the Fez+oxidatlon state of the heme Iron. The rate of converslon from Fescto the reduced Fez+ in myoglobin, under given electrophoretlc condltlons, followed first-order kinetlcs with a rate constant at 30 'C of 304 s-'. A second proteln, a-iactalbumln type I I I, demonstrated a conformational transltion that resulted In asymmetric peaks and slgmodlal mobillty plots versus temperature in the transltlon reglon.
INTRODUCTION High-performance capillary electrophoresis (HPCE) can be viewed as a complementary method to high-performance liquid chromatography (HPLC) for the analysis of peptides and proteins. Temperature can potentially be an important selectivity factor in HPCE as in HPLC. There are at least two aspects of the influence of temperature in HPCE that need to be considered: (1) the impact of temperature on the electrophoretic behavior due to changes in electroosmotic mobility (p,,) and electrophoretic mobility ( p e p )and (2) the effect of temperature on the structure of the protein, which may undergo thermally induced changes. A corollary of temperature manipulation is the Joule heat generated by the high electric fields. This heat must be actively dissipated; otherwise, the temperature of the buffer will be elevated ( I ) . In this paper, the influence of column temperature on the electrophoretic behavior of two proteins, horse heart myoglobin and bovine a-lactalbumin type I11 (calcium depleted), has been studied. Myoglobin is stable in the 20-45 OC temperature range and is typical of small globular proteins (2). However, in a recent paper sample degradation of myoglobin appeared to occur as the temperature was elevated from 20 to 50 OC (3). This anomaly is studied here. As a second example, a-lactalbumin type I11 is known to have a conformational transition near room temperature (4). In addition, conformational alteration of the protein has been studied in depth by chromatography (5). Conformational transitions may result in effective charge of hydrodynamic shape changes, or both, and such changes could result in variation in migration time and peak shape of the protein. This paper documents such behavior.
EXPERIMENTAL SECTION Commercial grade horse heart myoglobin and bovine a-lactalbumin type I11 (Sigma Chemical Co., St. Louis, MO) were used
* To whom correspondence should be addressed. 0003-2700/91/0363-1346$02.50/0
Table I. Relative Standard Deviation of Migration Time, Peak Area, and Peak Height as a Function of Column Temperaturea parameter migration time peak area peak height
myoglobin A t 20 "C
0.35 1.49 2.17
a-lactalbumin 0.56 1.28 3.73
At 35 "C migration time peak area peak height migration time peak area peak height
0.09
2.31 2.35 At 50 O C 0.25 0.09 1.21
0.04 1.45
1.18 0.23 1.17
2.10
OConditions as follows: 350 V/cm in 0.1 M borate buffer, pH
8.3, at the designated temperatures. Each set of experiments was repeated five times.
without further purification. Stock samples were prepared by dissolving the proteins in HPLC grade water at a concentration of 1.0 mg/mL, and aliquots were stored at -20 "C. (For other proteins, maintainence of native conditions may require dilute buffer concentrations in the sample solvent.) Under normal conditions,just prior to electrophoresis,the protein samples were diluted to 0.2 mg/mL in HPLC grade water. The electrophoretic bulk flow marker was 0.02% (v/v) mesityl oxide in water (Aldrich Chemical Co., Milwaukee, WI). Oxidized myoglobin (ferric) was prepared by bubbling oxygen through the sample for 1 h prior to use. Reduced myoglobin (ferrous) was prepared from the oxidized sample by sodium dithionite (Sigma), and the excess sodium dithionite was removed from the sample by gel filtration on a column of Sephadex G-25 (Pharmacia Fine Chemicals, Piscataway, NJ). HPCE conditions were as follows: a Beckman P/ACE System 2000 (Beckman Instruments, Palo Alto, CA) was employed throughout the study. Peaks were integrated with Beckman System Gold. The capillary had an effective length of 50 cm and a total length of 57 cm and was operated at defined fields of 250, 350, and 450 V/cm, respectively, unless otherwise specified. The electropherograms were UV monitored on-column at 214 or 410 nm. The buffer was 0.1 M tetrasodium borate, pH 8.3, unless otherwise noted. Samples were pressure injected from vials that were maintained at ambient temperature. The operation of the electrophoresis system was under computer control. The capillary was first rinsed under high pressure (25 psi) with 0.1 N NaOH for 0.5 min. The capillary was then rinsed under high pressure with electrophoresisbuffer for 0.7 min and allowed to equilibrate in the absence of field for 0.5 min. This wash and equilibration step was required for reproducibility (see Table I). Sample injection was conducted under low positive pressure (0.5 psi) for 3 s. Absorbance full scale was set at 0.05 AU. The detector rise time was 0.1 s, and the data collection rate was 5 Hz. Current data were simultaneously collected on data channel B. The system performance was primarily evaluated by examining the electroosmotic flow at each temperature. Additionally, the influence of temperature on the protein absorbance as well as on the volume injected at each temperature was examined. Each 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 83, NO. 14, JULY 15, 1991 1947
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Fbum 1. Elecboosmostlc flow (mesltyl oxlde) versus temperatwe for three electrlc fields: 250 V/cm (open triangles), 350 V/cm (open circles), and 450 V/cm (filled trlangles). The reclprocal viscostty of water as a function of temperature is shown as open diamonds (right
axis).
HPCE experiment was repeated five times at selected temperatures from which the mean value and the standard deviation were calculated. The number of different temperatures utilized were N = 9 for myoglobin and N = 10 for a-lactalbumin covering a temperature range from 20 to 45 or 50 "C, respectively.
RESULTS AND DISCUSSION Influence of Temperature on System Parameters. The electroosmotic flow at various temperatures from 20 to 50 "C at three electric fields, 250,350,and 450 V/cm, is presented in Figure 1. The operating power range for these experiments was from 0.2 to 1.25 W/m. The plots are parallel and linear for the three fields with correlation coefficients of 0.998,0.999, and 0.999, respectively. It can be concluded that the resistance is constant at a given temperature and that the temperature regulation is effective under these conditions. While successful as a measure of electroosmotic flow, mesityl oxide was somewhat problematical in this study because of volatility at the elevated temperatures. Other bulk flow markers such as riboflavin may also be employed. The viscosity of water decreases from 1.00 to 0.55 CPover the temperature range of 20 to 50 O C , i.e., by a factor of 1.82. The electroosmotic flow increased with temperature by a factor of about 1.75 over the same temperature range. Thus, it appears temperature-induced viscosity changes predominately account for the change in electroosmotic flow (p,) with temperature (see Figure 1) PeO = d / 4 7 w w (1) where c = dielectric constant, { = the zeta potential of the wall, and qw = the viscosity of the solution in the double layer a t the wall (6). Influence of Temperature on Myoglobin Electrophoresis. Figure 2 shows constant field (350 V/cm) electropherograms (corrected for electroosmotic flow) generated for horse heart myoglobin a t 214 nm and six column temperatures. The electropherograms demonstrate that, with increasing temperature, the migration, as expected, was faster. A standard curve of integrated area versus concentration of myoglobin was linear with the y intercept nearly at the origin, r2 = 0.999. It was therefore concluded that sample recovery from the capillary was good. Additionally, the integrated peak area with pressurized injection was found to increase with temperature, and the reason for this is discussed later. As already noted, we previously found sample degradation with temperature increase (3);however, those experiments were run a t constant current. By this procedure, the myoglobin sample remained in the capillary for roughly the same time as the temperature was varied. On the other hand, the time spent in the column continually decreased with increased temperature a t constant field, Figure 2.
Flgurr 2. Electropherograms of horse heart myoglobin (0.2 mg/mL) at 20, 30, 35, 40, 45, and 50 OC (ascendingdirection) at a field of 350 V/cm. Sample was dissolved in water and electrophoresis conducted In 0.1 M tetrascdium borate buffer, pH 8.3, and monitored at 214 nm. The 75" (i.d.) capillary had a effective length of 50 cm and a total length of 57 cm. The electropherograms have been corrected for electroosmotic flow. For more details, see Experlmental Section. We reexamined the constant current procedure, and the experiments at various temperatures are shown in Figure 3A (monitored at 214 nm) and Figure 3B (monitored at 410 nm). It can be seen that a t 20 "C a single peak was observed, whereas a second slow migrating peak appeared at 30 "C. The second peak grew a t the expense of the first at 40 "C, and at 50 "C, essentially only the second peak was observed. The identical pattern seen for 214 and 410 nm strongly suggests, as expected, that a significant conformational change has not occurred. Since the heme group is not covalently attached to the protein molecule, a conformational change typically results in the release of the prosthetic group (7,8). Another possible explanation for the behavior in Figure 3 is the reduction of Fe3+to Fe2+metal ion coordinated to the heme group. Physiologically, the iron is in the ferrous form in tissue (9). However, exposure to oxygen is known to result in oxidation to the ferric state (7). To test the possibility of the two forms being different oxidation states of iron, freshly prepared myoglobin in water was aerated with oxygen for 1 h. The sample was then electrophoresed at 200 V/cm, on a 50-cm effective length capillary a t 35 "C (conditions favorable for observance of the two forms of myoglobin; see Figure 3). A single sharp peak was observed in Figure 4. The oxidized sample was then reduced by the addition of 50 pg of sodium dithionite, and the excess sodium dithionite was removed by passing the reduced sample through a G-25 Sephadex column. The sample was then re-electrophoresed under identical conditions, and a single peak was again seen, but at a longer migration time, Figure 4. Finally, a mixture of four parta oxidized and one part reduced myoglobin was electrophoresed, and two peaks in the appropriate ratio were observed in Figure 4. The results of these experiments confirm that the observed forms of myoglobin represent the two oxidation states of iron, i.e., Fe3+ and Fe2+. The faster migrating peak corresponded to the more positive ferric form and the slower moving peak to the ferrous form. This order is expected on the basis of charge differences. Returning to Figure 3, it is clear that reduction of Fe3+to Fez+ was taking place within the column. The kinetics of conversion from Few to Fe2+were studied by varying the time that myoglobin spent in the column by electric field changes, i.e., the lower the field, the slower the migration time. Figure 5 presents electropherograms for myoglobin at 30 "C in 0.1 M Tris, 0.025 M boric acid, pH 8.6, under electric fields of 100,150,200,300, and 350 V/cm, respectively, and monitored
1348
NO. 14, JULY
ANALYTICAL CHEMISTRY, VOL. 63,
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Flgwe 3. (A) Influence of temperature on the electrophoretic behavior of horse heart myoglobin (0.2 mg/mL) under constant current conditions (9.8 pA) in 0.1 M Tris and 0.025 mM boric acid, pH 8.6. Eiectrophoresis was conducted at 20, 30, 40, and 50 OC, (ascending direction) at fields of 262, 213, 178, and 153 V/cm, respectively, and monitored at 214 nm. The 75-pm (i.d.) capillary had an effective length of 50 cm and a total length of 57 cm. (B) All conditions as in A except monitored at 410 nm.
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Flgure 4. Comparison of the electrophoretic mobility of the ferric (Fe3+) and ferrous (Fe2+)forms of myoglobin. The lower electropherogram is that of the ferric form; the center corresponds with the sodium dithionite reduced ferrous form. The upper is a mixture of four parts ferric and one part ferrous. Electrophoresisbuffer conditions were as in Flgure 3A; fieid 200 V/cm, 50-cm effective length, and 35 "C. Details given in text.
at 410 nm. Identical patterns were obtained at 214 nm. The migration time was linear with applied field, r2 = 0.969, and the total integrated area was constant (1.84 f 0.14). The rate of conversion of peak 1 into the slower moving peak 2 followed first-order kinetics with a rate constant of 304 s-l, 1.2 = 0.973. The possibility of applied field influencing the rate constant was studied by varying the column length and maintaining a constant electric field at various temperatures. Over the
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Figure 6. Comparison of the influence of temperature on the eiectrophoretic behavior of a-lactalbumin (0.2 mg/mL) at 20, 30, 35, 40, 45, and 50 "C (ascending direction) at a field of 350 V/cm. Conditions as in Figure 2.
range studies, field-induced changes appear not to take place because nearly identical profiles were observed at 150 and 200 V/cm using three temperatures (30,35, and 40 "C) and three effective column lengths (50,90 and 140 cm) (see Figure 6 for the 200 V/cm profiles). Approximating the rate constant under these conditions produced values in the same order of magnitude. The mechanism of reduction in the column is interesting to consider. One possibility is a reducing agent impurity in the buffer system. Another possible explanation may be autoreduction, in which an amino acid residue in myoglobin is the reducing agent. Such behavior is known to occur for other heme proteins (8). It is interesting that the rate of reduction appeared to be temperature dependent; see Figure 3. Both explanations would be consistent with such behavior. From the above data, sample handling in the case of myoglobin (as well as other heme proteins) would appear to be a significant consideration in HPCE analysis. Depending on handling and HPCE conditions, a single peak or two peaks may be observed. As in chromatography (9), the extent of interconversion of the two forms will be a function of the time spent in the column and the temperature of that column. Influence of Temperature on a-Lactalbumin. As noted, a-lactalbumin type I11 (calcium depleted) represents a model protein with which to evaluate temperature-induced conformational changes ( 4 , 5 ) . Figure 6 shows electropherograms generated at a constant field of 350 V/cm for a-lactalbumin at 214 nm at six column temperatures. Since the PI of a-
ANALWICAL CHEMISTRY, VOL. 63, NO. 14, JULY 15, 1991
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lactalbumin is 4.2-4.5 ( 4 ) , operation at 8.3 should lead to minimal adsorption of the protein on the column wall. At 20 "C, a single, relatively sharp peak was observed. As the temperature was raised to 30, 35, and 40 "C, the peak broadened substantially, in spite of the fact that migration was faster. Moreover, severe peak distortion was observed at 35 and 40 "C. At 45 and 50 "C, the peak again sharpened. This behavior of a-lactalbumin mimics that found in chromatography where a conformational change was observed (5, 10). In order to explore the electrophoretic behavior in more detail, we normalized the mobility of the protein to 20 "C in the following manner. First, the mobility of a-lactalbumin ( ~ c ~was ~ ) determined from the measured mobility (papp)as Clep
-- Papp - Cleo
(2)
In this study, the peak maximum was taken as a measure of migration time. The use of the centralized first moment did not significantly change this value. Next, we assumed the major bulk change due to temperature was buffer viscosity. We corrected pep at any temperature X to 20 "C by
(3) where 7 is the viscosity of water at the given temperature. A plot of peP2Ooc versus column temperature a t 350 V/cm is shown in Figure 7. The expected sigmodial curve is observed with an approximate transition temperature of 32-33 "C. Intrinsic fluorescence measurements under the same buffer conditions revealed a transition temperature of 31 "C (11). The results of Figure 7 are thus consistent with a thermally induced conformational change (12). Note that the unfolded state migrated with a faster mobility (more negative) than the folded state. While the unfolded state may be expanded, it is possible that a greater number of negative charges are exposed in this state. Evidently, the enhanced broadening in the transition temperature region, seen in Figure 6, is a consequence of a mixture of two conformational states with different migration times. As in chromatography, distorted peak shapes may reflect a transition region.
Influence of Column Temperature on the Amount Injected. If the column temperature is purposely to be varied to optimize separation, then one must also recognize that the amount injected will be temperature dependent, even if injection conditions remain constant. In this work, pressurized injection was employed, and we will now show the effect of temperature on the volume delivered to the column. The volume injected can be calculated from the well-known Poiseuille equation where V is the volume injected, AP the pressure difference between the ends of the capillary, r the internal radius of the capillary, t the injection time, 7 the viscosity of the column
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Figure 8. (A) Volume injected (open squares) calculated from the Poiseuille equation as a function of column temperatwe (left axis). The absorbance corrected normalized integrated area for a-lactalbumin (closed triangles) and for myogiobln (open circles) versus temperature (right axis) (see text for discussion). (B) Absorbance of a-lactalbumin (open circles) and horse heart myoglobin (open squares) against tem perature. These measurements were conducted by filling the 75-pm (i.d.) capillary with 0.2 mg/mL protein solutions maintained at ambient temperature. Absorbance readings were taken after 3mln incubatton, and the system was rinsed with buffer between readings and calibrated.
buffer at defined temperatures, and L the total length of the capillary. It can be seen that the volume is inversely proportional to the viscosity of the buffer solution, which is highly temperature dependent ( ~ 2 % /"C). Figure 8A presents the calculated volume as a function of column temperature from 20 to 45 "C, using the standard conditions for injection and the temperature variation of the viscosity of water. There is almost a 70% increase in the calculated volume injected over the given temperature range. We next measured the amount injected of the two proteins, myoglobin and a-lactalbumin, as a function of column temperature. The amount was determined from the integrated peak area after applying two corrections for temperature. First, the peak area was corrected for the change in solute velocity with temperature by dividing the integrated area by the migration time. Second, the change in extinction coefficient with temperature was corrected. Figure 8B presents the absorbance versus temperature data for myoglobin and a-lactalbumin at 214 nm. The absorbance of the protein solutions were determined by filling the capillary with the protein at the designated temperature and reading the absorbance after a 3-min incubation period. Myoglobin absorbance was essentially constant while a-lactalbumin exhibits an increase of about 1570,this latter change being related to the conformational change. The absorbance correction was calculated by multiplying the normalized area by the ratio of the absorbance at 20 "C to that determined at the given temperature. Returning to Figure 8A, the corrected peak area versus column temperature is presented. It can be seen that a-lact-
1350
Anal. Chem. 1991, 63,1350-1354
albumin followed quite closely to the expected variation in volume with temperature. In the case of myoglobin, there was also reasonable correspondence; however, the slope was slightly lower than the calculated volume change. Nevertheless, the results of Figure 8 clearly show a strong temperature dependence. A corollary of this study is that precision in the amount injected will depend strongly on the column temperature being maintained constant. Finally, the precision of the measurements with column temperature controlled is shown in Table I. The relative standard deviation in migration time was found to be 0.5% or less, that of the peak area 2.3% or less, and that of peak height 3.8% or less. It is interesting to note that good precision is obtained even at 50 "C. The values in Table I are comparable to those reported by other authors in HPCE (13).
CONCLUSIONS This study has been shown that column temperature can dramatically affect the electrophoretic pattern of proteins in HPCE. Reduction of iron in the heme protein, myoglobin, has been observed. Moreover, this on-column reduction is kinetically controlled, and column temperature affects the rate significantly. Thus, the specific electrophoretic pattern observed will be dependent on sample handling of myoglobin, column temperature, electric field, and column length, among other factors. In addition, we have observed the electrophoretic consequences of a conformational change in a-lactalbumin. In analogy to the behavior in HPLC, the temperature region where both the folded and conformationally altered species simultaneously exist yields an electrophoretic peak that is significantly broadened and somewhat asymmetric. The results point to the need for good temperature control in order to achieve a reproducible electrophoretic pattern.
Moreover, as in HPLC ( 5 9 ,IO), it must be recognized that broadened or multiple peaks do not necessarily mean that a protein sample is impure. Finally, since other proteins may be far less labile than those studied here, subambient temperature control is a desirable feature of HPCE equipment in the future. Operation at 4 "C is often recommended for protein separations on slab gels as well (14).
ACKNOWLEDGMENT We gratefully acknowledge support by Beckman Instruments, Inc., and the James L. Waters chair in analytical chemistry. We further thank Dr. Shiwen Lin and Dr. Peter Oroszlan for helpful discussions.
LITERATURE CITED Hjerten, S., Electrophoresis 1000, 1 7 , 665-690. Privalov, P. L. Adv. in Protein Chem. 1070, 33. 167-241. Nelson, R. J.; Pauius. A.; Cohen, A. S.; Guttman. A,; Karger, B. L. J. Chromatogr. 1080, 480, 111-127.
Kronman, M. J. Critical Reviews in Biochemistry 8 Molecular Biology; CRC Press: Boca Ratan, FL, 1969; Voi. 24, pp 566-667. Oroszian, P.; Blanko, I?.; Lu, X.-M., Yarmush, D.; Karger, B. L. J . Chromatogr. 1000, 500, 481-502.
Terabe, S.; Otsuka, K.; Ando. T. Ami. Chem. 1980, 6 1 , 251-260. Rothgeb, T. M.; Gurd, F. N. R. Enzymol. 1078, 52. 473-466. Mayo, S. L.; Ellis, W. R., Jr.; Crutchley, R. J.; Gray, H. B. Science 1086, 233, 948-942.
Lu, X. M.; Benedek, K.; Karger, B. L. J . Chromatogr. 1086, 359, 19-29.
Wu, S . L.; Benedek, K.; Karger, B. L. J . Chromatogr. 1086, 359, 3-17.
Oroszlan, P.; Karger, 8. L. Unpublished results. Kim, P. S.; Baidwin, R. L. Ann. Rev. Blochem. 1082, 51, 459-489. Morina, S. E.: Colburn. J. C.: Grossman. P. D.: Lauer. H. K. LC-GC 1900,-8, 34-46.
Chrambach, A. The Practice of Quant&thre Gel Electrophoresis; VCH: Deerfield Beach, FL. 1985: Chapter 5.
RECEIVED for review December 20,1990. Accepted March 14, 1991. This is contribution No. 421 from the Barnett Institute of Chemical Analysis and Material Science.
Preparation and Evaluation of a Bimodal Size-Exclusion Chromatography Column Containing a Mixture of Two Silicas of Different Pore Diameter David M. Northrop, R. P. W. Scott, and Daniel E. Martire* Chemistry Department, Georgetown University, Washington, D.C. 20057
Size-exclusion results from four dlfferent pore-sized silicas were used to select two of these silicas for use In a bimodal SlZe-excJudon cokmn. A mbttwe of 80- and 500-A poresized slllcas provided an excluslon curve of elutlon volume versus log molecular welght with a linear range from 5 X lo2 to 2 X 10' MW. The excluslon propertles were retained when a reversed-phase packlng was prepared by uslng the mlxed pore-slzed a k a . Column efficiency was found to be virtually Independent of pore slze.
INTRODUCTION Size-exclusion chromatography (SEC) is a liquid chromatographic method that provides molecular weight discrimination of samples. Conditions are chosen such that interactions between the solute and the stationary phase are mini-
mized. As a result, separations occur based on a solute's ability to migrate into and out of the pores of the stationary phase. This is largely determined by the solute's size, specifically the solute's hydrodynamic radius (the molecule and associated solvent) or the radius of gyration for macromolecules (the coiled molecule plus associated and entrained solvent) ( I ) , which are related to a solute's molecular weight and shape. Because there is essentially no interaction of solutes with the stationary phase, there is no retention; thus, each solute's elution volume is no greater than the total solvent volume of the column, V,. As described by Alhedai et al. (2), V , can be divided into several different regions including contributions from the various parts of the interstitial, Vi, and pore volumes, V,, where V , is the sum of Vi and V,. Nonionic molecules may sample as much of the total solvent volume as their size will permit. As the effective size of a solute increases, it is excluded
0003-2700/91/0363-1350$02.50/00 1991 American Chemical Society
