Isoelectric focusing by free solutioncapillary electrophoresis

ANALYTICAL BIOCHEMISTRY 2 0 6 , 84-90 (1992)

Isoelectric Focusing by Free Solution Capillary Electrophoresis Shiaw-Min Chen a n d J o h n E. Wiktorowicz Applied Biosystems, Inc., 850 Lincoln Centre Drive, Foster City, California 94404

Received March 27, 1992

A reproducible, quantitative isoelectric focusing method using capillary electrophoresis that exhibits high resolution and linearity over a wide pH gradient w a s d e v e l o p e d . R N a s e T1 a n d R N a s e b a a r e t w o prot e i n s t h a t h a v e i s o e l e c t r i c p o i n t s (pFs) at t h e t w o e x tremes of a pH 3-10 gradient. Site-directed mutants of the former were separated from the wild-type form and p/'s determined in the same experiment. The p/'s of R N a s e T~ w i l d - t y p e , its t h r e e m u t a n t s , a n d R N a s e b a w e r e d e t e r m i n e d f o r t h e first t i m e as 2 . 9 , 3 . 1 , 3 . 1 , 3 . 3 , and 9.0, respectively. The paper describes the protocol f o r i s o e l e c t r i c f o c u s i n g b y c a p i l l a r y e l e c t r o p h o r e s i s , as well as presenting data describing the linearity, resolution, limits of mass loading, and reproducibility of the method.

© 1992 Academic Press, Inc.

Isoelectric focusing (IEF) 1 is a method for purification and analysis t hat causes charged molecules to migrate electrophoretically through a pH gradient until they experience a p H at which they exhibit zero net charge. At the time of its introduction (1), IEF was accomplished in a density-stabilized, vertical column of ampholytes. These ampholytes were small zwitterions t h a t exhibited sufficient buffering capacity in order to maintain a stable pH gradient. Sample was dissolved in the ampholyte solution, and electrophoresis was performed in free solution, i.e., without the use of gels or solids as anticonvective agents. Partly because of the attractiveness of IEF as a first dimension for two-dimensional electrophoresis (2) and the need therefore for a semisolid matrix, but mostly because convective currents generated by Joule heating limited the resoluAbbreviations used: IEF, isoelectric focusing; CE, capillary electrophoresis; CCK, cholecystikin; RNase A, ribonuclease A; RNase T1, ribonuclease T~; RNase ba, ribonuclease from Bacillus amylofasciens; DB-1, dimethyl polysiloxane; T E M E D , N,NJVJV-tetramethylethylenediamine; DB-17, dimethyldiphenylpolysiloxane. 84

tion achievable by this system, IEF was further developed using a cross-linked polyacrylamide matrix. Because of this development, standard detection strategies and gel handling mechanisms were quickly applied to IEF analysis of proteins and peptides. The high degree of resolution led to its popularity as an analytical technique for the measurement of pFs as well as another method for gauging purity and establishing identity. Commercial systems designed to partially automate the separation and detection became available (Phast systems, Pharmacia, Piscataway, NJ). However, the limitations of gel-based separations, in addition to the semiquantitative nature of current staining strategies, hinder the realization of the full potential of IEF; e.g., loss of resolution due to Joule heating, inability to accurately quantify the am ount of analyte in bands, long analysis time, labor intensive preparation, postseparation handling, and large sample mass requirements. T h e most recent developments in gel-based IEF address some of these limitations, such as the application of silver staining to decrease mass loading requirements, t hi nner gels to permit more efficient heat dissipation, or shorter gels to decrease analysis time, but all achieve their benefit through some sacrifice, either in expense, resolution, gel fragility, etc. The most recent IEF development, using capillary electrophoresis (CE-IEF) as the separation strategy (3-9), maintains and enhances all the advantages of gel-based IEF but suffers none of the disadvantages. This method rediscovers the merits of free solution electrophoresis without the detrimental effects created by convection by accomplishing separations in a 50-#m diameter fused-silica capillary. T h e high efficiency of heat dissipation of the smaller diameter tube permits high field strengths (volts per unit length) and therefore higher resolution and shorter analysis times. T he narrow diameter capillary permits analysis with minute mass requirements (pg). In addition the uv transparency of the fused silica permits online detection with precise and accurate quantification. Finally the free solution format eliminates the need for 0003-2697/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

CAPILLARY ELECTROPHORESIS-ISOELECTRIC FOCUSING polymerization steps in preparation for electrophoresis and permits complete control over the composition of the separation buffer. In general, C E - I E F poses two challenges. Since detection is on-line, focusing must be performed in the absence of endosmotic flow; however, once established, the ampholyte gradient must be mobilized past the online detector in order to observe the focused proteins and pH gradient. Various strategies have been published (3,5,6,8,9), and the yardstick for effectiveness is (or should be) the demonstration of the linearity of the pH gradient, the accuracy and precision of the pI estimation, and the degree of resolution achieved. Indeed, the majority of the literature on C E - I E F is dedicated to the resolution of these two challenges (see Discussion for more complete comparison). This paper describes capillary electrophoresis isoelectric focusing and demonstrates its utility in the measurement of the pI of proteins at the extremes of a pH 3-10 gradient. In addition, the linearity, accuracy, precision, and resolution observed for this technique at the extremes of the pH gradient is discussed. MATERIALS AND METHODS In this study Servalyt 3-10 was purchased from Serva (Serva Fine Chemicals, Paramus, NJ), and methylcellulose (1500 cps at 2%) from Sigma (Sigma Chemicals, St. Louis, MO). Carbonic anhydrase (pI 5.9),/~-lactoglobulin A (pI 5.1), and ribonuclease A (RNase A, pI 9.45) were obtained in the highest purity from Sigma. Unsulfated cholecystikinin (CCK) flanking peptide (pI 2.75) was obtained from Peninsula Labs (Belmont, CA.). Ribonuclease T1 (RNase TO and mutants from Aspergillus oryzae and ribonuclease from Bacillus amylofasciens (RNase ba) were kind gifts of Drs. C. Nick Pace and Bret Shirley at Texas A&M University. All other reagents used were of analytical grade. Capillary electrophoresis was performed on the Applied Biosystems Model 270A-HT capillary electrophoresis system (Foster City, CA) using a dimethylpolysiloxane (DB-1)-coated capillary with 50 #m internal diameter and 0.05 #m coating thickness (J&W Scientific, Folsom, CA). The total length of the capillary used in this study was 72 cm and the length from the sample injection end to the detector was 50 cm. The output of the 270A-HT was plotted and integrated by a HewlettPackard Model 3396 integrator. The protocol used to achieve the separations requires the accurate application of precise vacuum to the capillary in order to fill it with the correct amount of electrode buffers, ampholytes, and sample. The system was programmed to first fill the capillary with 20 mM NaOH in 0.4% methylcellulose (8 min of 20 in. Hg vacuum), followed by a section of 0.5% Servalyt 3-10 in 0.4% methylcellulose (4.5 rain of 20 in. Hg vacuum). An ali-

85

quot of sample and marker (dissolved in Milli-Q water) was loaded separately into the capillary (30 s of 5 in. Hg vacuum each) and another section of 0.5% Servalyt 3-10 with 0.4% methylcellulose (0.1 min of 20 in. Hg vacuum) was added to the capillary. As illustrated in Fig. 1, at this point the interface between the NaOH solution and Servalyt solution was positioned on the sample side of the detector. High voltage, usually 30 kV, was then applied for 6 min to focus the ampholyte and proteins. After focusing, the system was programmed to apply a precisely regulated vacuum (5 in. Hg), while maintaining the high voltage. The applied vacuum caused the focused zone of proteins in the capillary to flow pass the detector while the voltage maintained the pH gradient and zone sharpness even in the presence of the distorting effects of laminar flow. The protein bands were detected at 280 nm as they passed the detector under the influence of vacuum and voltage. RESULTS

Linearity of the pHgradient. The linearity of the pH gradient was determined by running RNase A, carbonic anhydrase, ~-lactoglobulin A, and CCK-flanking peptide as markers. Figure 2 shows the electropherogram of a 20-nl injection of 100 #g/ml of each protein (2 ng each loaded). The proteins were focused and mobilized as described under Materials and Methods. After mobilization and detection at 280 nm, the relative mobility of each protein was calculated (see below) and plotted against the published isoelectric point (Fig. 2, inset). The double peak before the RNase A in Fig. 2 represents the interface between cathodic buffer and ampholyte solution. The peak after CCK-flanking peptide is the interface of anodic buffer and ampholyte solution. These two interface peaks are characteristics of this procedure and can be used to indicate the beginning and the end of the pH gradient. The times at which these peaks appear after mobilization is initiated (mobilization vacuum and voltage applied) is used to calculate the relative mobility (Rm) as follows: Rm

=

to t f - to

tp

where tp is the mobilization time of the protein peak of interest, t o (origin) is the mobilization time of the cathodic buffer interface, and tf (front) is the mobilization time of the anodic buffer interface. The linearity of the pH gradient was determined by plotting the pI vs relative mobility (Fig. 2, inset). This plot shows this method can provide linear range between pH 2.75 and 9.5 (CCK-flanking peptide and RNase A, respectively). Resolution. RNase T1 from A. oryzae (wild-type) is a protein with a calculated pI of 2.9. Because of its ex-

86

CHEN AND WIKTOROWICZ Detector

~

Hydrodynamic flow LOAD CATHOLYTE

NaO

Detector

NaOH

~

~:)7..~:~.~7~'..~:~`~.7.;:.:~.~.`~.~:~`~i..;?.:~:.('~:~.ii/~:~.~i~:~.~:.:'.~:'?~.!.~Ampholyte :..~7.~.~`~(~(~.AMPHOLYTE ::.LOAD 7i:~.~.~: Detector

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Hydrodynamic flow

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:::::::::::::::: ::::::::::::;::.':::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::.',::;::.~?::::= ;:::::::::::;:::: . ........... ' : : . , v : : t "............. . ' . , ' : : . , , I.:::.,'.':.'.:.~ . . . . . . . . . . . . . . :.': ! ".'.'.'.'.,'::.:. . . . . . . . ...................... ............. ".:.:.:<~ .....: ...................................... ...................... ...........

li

F I G . 1.

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MOBILIZE

Capillary isoelectric f o c u s i n g procedure.

t r e m e acidic character, the m e a s u r e m e n t of its p I has b e e n difficult. In addition to the wild-type form, we have o b t a i n e d t h r e e site-directed m u t a n t s (courtesy of Dr. C. N. Pace). T h e first m u t a n t (Gln 25, Lys), r e p r e s e n t s a difference of + 1 from the wild-type e n z y m e and is design a t e d as m u t a n t I. A n o t h e r m u t a n t of interest contains the s u b s t i t u t i o n of Glu 58 to Ala, also r e p r e s e n t i n g +1 difference f r o m the wild type, and is designated as mut a n t II. T h e double m u t a n t , r e p r e s e n t s a difference of +2 f r o m the wild-type, is designated as m u t a n t III. Previous studies of these e n z y m e s have shown t h a t at neutral pH, all four forms m a y be s e p a r a t e d by capillary electrophoresis (10) despite the a p p a r e n t similarity in charge of the two single m u t a n t s . However, since the p I ' s of t h e s e m u t a n t s have n o t b e e n d e t e r m i n e d , interp r e t a t i o n of the different behaviors of the m u t a n t s in native free-solution CE was difficult. Nevertheless, f r o m the substitutions, one would expect the p I of mut a n t III to be the m o s t alkaline with respect to the wild type, while the p I ' s of m u t a n t s I a n d II should be slightly higher t h a n the wild-type R N a s e T 1. At the o t h e r p H e x t r e m e , R N a s e ba exhibits an alkaline p I p r e d i c t e d f r o m s t r u c t u r a l studies to a p p r o x i m a t e

9.0. T h e difficulty in m e a s u r e m e n t is similar to the difficulty in m e a s u r e m e n t of the R N a s e T1 isoforms. W e have used b o t h enzymes as a m e a n of exploring the limits of the s e p a r a t i o n power of C E - I E F . B o t h model proteins were focused in the presence of the four s t a n d a r d proteins (Fig. 3a). As expected wildtype R N a s e T1 exhibited the m o s t acidic p/, followed by the single m u t a n t s , I and II, and the double m u t a n t , III, with the least acidic pL T h e e l e c t r o p h e r o g r a m showed these four p r o t e i n s were s e p a r a t e d into three baselineresolved peaks. M u t a n t s I and II, however, focused as a single peak. T h e p I of R N a s e T1 a n d m u t a n t s was d e t e r m i n e d from the s t a n d a r d plot derived from the mobilization times of the s t a n d a r d p r o t e i n s (Fig. 3b). T h e p I of the R N a s e T1 wild-type was calculated to be 2.9, while the p I ' s of m u t a n t s I, II, and III are 3.1, 3.1, and 3.3, respectively. F r o m the same e l e c t r o p h e r o g r a m , R N a s e A a n d R N a s e ba were well-resolved from each o t h e r at the high end of t h e p H range as showed in Fig. 3a. F r o m the s t a n d a r d curve in Fig. 3b, the p I of the R N a s e ba was d e t e r m i n e d to be 9.0.

CAPILLARY ELECTROPHORESIS-ISOELECTRIC

FOCUSING

87

Linearity of pH Gradient 10.

8.

=

.

.

.

4

~o.0..... 0'2

0~

0.'6

0'8

10

Relative mobility (Rm)

I

I

I

I

I

I

I

I

I

I

5

I

I

I

I

I

10

I

I

I

I

I

15

I

I

20

Mobilization Time (min) F I G . 2. C E - I E F calibration. R N a s e A, carbonic a n h y d r a s e , B-lactoglobulin A, a n d C C K - f l a n k i n g p e p t i d e (100 # g / m l ) were injected for 30 s u s i n g 5 in. v a c u u m (2 n g each loaded). F u r t h e r details are given in t h e text. T h e linearity of t h e p H g r a d i e n t w a s m e a s u r e d by p l o t t i n g t h e p I of t h e s t a n d a r d p r o t e i n s a g a i n s t relative mobilities (as defined in t h e text). T h e e q u a t i o n s h o w n is t h e b e s t - f i t f i r s t - o r d e r linear regression.

Quantification. Q u a n t i f i c a t i o n a n d d y n a m i c r a n g e of the C E - I E F m e t h o d is d e m o n s t r a t e d in Fig. 4. S a m ples of fixed c o n c e n t r a t i o n (200 ~g/ml) were injected for various l e n g t h s of time. W h e n t h e p e a k a r e a as determ i n e d b y t h e i n t e g r a t o r was p l o t t e d v e r s u s the length of injection t i m e (Fig. 4), t h e p e a k a r e a i n c r e a s e d linearly

Ai I 2

I

I

with increasing injection t i m e over a n eightfold r a n g e ( f r o m 10 to 80 s, e q u i v a l e n t to 1.3 to 10.7 ng, respectively). Also, it should be n o t e d t h a t , within t h e e r r o r in the e x p e r i m e n t , the four lines c o n v e r g e d at t h e origin of t h e plot, testifying t h a t no p r o t e i n e n t e r e d t h e c a p i l l a r y unless v a c u u m was applied. T h e v a r i a b l e slopes exhib-

A I 5

I

I

I 8

I

I

J 11

I

I I

I

I

14

I

I 17

I

I 20

I

I

I 23

Mobilization Time (min) F I G . 3 a . Capillary isoelectric focusing of R N a s e T1 a n d R N a s e ba. S t a n d a r d p r o t e i n s were rejected as i n d i c a t e d in t h e legend to Fig. 2. R N a s e T 1 a n d m u t a n t s (200 ~ g / m l ) were injected for 30 s e a c h (4 ng). F u r t h e r details are given in t h e text.

88

CHEN AND WIKTOROWICZ 10.09.0-

pI = -8.51 Rm + 9.96 R2 = 0.998

8.0. 7.0

6.02 pi=3.3

5.0

~

/

RNaseT1-Wildtype p|=2.9

4.0 3.0 2.9 , , , , . . , 0.0 0.1

0.2

,,,,~,.,., 0.3 0.4

0.5

,..~.,,,'',,'' 0.6 0.7 0.8

0.9

, 1.0

Relative Mobility (Rm)

3b. Measurement of the isoelectric points of RNase T 1 and RNase ba. The linear plot was constructed to determine the pI of unknown proteins. The pls were calculated as described in the text and legend for Fig. 2 inset. FIG.

ited by the different e n z y m e s reflect their different extinction coefficients at t h e i r pI's. Reproducibility. In order to define t h e reproducibility of C E - I E F , five separations of the R N a s e T1 isoforms were p e r f o r m e d with i n t e r n a l p I s t a n d a r d s using four i n d e p e n d e n t l y p r e p a r e d solutions. T h e data are s u m m a r i z e d in T a b l e 1. R u n s 1 and 2 were p e r f o r m e d with the same solution, while runs 3, 4, and 5 were acc o m p l i s h e d in s e p a r a t e l y p r e p a r e d solutions. T h e p I ' s of the p r o t e i n s in each r u n were d e t e r m i n e d from the linear calibration plot of t h a t p a r t i c u l a r run. T h e data indicate a reproducibility of +0.1 p H unit, with relative s t a n d a r d deviations of the calculated p I ' s less t h a n 3% of the means. Capillary Stability. Stability of the capillary coating was d e t e r m i n e d over the course of 2+ months. During this period, C E - I E F was p e r f o r m e d each day with the e x c e p t i o n of weekends. T h e data p r e s e n t e d in Fig. 5 repr e s e n t a selection of s t a n d a r d curves run during this time period on Days 1, 19, 37, a n d 64, and d e m o n s t r a t e the stability of the coating u n d e r the conditions of C E I E F for a l m o s t 420 consecutive runs. T h e e x p e r i m e n t could n o t be e x t e n d e d for a longer period of time as the capillary was b r o k e n during handling. As can be seen f r o m Fig. 5, t h e linearity of each of the lines, quantified by the c o r r e l a t i o n coefficients of the least-squares fits, is quite precise, ranging from 0.993 on Day 64 to 0.998 on Days 19 a n d 37. DISCUSSION T h e two m o s t i m p o r t a n t steps in the capillary isoelectric focusing are the s t a t i o n a r y focusing step and the s t e a d y - s t a t e mobilization step. T h e s t a t i o n a r y step was

accomplished using a DB-1 b o n d e d - p h a s e capillary, in which endosmosis is sufficiently minimized. O t h e r c o a t e d capillaries have been described, such as polyacrylamide (3) a n d DB-172 ( J & W Scientific), which abolish endosmotic flow and m a i n t a i n the p H gradient s t a t i o n a r y during the focusing step. In this study, we f o u n d t h a t the DB-1 b o n d e d - p h a s e capillary m a i n t a i n s an adequately low endosmotic flow for the purposes of C E - I E F for over 2 m o n t h s (>400 runs). T h i s is based on the observation t h a t over this period of time, the linearity of the p H gradient remains u n c h a n g e d (Fig. 5). T h e logic t h a t relates endosmotic flow a n d p H gradient linearity is based u p o n the fact t h a t the loss of coating would result in increased e n d o s m o t i c flow. T h i s increase would v a r y during the mobilization step, since endosmotic flow is d e p e n d e n t u p o n p H and ionic strength. Since b o t h p H and ionic s t r e n g t h change during mobilization (as a result of a m p h o l y t e focusing and increasing a m o u n t of anode electrolyte drawn into the capillary, respectively), the loss of coating would result in n o n l i n e a r d e p e n d e n c e of migration time as a function of pH. Since linearity is m a i n t a i n e d , the coating is stable. This is in c o n t r a s t to alternative coatings used for I E F t h a t exhibit significant instability to the ext r e m e s o f p H i n h e r e n t in C E - I E F (11,12). It is not possible to rule out, however, t h a t the coating applied to the DB-1 capillary might be s o m e w h a t labile to the ext r e m e s of pH, b u t if so, the loss of coating is slight e n o u g h to p e r m i t the methylcellulose to mask any exp o s e d sites. Since methylcellulose is always in the amp h o l y t e mixture, the p r e s u m e d loss of covalent coating n e v e r reaches the p o i n t at which resolution and p H gradient linearity b e c o m e s adversely affected. In 1985 H j e r t e n (3) p i o n e e r e d the application of I E F to the capillary f o r m a t in a free solution mode. H e was able to abolish endosmotic flow by coating the capillary surface with a m o n o l a y e r of n o n c r o s s l i n k e d polyacrylamide. H j e r t e n a n d Zhu (4) accomplished mobilization of the focused p r o t e i n zone by cathodic (or anodic) mobilization by changing cathode (or anode) buffer to m a t c h the anode (or cathode) buffer. Later, H j e r t e n et al. (5) added N a C l to the cathode (or anode) buffer to induce mobilization. However, these mobilization m e t h o d s did not p r o d u c e linear p I vs migration plots. Moreover, the range of the focused p H gradient was limited. Using the a p p r o a c h of Y a o - J u n and Bishop (13), Zhu et al. (6) e x t e n d e d the range of the p H gradient to between p H 4 a n d b e y o n d 8.6 by incorporating various additives ( T E M E D ) in the a m p h o l y t e and mobilization buffer (zwitterions). T h e effect on the linearity of the gradient was not discussed, however. 2 Chen, S. M., Shively, J. E., and Lee, T. D. Analysis of antibodyantigen complexes and avidin-biotin complexes using capillary electrophoresis., in Proceedings, Second Symposium of The Protein Society, Aug. 13-17, 1988.

CAPILLARY

ELECTROPHORESIS-ISOELECTRIC

89

FOCUSING

300 -,

250

200

•

R N a s e ba

O

RNase TI-III

•

RNase TI-I and II

Q

RNase T1 wild type

150

100 -

50

0

0

10

20

~ 30

40

50

60

70

80

Injection Time (sec) FIG. 4. Quantification and dynamic range. Samples of 200 ~g/ml were injected for various lengths of time. Protein peaks were detected and the peak areas integrated as described in the text. The four proteins showed a linear dynamic range over the range of 10 to 80 s of injection, w h i c h c o r r e s p o n d s t o 1.3 t o 10.7 n g o f p r o t e i n .

Hydrodynamic mobilization was described by Hjerten and Zhu (4) and their results suggested no large difference in the separation pa t t e r n compared to electrophoretic mobilization. However, no further work was published on this mobilization strategy, presumably due to the requirement for precise and constant hydrodynamic flows. Recently, Mazzeo and Krull (8) used an uncoated capillary for IEF, in which the endosmotic flow was c o n trolled by the addition of methylcellulose to the sample-ampholyte mixture, while T h o r m a n n e t al. (9), in a similar approach, used hydroxypropyl methylcellulose TABLE 1 Reproducibility of p I D e t e r m i n a t i o n R u n no.

RNase ba

RNase T 1 K25, A58

RNase T 1 Q25, A58

R N a s e T1 K25, E58

R N a s e T~ Q25, E58

1 2 3 4 5

9.0 9.0 8.9 9.0 9.0

3.3 3.3 3.2 3.2 3.4

3.1 3.1 3.0 3.0 3.2

3.1 3.1 3.0 3.0 3.2

2.9 2.9 2.8 2.8 2.9

Mean % SD

9.0 0.5

3.3 2.5

3.1 2.7

3.1 2.7

2.9 1.9

Note. T h e p I o f e a c h p r o t e i n w a s c a l c u l a t e d b y u s i n g t h e p I v s r e l a t i v e m o b i l i t y p l o t f r o m e a c h r u n . T h e s o l u t i o n s u s e d in e a c h r u n were prepared separately and run on different days with the except i o n o f r u n 2, w h i c h u s e d t h e s a m e s o l u t i o n s p r e p a r e d f o r r u n 1.

as a dynamic coating agent. T h e residual endosmotic flow means t hat mobilization is initiated at the beginning of electrophoresis and therefore, in order to obtain a linear relationship between migration time and pH, a constant endosmotic flow is required. However, because of the pH gradient in the capillary, an increasing length of anode buffer (due to endosmosis), and decreasing length of cathode buffer, the flow rate is not a constant value. As indicated (8), this resulted in non linearity of the p H gradient. Understanding the two i m port ant factors of capillary isoelectric focusing, we took advantage of a well-regulated vacuum system, with a stable capillary surface, to obtain a stable hydrodynamic flow rate. However, because laminar flow diminishes resolution by inducing band-broadening, simultaneous application of voltage was necessary in order to keep the protein peaks focused. Normally, small peptides cannot be studied by conventional acrylamide IEF methods due to the loss during fixing prior to staining. Because on-line detection is used, the pI's of peptides may be measured provided there is a chromophore with an absorbance maximum of 250 nm or greater. This limitation arises because of the large absorbance of ampholytes below 250 nm. T h e online detection using a uv spectrophotometer, however, does permit the labeling of residues with specific agents exhibiting unique absorption spectra in the effort to separate and identify critical peptides or proteins. In this

90

CHEN AND WIKTOROWICZ 10-

7

d e t e r m i n e d t o b e 0.2 a n d 0.4 p H u n i t h i g h e r t h a n t h e w i l d t y p e a n d w e r e all b a s e l i n e r e s o l v e d . The separation of the single mutants at native pH has b e e n r e p o r t e d (10). T h e c u r r e n t e v i d e n c e s u g g e s t s t h a t t h i s s e p a r a t i o n is a c c o m p l i s h e d t h r o u g h t h e i n f l u e n c e o f a h i s t i d i n e i n m u t a n t II, w h i c h e x h i b i t s a p K n e a r t h e n a t i v e p H , w h i l e in m u t a n t I, t h i s h i s t i d i n e e x h i b i t s a higher pK. The behavior of both histidines can be und e r s t o o d in t e r m s o f t h e p r o t e i n f o l d i n g n e a r t h e s e s i t e s . T h e c o m i g r a t i o n o f t h e s i n g l e m u t a n t s in C E - I E F c a n be reconciled with their separation at native pH by understanding that at their pI's, the histidines are fully charged and therefore contribute equally to the pI. Also at their pI, both proteins may exhibit some degree of denaturation which may abolish the folded structure which permits their separation at a native pH. I n c o n c l u s i o n , i t is h o p e d t h a t t h i s I E F a l t e r n a t i v e will encourage further study on proteins which have pI values beyond the range measurable in the past, as well as protein populations for which rapid, high resolution measurements are necessary.

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ACKNOWLEDGMENT I

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The authors thank Drs. C. N. Pace and Bret Shirley for providing RNase T1, its site-directed mutants, and RNase ba.

Relative Mobility

FIG. 5. Capillary coating stability. IEF was performed as described in the text over a period of greater than 2 months. Standard curves were obtained at regular intervals during this period. Shown are the curves obtained from Days 1 (Q; R2 = 0.997), 19 (©; R2 = 0.998), 37 (I; R 2 = 0.998), and 64 (A; R2 = 0.993).

study, CCK-flanking peptide, a nine-residue peptide (containing two tyrosines) with a molecular weight of 1073 D a , w a s u s e d a s a m a r k e r . I t s u t i l i t y w a s e s t a b lished after multiple analyses demonstrating agreement between the CE-IEF-derived pI and its calculated pI (data not shown). N e i t h e r R N a s e T1 a n d i t s m u t a n t s n o r R N a s e b a h a d pI's estimated by conventional IEF due to the extreme values expected from their sequences. Since the method d e s c r i b e d h e r e is a b l e t o p r o d u c e a w i d e r a n g e , l i n e a r p H gradient, it was decided to qualify the method with these proteins. The pI's of these proteins were determined to b e s i m i l a r t o t h e p I ' s p r e d i c t e d f o r t h e R N a s e T1 a n d RNase ba from sequence calculations and isoionic point d e t e r m i n a t i o n (14). T h e m u t a n t s o f t h e R N a s e T1 w e r e

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11. 12. 13. 14.

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