ANALYTICAL

BIOCHEMISTRY

197,

197-207

(1991)

Correlation of Electrophoretic Mobilities from Capillary Electrophoresis with Physicochemical Properties of Proteins and Peptides E. C. Rickard,**l

M. M. Strohl,*

and R. G. Nielsen?

*Eli Lilly and Company, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana 46285, and THybritech, Inc., Manufacturing and Technical Support, P. 0. Box 269006, San Diego, California 92126

Received

March

11, 1991

Excellent correlation was observed for the electrophoretie mobilities measured by capillary zone electrophowhere q is the calculated charge resis versus q/MW2”, and MW is the molecular weight. Mobilities of a set of 33 diverse peptides from enzymatic digests and 10 intact proteins were measured for separations at pH 2.35, 8.0, and 8.15 with constant ionic strength, temperature, and viscosity. The correlation suggests that the frictional drag is proportional to the surface area of a sphere that has a volume proportional to the MW. The correlation of electrophoretic mobility with physicochemical properties will facilitate the elucidation of optimum separation strategies for protein and peptide mixtures. 0 1991 Academic Press, Inc.

Capillary electrophoresis has proven to be quite useful in the separation and characterization of many biomolecules (l-6). Although several modes of operation are possible, much of the work has used the open tubular mode referred to as capillary zone electrophoresis (CZE)2 or free solution capillary electrophoresis (FSCE). CZE exhibits simplicity, high separative power, ease of quantitation, and the ability to perform rapid, automated analyses. We exploited those characteristics to separate and quantitate biosynthetic human insulin (BHI) and human growth hormone (hGH) in the presence of closely related species that could originate as impurities or as degradation products (7-9). We also utilized CZE for separations of enzyme digests in map’ To whom correspondence should be addressed. * Abbreviations used: CZE, capillary zone electrophoresis; FSCE, free solution capillary electrophoresis; BHI, biosynthetic human insulin; hGH, human growth hormone; hP1, human proinsulin; IGF-II, human insulin-like growth factor II; bST, bovine somatostatin. 0003-2697/91

$3.00

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

ping experiments (g-10). However, the future utility of this technique will depend upon the rapid development of optimized separations. Thus, we have looked at a twofold strategy to accomplish this objective. We first reported a systematic identification of crucial separation variables that included pH, buffer concentration, ionic strength, and morpholine concentration; morpholine is a mobile phase additive to minimize wall interactions. These variables were then optimized empirically to give the best separation of hGH digest fragments (11). This paper reports the second strategy, correlation of electrophoretic mobilities to charge and size for disparate separation conditions. The relationships developed from this work could then be used to guide the selection of the optimum pH range for separation. Additional insight also is given by understanding the effects of ionic strength, temperature, and viscosity on separations. The ability to both identify experimental variables that affect separation and then to predict optimum ranges for those variables would facilitate the expeditious development of CZE separations. Only a relatively few reports correlate electrophoretic mobilities of peptides and proteins with physicochemical properties. This originates partly from the lack of a well developed theory for the expected relationships so that simplifying assumptions have to be made. It also arises from the difficulty involved with estimation of variables such as charge and size. Consequently, divergent results have been reported. For example, Jokl(12) used data from paper electrophoresis to correlate the electrophoretic mobilities divided by charge with the inverse square root of molecular weight on a series of small ionic species (no amino acids or peptides were included). The coefficient of variation (relative standard deviation) was 8.6% but there was a non-zero intercept. Offord (13) considered the same problem and concluded that mobility should be related to the inverse f power of 197

198

RICKARD,

STROHL,

molecular weight. He plotted paper electrophoretic mobilities of more than 100 peptides vs molecular weight on a log-log plot. These plots produced a series of straight lines (one for each value of charge) whose slopes were almost exactly --i at both pH 6.5 and 1.9 when peptides containing histidine (pH 6.5 data) and cysteic acid (both pH sets) were excluded. Nyberg et al. (14) reports the linear correlation of relative migration times vs the quantity of molecular weight to the $ power divided by the calculated charge for a series of 6 peptide fragments from Substance P analyzed by CZE in a 0.02 M, pH 2.6 phosphate buffer. This correlation is equivalent to that used by Offord for paper electrophoresis. Deyl et al. (15) reports a similar correlation with excellent results for relative migration times from CZE (2.5 mM, pH 10.5 borate) for a series of seven cyanogen bromide cleavage fragments from collagens. Finally, Grossman et al. (16) developed a correlation for CZE electrophoretic mobilities in a pH 2.50 buffer (where all species are positively charged) with the logarithm of the quantity (q + 1) divided by the number of peptide residues to the 0.43 power for 40 peptides with charge, q, of 0.33 to 14.0 and size between 3 and 39 amino acid residues. We first examined the assumptions underlying the available theoretical predictions of electrophoretic mobility and then explored the ability to correlate electrophoretic mobility with physicochemical properties of the analytes and the separation buffer. These properties included the charge and size of the species and the properties of the separation buffer, such as ionic strength, temperature, and viscosity. Mobility data were obtained for both low pH (pH 2.35, 0.1 M glycine) and moderate pH (0.1 M, pH 8.15 tricine) separations of BHI and hGH and their related substances, human proinsulin (hPI), human insulin-like growth factor II (IGF-II), and bovine somatostatin (bST). Data also were obtained in both buffers for the peptide fragments generated by digestion of hGH with trypsin and by digestion of IGF-II with pepsin; data from a pH 8.0 separation also were acquired for the hGH digest mixture. Our data span a diverse set of chemical species for pH values that produce differences in ionization. The intact proteins range from a size of 5808 Da for BHI to 22,818 Da for bST and from relatively hydrophilic to quite hydrophobic. The peptides produced by digestion are equally diverse. Trypsin, used to digest hGH, cleaves at the carboxy terminus of basic amino acids (Lys and Arg) whereas pepsin, used to digest IGF-II, cleaves at the carboxy terminus of aromatic amino acids (Phe, Trp, and Tyr), Leu, and sometimes others. Digest fragments range from 3 to 32 amino acid residues (data from fragments with only a single amino acid residue were excluded) and from very hydrophobic to very hydrophilic. Thus, the peptides produced from these digestions are diverse in structure, size, and properties and have different termini. Furthermore, low pH values cause pro-

AND

NIELSEN

tonation of all ionized groups (i.e., neutral or positively charged residues only) whereas moderate pH separations include positively, negatively, and partially ionized residues. The experimental results demonstrate that electrophoretic mobilities from CZE are quantitatively correlated with charge and size when variables such as ionic strength, temperature, and viscosity are maintained constant. This correlation holds even across the use of different buffer components and a wide pH range. Thus, the electrophoretic mobilities and separations are predicted from physicochemical properties of the peptide and protein species. The application of this knowledge can then be combined with other techniques for method optimization in CZE (11) to rapidly develop optimized separation methods. EXPERIMENTAL

Reagents and Materials Biosynthetic BHI, hGH, hP1, IGF-II, and bST were obtained from Eli Lilly and Co. (Lilly Research Laboratories, Indianapolis, IN). Related substances for BHI and hGH were obtained as described previously (7). Their properties are given in Table 1. Morpholine and glycine were purchased from Fisher Scientific Co. (Pittsburgh, PA), tricine and pepsin were purchased from Sigma Chemical Co. (St. Louis, MO), and mesityl oxide was from Aldrich Chemical Co. (Milwaukee, WI). Tris(hydroxymethyl)aminomethane (Tris) was purchased from Boehringer Mannheim Biochemicals, Inc. (Indianapolis, IN). Trypsin (TPCK treated, 267 units/ mg protein, 98% protein) was purchased from Cooper Biomedical, Inc. (Malvern, PA). Purified water obtained from a Milli-Q purification system from Millipore Corp. (Bedford, MA) was used to prepare all solutions. All other reagents were analytical grade and were used without further purification. Polyimide-coated, fused silica capillaries, 50 pm i.d. and 360 pm o.d., were purchased from Polymicro Technologies, Inc. (Phoenix, AZ). Tris-acetate buffer, used for the trypsin digestion of hGH, was prepared by adjusting the pH of a 0.05 M Tris solution to pH 7.5 with acetic acid. The pH 2.35 separation buffer was prepared from a 100 IIIM glycine solution. The pH 8.15 separation buffer contained 100 mM tricine, whereas the pH 8.0 separation buffer contained 10 IIIM tricine, 45 IIIM morpholine, and 20 IIIM NaCl. Minor adjustments of the pH of the separation buffers were made with 1 M HCl or 1 M NaOH as necessary. Methods The trypsin digestion of hGH was carried out according to reported methods using nonreducing conditions

CORRELATIONS

OF

ELECTROPHORETIC

MOBILITIES TABLE

Properties

Protein BHI Arginyl-A0 BHI Diarginyl-B31-B32 BHI Desamido-A21 BHI hGH Desamido-149 hGH Didesamido-149-152 hGH hPI IGF-II bST a Calculated * Calculated

Isoelectric point” 5.52 6.23 7.06 4.95 5.25 5.08 4.95 5.26 6.75 6.11

199

ELECTROPHORESIS

Proteins Molecular weight

Hydrophobicity* 144 144 143 146 448 450 452 201 152 478

so that both the correct amino acid sequence and the presence of the correct disulfide linkages could be confirmed (17). Nonreducing conditions were also used for the IGF-II digestion (18). A l-ml aliquot of a 1.5 mg/ml solution of pepsin in 0.01 M HCl was added to about 30 mg of IGF-II, then the solution was diluted to 5 mg/ml IGF-II with 0.01 M HCl. The digest was allowed to proceed for 48 h at room temperature. Aliquots of the digest mixtures were frozen (-20°C) for use at a later time. The IGF-II digest mixture was examined by a gradient procedure on reversed-phase high-performance liquid chromatography (RP-HPLC). A Beckman (Beckman Instruments, Palo Alto, CA) Ultrasphere ODS 25 cm X 4.6 mm column operated at 40°C and 1 ml/min flow rate was used for the separation. The injection volume was 10 ~1; detection was at 214 nm. The gradient consisted of 4% solvent B initially, followed by increasing B at 05%/min for 40 min, then increasing B at 0.75%/minute for an additional 40 min followed by a 5-min hold at 54% solvent B. Solvent A was a mixture of 500 ml of phosphate buffer, 460 ml purified water, and 40 ml acetonitrile; solvent B was a mixture of 500 ml of phosphate buffer, 150 ml purified water, and 350 ml acetonitrile. The phosphate buffer was 0.3 M monosodium phosphate adjusted to pH 4.0 with 85% phosphoric acid. Preparative separation of IGF-II digest fractions were carried out in a similar manner using a Vydak (Separations Group, Hesperia, CA) protein and peptide Cl8 25 cm X 10 mm semipreparative column at a flow rate of 2 ml/min and an injection volume of 500 ~1. The gradient was modified to 0% B for 25 min, followed by increasing B at O.S%/min for 25 min, then increasing B again at l.O%/min for 30 min. Aliquots were manually collected in polypropylene containers and evaporated to dryness on a Speed-Vat concentrator (Model SVC lOOH, Savant Instruments, Inc., Farmingdale, NY). Fractions were desalted by dissolution of the dried fraction in 2 ml of purified water followed by injection onto a

CAPILLARY

1

of Intact

from a comnuter program based on Skoog and Wichman according to Meek and Rossetti (34). -

IN

5808 5964 6120 5809 22125 22126 22127 9389 7470 22818

Amino acid residues 51 52 53 51 191 191 191 86 67 199

Number chains/ disulfides 2/3 2/3 2/3 213 l/2 l/2 lJ2 l/3 l/3 l/2

(23).

SepPak Cl8 cartridge (Waters, Milford, MA). The cartridges were prerinsed with 20 ml methanol and 20 ml purified water, the sample was applied, the salt was removed by rinsing with 50 ml purified water, and the material was eluted with 5 ml of a 1:l acetonitrile:water mixture into a polypropylene tube. The final eluant was evaporated to dryness using the Speed-Vat concentrator. These purified fractions were examined for purity using the analytical RP-HPLC procedure and were subsequently used for the spiking experiments to determine the peak identities in the CZE electropherograms. The identification of the digest fragments for hGH at pH 8.15 has been previously reported (10); the identification at pH 2.35 was performed in an analogous fashion. The structure of hGH and the composition and properties of the digest fragments have been given previously (10-11). The identification of the digest fragments for IGF-II for both pH conditions was performed in a manner similar to the procedure used for hGH by spiking isolated fractions into the unseparated digest mixture. The structure and number system for the digest fragments have been given (18); the composition and properties of the fragments are given in Table 2. The concentration of the analyte in all studies was about 1 mg/ml total protein except for the IGF-II digest which was about 0.25 mg/ml. Protein samples were diluted in the separation buffer; the digests were thawed and used directly. The purified digest fragments were dissolved in purified water and aliquots diluted with the separation buffer. Data were obtained on an Applied Biosystems Inc. (Santa Clara, CA) Model 270A instrument. The sample (approximately 8-16 nl as estimated from the Poiseuille equation for a 2.5- to 5-s injection) was introduced by applying vacuum (5 in. of Hg) to a capillary that was approximately 100 cm in length with 80 cm to the detector. For low pH separations, the column was rinsed with mobile phase between injections or successively with 0.1

200

RICKARD,

STROHL,

AND NIELSEN

TABLE Composition Fragment number

and Properties

Isoelectric point”

of Fragments

2

from Pepsin

Hydrophobicity”

Digestion

of Insulin-Like

Molecular weight

Amino acid residues

1 2-7

6.36 3.97

3.6 28.3

936 1436

8 13

2A-7

3.97

36.1

1650

15

2%7

3.97

37.5

1549

14

3 3A 4-10

3.35 3.35 5.99

-3.4 -0.9 31.5

575 446 1867

5 4 17

4-10A

4.64

30.7

2097

19

4B-10

5.99

37.7

2030

18

5 5A 6 8 9

13.10 10.10 3.85 5.70 3.85

-4.1 -2.2 0.3 7.0 -3.9

1619 664 359 315 361

14 6 3 3 3

Growth

Factor II Amino acid sequence’

AYRPSETL 2: CGGEL 7: ECCFRSCD 2A: TLCGGEL 7: ECCFRSCD 2B: LCGGEL 7: ECCFRSCD VDTLQ VDTL 4: FVCGDRGF lo: YCATPAKSE 4: FVCGDRGF 10A: E’NCATPAKSE 4B: FVCGDRGFY lo: YCATPAKSE FSRPASRVSRRSRG FSRPAS IVE LAL LET

’ Calculated from a computer program based on Skoog and Wichman (23). * Calculated according to Meek and Rossetti (34). ’ Single-letter code for amino acids used.

M sodium hydroxide and mobile phase when the mobile phase composition was changed. For moderate pH separations, the column was rinsed for 3 min with 1 M NaOH followed by a 6-min rinse with the separation buffer between each injection. Separation conditions were 30 kV applied voltage and 30°C. The components were detected by uv absorbance at 200 nm. Analog data were collected directly from the absorbance detector on an in-house centralized chromatography computer system based on the Hewlett-Packard Model 1000 minicomputer that has storage, manipulation, and graphics capabilities. The electrophoretic mobility was determined by measurement of the migration times relative to that of a neutral marker, mesityl oxide, for the pH 8.15 separations. For the pH 2.35 separations, the electrophoretic mobility of hGH was determined compared to mesityl oxide and the mobilities of the analytes were determined relative to hGH. Equations [l] and [2] describe the relationship between electrophoretic velocity (V,,, cm/s), electroosmotic velocity (V,,, cm/s), total velocity of the analyte (V,, cm/s), and electrophoretic mobility (p,, cm’/Vs) where t, is the migration time of the analyte (set), t,, is the migration time of the neutral marker (set), 1 is the length of the capillary to the detector (cm), E is the electric field strength (V/cm), L is the total length of the capillary (cm), and V is the separation voltage (V).

v,, = v, - v, = 1/t, - 1/t,,

PI

p, = V,,/E = V,, x (L/V).

PI

The ionic strength of the buffers was calculated from the following values: glycine, pKal, 2.35 (carboxylate), pK,z 3 9.78 (amine); tricine, pK,,, 2.33 (carboxylate), pK,,, 8.15 (amine); morpholine, pK,, 8.40. The calculated ionic strength includes contributions from all species with a net charge; concentrations of buffer components were determined using the acid dissociation constants without consideration of activity effects.

RESULTS

AND

DISCUSSION

The correlation of electrophoretic mobility with physicochemical variables is complicated both by the lack of a well developed theory for the expected relationships and by the difficulties in evaluating the physicochemical parameters. The theory must account for the movement of a charged ion of finite size through an ionic media in the presence of an electric field. However, that field is distorted by the presence of the ions and its effective strength is influenced by the dielectric constant of the media. The charge and size of the analyte must be determined but both of these are affected also by the pH of the media and its composition (e.g., ionic strength). Fi-

CORRELATIONS

OF

ELECTROPHORETIC

MOBILITIES

IN

CAPILLARY

ELECTROPHORESIS

201

nally, the transport phenomena through the media must be considered. Many of these interrelationships have been discussed theoretically by Van Holde (19). In contrast, most conclusions from experimental explorations have necessarily invoked simplifying assumptions or focused on a single aspect. For instance, the species were usually considered to be spherical and Stoke’s law was used to estimate the frictional forces. We will briefly review the factors that affect electrophoretic mobility-charge and size of analyte and the pH, ionic strength, viscosity, and temperature of the separation buffer-and the underlying assumptions as we present the experimental results.

strengths produce a diffuse and extended atmosphere. Mathematically, Henry’s function varies from 1.0 to 1.5 in a sigmoidal fashion as the quantity KR varies from zero (zero ionic strength) to infinity (large ionic strengths). The corresponding values for the correction term are 1.0 to 0.0. That is, the electric field experienced by the ion is effectively reduced due to shielding from its ionic atmosphere as the ionic strength is increased. The mobility, however, would still be directly related to the charge and inversely related to the size (radius) of the analyte for a given set of experimental conditions. Although this reduction in electric field strength is important, the ionic character of the media produces other effects that alter the electrophoretic mobility. For Electrophoretic Mobility example, the charged analyte interacts electrostatically Electrophoretic migration occurs when a species with with the surrounding charged ions of the separation a net charge is placed in an electric field. The force exbuffer. The surrounding ionic atmosphere also effecerted by the electric field is proportional to the charge tively increases the apparent size of the analyte ion and on the species. For example, the force exerted on a produces microscopic anisotropy in the electric field. charged (gaseous) ion moving through a vacuum is proHowever, it is difficult to quantitatively account for portional to the charge since there is no resistance to these additional effects. movement. When the species is placed in a medium, the Offord’s (13) treatment of this problem for paper elecnet force must include an additional term related to the trophoresis assumes that an ion moving through a confrictional drag. The net electrophoretic mobility is ducting media would experience a retarding shear force achieved when the force due to the electrostatic attracthat would be proportional to the surface area of the tion is exactly balanced by the frictional drag of the meanalyte. This treatment predicts an electrophoretic modium. For a charged spherical particle moving slowly bility that is inversely proportional to the square of the through a nonconducting medium, one may use Stoke’s radius (rather than the first power as given by Stoke’s law for the frictional drag to obtain Eq. [3] (19-20) law). Offord then used his relationship to show that data from a wide variety of peptides would fit this 131 model. Most other treatments have relied upon experipep = ql&r~R, mental approaches to estimation of size with a relationwhere q is the net charge on the species (equal to the ship of the form of Eq. [3]. Since the effect of size is electronic charge times the units of charge), 17is the vismathematically inseparable, at least in simple models, cosity of the separation buffer, and R is the radius of the from the type of interaction that is theoretically prespecies. dicted by the various models (e.g., radius dependent However, the situation is more complicated when the drag or surface area dependent shear force), we shall media is a conducting solvent, e.g., an aqueous buffer. In consider effects from size and shape before discussing this case, the migrating species is surrounded by an our experimental results. ionic atmosphere of the opposite charge that modifies the electric field. The magnitude of the electric field corSize and Shape rection can be estimated by the Debye-Htickel theory; it is dependent upon the ionic strength (1, mol/liter), the When calculating electrophoretic mobilities, the dielectric constant of the medium (D), and the temperashape of chemical species traditionally has been asture (T, “K). The corrected electrophoretic mobility is sumed to be spherical, perhaps for the following reagiven by Eq. [4] where X(KR) is Henry’s function and K is sons. First, the rapid changes in shape and orientation the reciprocal ion-atmosphere radius defined by Eq. [5] of small molecules would tend to reduce any shape efwhere N is Avogadro’s number, k is Boltzman’s con- fect unless there were a net overall orientation of the stant, and e is the electronic charge (19). molecule in an electric field. In those cases, it is relapep = (q/6avR) K

X X(KR)/(~

+ KR).

= [ (8aNe’I) /( 1000DkT)]l’z.

[41 [51

For large ionic strengths, the ionic atmosphere is shrunk tightly around the ion whereas low ionic

tively straightforward to correct for a different shape if that shape is known (21). For example, Grossman and Soane (20) considered the orientation of rod-shaped molecules during a CZE separation. Second, the solvation sphere and ionic atmosphere that surround ions in solution will tend to smooth out irregular shapes. Fi-

202

RICKARD,

STROHL,

nally, it is difficult to experimentally elucidate the shape of large proteins in solution. However, it is likely that a spherical shape is only rarely correct in a quantitative manner. For instance, polypeptides and proteins long enough to have secondary and tertiary structure have severe constraints on the shapes that they may assume. Nevertheless, we shall assume a spherical shape in this work. The volume of protein and peptide molecules is proportional to their molecular weight (mass) if the density is constant. Thus, the radius of a sphere containing an equivalent volume @rr3) will be proportional to the cube root of the molecular weight. A mobility model that predicts that frictional drag is related to the radius of the species will be proportional to the f power of the molecular weight. Similarly, models that use the surface area of that sphere (47~~) predict a mobility that is proportional to the f power of the molecular weight. Note that the cross-sectional area (nr”) is proportional to the $ power also. Finally, extensive studies of synthetic polymers have shown that the average radius of gyration is proportional to the square root of the number of polymer units times the length of a single unit (22). If the frictional drag were proportional to the radius of gyration, it would be proportional to the square root of the number of residues (approximately the square root of molecular weight). With the above assumptions, we would predict the following relationships for the dependence of electrophoretic mobility in solution on molecular weight:

AND

NIELSEN

$ 5 e % 9 5

D

800

1100

Time FIG. 1. buffer; (11).

Electropherogram other separation

1400

1700

2000

(set)

of hGH digest in pH 2.35,0.1 conditions given in text. Reprinted

M glycine from Ref.

not shown). The correlation of electrophoretic mobility with the radius, Eq. [6a], is clearly the worst and cannot be supported. In all cases, the fit for Eq. [6c] is better than that for Eq. [6b] although the difference was minor in some instances. Based on these results and with the support from Offord’s theoretical treatment, Eq. [6c], the model that predicts that drag will be proportional to the surface area (I power of the molecular weight), was chosen for further testing. Charge

Charge is the final component to electrophoretic mobility. The charge is highly dependent upon the pH of Radius of gyration (square root of chain length): MW-‘” Ml the separation buffer due to the ionization of the acidic Offord’s relationship (surface area): MW-2’3. 16~1 and basic side chains as well as the carboxy and amino terminal groups of peptides and proteins. Thus, selecAs described in the introduction, other workers have tion of pH is one of the most important factors that can applied all three models to experimental data with some be manipulated to alter selectivity for protein and peptide separations. Conversely, accurately predicting the success. In this work, we examined the experimental mobilities obtained for the hGH and IGF-II digests at change in charge as the pH is varied is crucial for preboth low and moderate pH values to determine the best dicting selectivity. That is, once the charge relationship correlation of mobility with size. Data were plotted as is known, then changes in electrophoretic mobilities as the observed electrophoretic mobility vs the quantity of the pH is changed could be predicted. The calculation of the net charge of an ionic species calculated charge divided by the molecular weight to the various powers. using the Henderson-Hasselbalch equation is quite simple if the pH of the surrounding media and the ionizaThe electropherogram for the separation in the pH 2.35 buffer is given in Fig. 1. The corresponding results tion constants are known. The charge on each group is for the correlations obtained using Eqs. [6a], [6b], and assumed to be independent of that contributed by other [6c] are given in Fig. 2. The equations and correlation groups in the molecule. The pl (isoelectric point) of a coefficients for the least squares linear fit are given. It protein can be calculated from the same equations using can be seen that best fit is obtained with Eq. [6c], Of- an iterative mode until the sum of all of the charged ford’s relationship. Electropherograms for the pepsin residues approaches zero. That approach was used in this work to calculate charges and isoelectric digests of IGF-II are given in Figs. 3 and 4; the digest fragments are indicated. These data and the pH 8.0 points (23). However, the estimation of the appropriate ionization hGH digest data were examinedvs the alternative mobility models with the results obtained in Table 3 (raw data constants is quite difficult. Although the ionization conNonconducting

media

(Stoke’s

law,

radius):

MW-‘”

lsal

CORRELATIONS

OF

ELECTROPHORETIC

MOBILITIES

IN

CAPILLARY

203

ELECTROPHORESIS

4.OOe-4

a

W

o.ooe+o~..~-. 0.10

0.15

'I 0.20

W

......r...s...I 0.25

0.30

0.35

o.ooe+oI

0.40

0.02

0.04

0.06

q I MWf’113

0.06

0.10

0.12

0.14

0.16

q 1 MWA112

o.ooe+oJ

4

“1’ 0.00

0.01

0.02

0.03

0.04

0.05

0.06

q I MWA2/3 FIG. 2. Fit of electrophoretic mobility (cm*/Vs) Equations and correlation coefficients for the linear pK, values.

vs charge to size parameter for hGH digest separated in pH 2.35, 0.1 M glycine buffer. least squares lines are given. Data are plotted for various mobility models using Shields’

(a)

mobility

vs q/MWL’3

y = 9.09e - 4x - 3.17e - 5

r = 0.768

(b)

mobility

vs q/MW”’

y = 2.58e

- 5

r = 0.955

(c)

mobility

vs q/MW2’3

y = 6.5Oe - 3x + 2.45 e - 5

r = 0.989

stants for isolated amino acids are well known, they are shifted for all species larger than a single amino acid. There are three primary reasons for these shifts. The most important is that formation of the first peptide bond induces an electrostatic change in the charge on the neighboring amino and carboxy groups. The net result is that the terminal carboxylic acid becomes about lo-fold weaker (pK, shifts from about 2.2 to about 3.2) and the terminal amine is even more affected (pK, shifts from about 9.5 to about 8.1). This effect is somewhat dependent upon the specific neighbor. For example, Field et al. (24) characterized the effect observed when the neighboring amino acid was basic. In contrast, the ionization constants of the side chains remain at similar values as for the free amino acids. The second reason for the shift in ionization constants is the multitude of microenvironments found within proteins (25-26). That is, most charged groups are exposed to an aqueous environment since they tend to occur at the surface of the

- 32 - l.lle

protein. However, some are found deep enough within the protein that they may have only limited exposure to the media. Those groups, especially those associated with biologically active sites, reside in environments where their ionization constants may be dramatically altered. The relative degree of hydrophobicity of their environment (more hydrophobic environments favor uncharged species over charged species), changes in the local dielectric constant (from 78 in water to about 3-5 in the interior of a protein; lower dielectric constants favor uncharged species over charged species), and induced effects from peptides held in close proximity by conformational restraints are other factors that produce a shift in the ionization constants. The effect of microenvironments on mobilities in CZE was demonstrated convincingly by Wiktorowicz and Colburn (27) for site-directed mutants ofAspergillus oryzae ribonuclease Tl. Finally, ionization constants, like all equilibria, are subject to ionic strength effects in the media. In

204

RICKARD,

STROHL.

-I

900

1260

1620

Time FIG. 3. Electropherogram buffer; other separation

1960

2340

2700

AND

NIELSEN

are nearly completely protonated. However, it is much more difficult to calculate accurate charges for the pH 8.15 data, especially for species that contain histidine. The ionization constant of the histidine side chain is quite variable and, moreover, typically lies near pH 6 but may vary from 5 to at least 8 depending upon its environment (30). Tanokura et al. (31) estimated the ionization constants for histidine in model di- and tripeptides by NMR; his best estimates were 6.4 to 6.8 which are in good agreement with the value of 6.2 that we used. Thus, our values seem to be consistent with average values from the literature and avoid the necessity of measuring values for each species.

(set)

of IGF-II digest in pH 2.35,0.1 conditions given in text.

M glycine

summary, the ionization constants of ionizable groups in peptides and proteins differ from those in the corresponding free amino acids in a very complex manner such that it would be nearly impossible to determine the correct ionization constant for each group in a protein. Thus, the following approach was used in this paper. First, we tested the conclusion that the ionization constants for the free amino acids were inappropriate. These data are shown in Fig. 5 where the electrophoretic mobilities for the pH 2.35 hGH digest fragments are plotted vs the Offord relationship using a charge calculated from the dissociation constants for free amino acids. Although these data fit the relationship, there is a significant lack of fit that can be observed visually and by comparison of the correlation coefficient for this plot compared to that for Fig. 2c; the charge for Fig. 2c was calculated using the ionization constants appropriate for peptides and proteins. Second, we attempted to find ionization constants from the literature. Many values for specific groups in individual proteins, but relatively few average values, have been reported. Hirokawa et al. (28) estimated the N-terminal pK, values for a series of 28 dipeptides by isotachophoresis. Sillero and Ribeiro (29) used values compiled by Matthew (26) for the side chains and values of 3.2 and 8.2 for the carboxy and amino terminal pKvalues, respectively. We chose to use a set of values developed at Lilly by Shields (personal communication); they were chosen to fit the behavior (not electrophoretic) of BHI and hGH and are similar to those used by Sillero and Ribeiro and found by Hirokawa et al. These values are given in Table 4. The calculated charge at very low pH (below 1.5-2) is nearly insensitive to the exact values of the ionization constants. Even at pH 2.35, one would expect very good agreement of calculated charge vs actual charge (if that could be measured) since the carboxy ionization constants are relatively uniform and most carboxy groups

Separation

Buffer

The last set of parameters to be considered are those of the separation buffer. It is well known that the electrophoretic mobility depends upon temperature, a value of about 2%/“C is usually assumed. Therefore, no investigation of temperature effects were made. The effects of viscosity and ionic strength are less well characterized. Furthermore, changes in ionic strength change the electroosmotic flow velocity (11) and dielectric constant (above). Burgi, Salomon, et al. (32,33) considered the interrelationships of viscosity, ionic strength, and dielectric constant as organic solvent (methanol) was added to the separation buffer. Among other effects, adding methanol gave lower electrophoretic mobilities due to the increased viscosity of the separation buffer. However, there was still a decrease in the product of electrophoretic mobility times viscosity as the amount of methanol was increased whereas Eq. [3] predicts that mobility times viscosity should be constant. They ascribed this residual effect to a change in the ionization constant resulting from a lower dielectric constant as methanol was added. A lower dielectric constant favors

I

I

I 400

660

960

Time FIG. 4. Electropherogram of IGF-II tricine, and 0.02 M morpholine; other text.

1240

1520

1800

(set) digest in pH 8.15 buffer, 0.1 M separation conditions given in

CORRELATIONS

OF

ELECTROPHORETIC

MOBILITIES TABLE

Linear Least Squares

IN

CAPILLARY

3

Fit of Experimental

Mobilities

to Mobility

Models

Correlation Separation buffer

Digest pH pH pH pH pH

hGH hGH hGH IGF-II IGF-II

q/MWlf3

2.35 glycine 8.0 tricine/morpholine/NaCl 8.15 tricine/morpholine 2.35 glycine 8.15 tricine/morpholine

0.768 0.909 0.923 0.785 0.863

the neutral species and leads to a lower net average charge. We looked at the effect of ionic strength on the separation of the hGH digest at pH 8.15 (Fig. 6). In these data, the concentration of morpholine was held constant and the amount of tricine varied. As the ionic strength increased, the electroosmotic flow velocity decreased (as measured by the elution of mesityl oxide). In addition, the selectivity was relatively unchanged but the electrophoretic velocity obviously decreased as the ionic strength increased. All species were affected similarly as shown by plots of electrophoretic mobility vs ionic strength. This uniform response is not consistent with a shift in the dissociation constants. The effect is qualitatively in agreement with a decrease in effective electric field strength as predicted by Eqs. [4] and [51. However, the change was larger than that predicted. Other contributing factors could have been changes in viscosity with buffer concentration or temperature. Higher buffer concentration would increase viscosity and give lower mobilities, but increased temperatures (from higher currents at high buffer strengths) would increase

o.oofl+o~ 0.00

1 0.01

0.02

q I MW*2/3

0.03

from amino

0.04

- 3x + 4.21e

- 5

coefficient

q/MW’” 0.955 0.938 0.955 0.864 0.925

q/MW’” 0.989 0.940 0.963 0.923 0.933

mobility by decreasing viscosity. That is, an increased buffer viscosity would augment the effect of ionic strength but increased temperature would counteract the effect of increased ionic strength in Eq. [5]. Thus, both a shielding of the electric field and an increase in viscosity could decrease the electrophoretic mobility of a species in a relatively uniform manner. Correlation across Separation Conditions Based on the above considerations, it should be possible to correlate the electrophoretic mobilities with the quantity q/MW2j3 for different species and at different pH values as long as variables such as ionic strength, temperature, and viscosity remain constant. The data collected in our characterization of the digests are suitable for testing this hypothesis. Furthermore, the data from the intact proteins represent an important addition in that these species have more rigid structure and the charged residues may not be entirely exposed to the solution. The data from this correlation are plotted in Fig. 7. An excellent correlation was obtained with these data from all conditions fitting on the same least squares line. Furthermore, the observed electrophoretic mobility is approximately zero when the calculated charge is zero. That is, the intercept of the line is approximately zero in spite of practical experimental difficulties in measuring absolute mobilities. These data have been gathered over a period of about 2 years and recent checks of the old data produced experimental mobility values in excellent agreement with the old data. However, it has not been possible to check other limitations to this approach due to the lack of published absolute mobilities for proteins. For example, one might find that larger proteins or those with extreme pl values would not fit this model.

acid pKa

FIG. 6. Fit of electrophoretic mobility vs q/MWZ’3 for hGH separated in pH 2.35, 0.1 M glycine buffer. The equation and tion coefficient for the linear least squares line is given; pK, are from isolated amino acids. y = 7.71e

0.05

205

ELECTROPHORESIS

r = 0.956.

digest

correlavalues

CONCLUSION

The results show that the electrophoretic mobilities measured by capillary electrophoresis for a highly diverse set of peptides and proteins are linearly correlated with the quantity q/MW213 where q is the net charge on

206

RICKARD,

STROHL,

AND

TABLE Values

of Ionization

Constants

Adjusted Amino acid residue Ala Arg Asn ASP

(A) (R) (N) CD)

CYS (0

Gin Glu

(Q)

Gly

(G)

His Ile Leu LYS Met Phe Pro Ser Thr Trp Tyr Val

(H) (I) (L) W (M) (F) (P) (S) (T) Of’) (Y) (V)

(E)

’ Calculated

from

values

N-terminal

3.20 3.20 2.15 2.75 2.75 3.20 3.20 3.20 3.20 3.20 3.20 3.20 3.20 3.20 3.20 3.20 3.20 3.20 3.20 3.20

8.20 8.20 7.30 8.60 7.30 7.70 8.20 8.20 8.20 8.20 8.20 7.70 9.20 7.70 9.00 7.30 8.20 8.20 7.70 8.20 by Shields

4

Used

for

the

Calculation

of Charge

values”

C-terminal

given

NIELSEN

(personal

Isolated Side-chain

amino

C-terminal

N-terminal

2.34 1.91 2.06 2.02 1.93 2.17 2.15 2.35 1.79 2.34 2.35 2.17 2.28 2.37 1.98 2.20 2.09 2.40 2.20 2.30

9.87 9.02 8.82 9.85 10.40 9.13 9.57 9.78 9.18 9.72 9.67 9.06 9.24 9.21 10.62 9.18 9.10 9.42 9.11 9.68

12.50 3.50 10.30 4.50 6.20

10.30

10.30

acid Side-chain

12.48 3.82 8.26 4.18 6.08

10.66

10.11

communication).

the species and MW is its molecular weight; that is, the frictional forces opposing the electrophoretic migration appear to be proportional to the surface area of the species. Furthermore, the surface area could be well approximated by assuming that the analyte is a sphere whose radius is proportional to the cube root of the molecular weight. Finally, the net charge on the species could be calculated from the amino acid composition when the acid dissociation constants, pK,, , were chosen

to be consistent with those of typical peptides and proteins. The ability to predict changes in electrophoretic mobilities across wide variations in pH will allow one to guide the selection of optimum separation conditions. This correlation gave excellent results when the ionic strength, temperature, and viscosity were maintained relatively constant. Thus, predictions of the effect of

*

3.000e-4

g % z y a, s 5

l.OOOe-4

-1.000e-4

z i Ill

I 400

640

660

Time

1120

1360

-3.000e-4

-0.04

-0.02

-0.00

0.02

0.04

0 .( 16

q I MWA2/3

1600

(set)

FIG. 6. Ionic strength effects on the electrophoretic mobility for the hGH digest. pH 8.15 separation with 20 mM morpholine plus (A) 20 mM tricine, p = 17 mM; (B) 60 mM tricine, p = 37 mM; (C) 100 mM tricine, 1 = 57 mM; and (D) 200 mM tricine, p = 107 mM.

FIG. 7. Fit of electrophoretic mobility vs q/MW” for all data. Data include hGH digest (pH 2.35, 8.0, and 8.15) and IGF-II digest, BHI, hGH, IGF-II, hP1, and bST (pH 2.35 and 8.15). The equation and correlation coefficient for the linear least squares lines is given; Shields’ pK, values are used. y = 7.08e - 3x - 1.65e - 5

r = 0.948.

CORRELATIONS

OF

ELECTROPHORETIC

MOBILITIES

physicochemical variables, especially pH, can be cornbined with empirical determination of critical separation variables, such as buffer composition. This appreach will lead to rapid development of optimum separations when the effects of ionic strength, temperature, and viscosity are understood. ACKNOWLEDGMENT We acknowledge

the technical

assistance

given

by P. A. Farb.

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