Capillary Electrophoresis A Fast and Simple Method for the Determination of the Amino Acid Composition of Proteins Paul L. ~ e b e r and ' Daniel R. Buck Briar Cliff College, Sioux City, IA51104 Capillary electmphoresis (CE) is a new analytical tool applied to the separation and quantitation of a wide variety of substances. The extremely high efficiencies of CE compared to high performance liquid chromatography (HPLC) and other separation methods coupled with the low sample volume requirements (typicallx less than 5 nL)have made it a popular technique among researchers both in the academic and industrial sectors. Also, CE is beginning to find use as an instrument employed in routine analysis in the industrial laboratory, complementingthe existing array of instrumentation. A comprehensive, general review of CE has appeared recently in the literature ( I ) . On the other hand. CE is not nvrentlv one of the common instrumental techniques used by students in educational laboratories at colleges and universities. In terms of development and use in this setting, CE seems to be at a historical point similar to that of HPLC a decade or so ago. Just as HPLC joined the ranks of the standard instrumental techniques encountered in undergraduate education then, so too, CE is poised to become the newest member of the team. Laboratory instructors knowledgeable in the area of HPLC are likely to feel comfortable with CE. Also, there are a number of facton that make CE a technique more suitable than HF'LC to student labs and give it the potential of becomine ., a common feature of the future undergraduate laboratory experience. It has a relatively low initial cost (unlike cradient HPLC): low cost of consumable~. because little of a n y chemical i s used and columns have long lives; little generation of waste materials (a major concern for educational laboratories in light of OSHA rules); very small sample requirements, even less than those of HPLC (compatible with today's trend toward micmscale laboratories); ease of maintenance; and simplicity of design and use, with no seals to replace or fittings to clog (always important for any student lab). We have o ~ t e d to develo~an exoeriment that uses CE in a biochemistry laboratory. Because biochemistry experiments often are oerformed in labs offered throueh chemistry, biochemis&, and biology departments, a EE experiment involving biochemistry would have the widest audience. Also, many undergraduate biochemistry laboratories use traditional forms of electrophoresis already, making the transition to CE a natural step. Indeed, the theoretical backgmund for both techniques overlap. Within the realm of biochemistry, amino acid analysis of a peptide or a protein seems to be an experiment that has been accomplished traditionally by other means yet can be done using CE. lkaditionally, amino acids are separated by paper chromatography (2) or thin-layer chromatography (3). Both methods are time-consumingtechniques, requiring about an hour for preparation and an hour for staining and analysis, not to mention the extensive time required 'Author to whom correspondence should be addressed.
NDA
CK
Figure acid.
1.
N-CH R
General reaction for the NDA derivatization of an amino
for chromatographic development. Also, these techniques are only semi-quantitative, a t best. An undergraduate lab in which the amino acids in an unknown dipeptide are identitied by HPLC has been described (4). However, besides the general disadvantages of HF'LC mentioned above, there are reported problems with the separation (4). A laboratory procedure for determining the amino acid composition of a protein using gas chromatography-mass spectrometry (GC-MS) has been reported (8,but it is a difficult and time consuming experiment. Thus, the experiments described here use CE to determine the amino acid composition of proteins. Amixture of amino acids is obtained fmm the add-catalyzed hydrolysis of a protein. After the addition of an internal standard, the amino acids are converted to the naphthalene-2,3-dicarboxaldehyde (NDA) derivative (Fig. 1). NDA reacts with primary amines to form fluorescent products that can be detected by absorbance of light a t 420 nm. The derivatives are then seoarated bv CE. Asimilar orocedure is aoolied to stock soluions of a&no acids to geierate migratibk times for identification and relative resoonse factors for ouantitation. These, in turn, are used determine theBmino acid composition of the protein. The mechanism by which the amino acid derivatives are separated in these experiments involves not only electrophoresis but also partitioning between the running buffer and micelles. A good discussion on this form of CE, commonly referred to as micellar electrokinetic capillary chromatography (MECC), has been published (6). Experimental Amino acids, peptides, and proteins were obtained from Simna Chemical Comoanv (St. Louis. MO.1 Potassium cvauize (99.5%) was acqkr& from ~ l & achemical corporation (Ronkonkoma. NY) and NDA was ~urchasedfrom Oread Laboratories !Lawrence, KSJ. sod~umtetraborate decahydrate tACS reagent, was obtained from Aldrich Volume 71 Number 7 July 1994
609
Chemical Company, Inc. (Milwaukee, WI) and sodium dodecyl sulfate (SDS) (Electrophoresis Purity Reagent) was acquired from Bio-Rad Laboratories (Richmond, CAI. Water was deionized and distilled twice before use. Protein Hydrolysis A number of standard protocols for acid-catalyzed protein hydrolysis exist but the following was chosen for convenience a t this institution. Approximately 6 mg of an unknown protein (cytochrome c or lvsozvme) is dissolved in 0.5 mL of 6 M HC1 in a small vial. Then helium gas is bubbled through the solution for 1 min to remove atmospheric oxygen. The bulk of the solution is immediately aspirated by a needle into a 17 x 55 mm evacuated glass tube with a septum. Another 0.5 mL of 6 M HCl is added to the remainder and aspirated into the vial to ensure a quantitative transfer. The glass tube and contents are placed in a hot oil bath at 10Cb110 "C for 18 to 24 h for hydrolysis. After allowing the tube to cool to room temperature, the septum of the tube is removed. breakine the vacuum. and t h i contents poured into a 2 5 - m ~rouxkbottomed flask. Eva~orationof the HC1 is accomplished bv gentle heatine of the sample under a vacuum applied by a faucet aspirator. Evaouration is complete in 20 to 30 min. The sides of the tube-are rinsed witGlmL of water, the contents poured into the 25-mL flask and then evaporated. The sample in the flask is reconstituted in 1.0 mL of 0.05 M NaOH. Samples that appear cloudy at this point are filtered with a 0.45 micron syringe filter.
--
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Sample Derivatization The sample derivatization procedure is a modification of that given by Lunte and Wong (7). For derivatization of protein hydrolyzates, 10 & of reconstituted hydrolyzate (approximately 50 mM amino acid content) is mixed with 345 pL of 20 mM sodium borate (pH 9.0) in a small screwcap vial. Next, the following three solutions are added and mixed sequentially: (a) 1 0 i of'5 ~ mM internal standard, n-amlnoadi~icacid. (b160ul, of 10 mM KCN. and rc, 50 uL of'10 m M N f l ~ i nacetonithle. he appearance ofa fluorbscent yellow-meen color withina few minutes indicates that the reaction-is proceeding. After 25 min, 25 WLof 1.5 M ammonia is added to react with excess NDA that would otherwise appear as a large contaminant peak in the ensuing electro~horesis.The total volume is 500 uL. M e r 15 m& the sample is ready to inject. Standard solutions containing up to 15 amino acids and the internal standard are derivatized in a similar way except that larger amounts of amino acids (25 nmol of each amino acid, are derivatized requiring that slightly larger volumes of KCN t90uL1and NDAr75 uL, be used. Deoending on the skill of the students undertaking this lab, all volumes can be scaled down proportionally to give a total sample of 75 &with no adverse affect.
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Capillary Electrophoresis and Data Acquisition and Analysis The instrument used in this work was an ISCO Model 3850 Capillary Electropherograph. This electropherograph is typical of commercial electropherographs in that a number of safetv features are incornorated into the design of the instrumek so that thehigh;oltages encountered present little danger to the operator. However, the instructions of the equipment manufacturer should be followed carefully to minimize any possible risk. The columns used were uncoated silica capillary columns with an inner diameter of 50 pm and of various lengths. The buffer composition in the columns and the reservoirs was 50 mM SDS in 20 mM sodium borate (pH 9.0). Operating voltage was ~
610
Journal of Chemical Education
~~
~
~~~
~~~~
20 to 24 kV These conditions represent a slight modification of those eiven bv Ueda et al. used for similar seoarations(81.~he'detect&was set at 420 nm with a sensiiivity of 0.005 AUFS. Althoueh the ISCO Model 3850 is soecified for use from 190 to 386 nm,the instrument has sifficient lamp energy a t 420 for the experiment. Use of 420 nm allows for selective detection of NDA derivatives. Arise time of 0.8 s was used. Two types of injection systems were employed. The splitflow iniection svstem is the t v ~ esuodied with the base electr&hero&h. Using this "&stem','a 5 &volume of solution was injected into the system with a split-flow ratio of 1:1930 installed, thus giving a one-column injected volume of 2.6 nL. The vacuum injection system is available as an accessory to the instrument. In this method a vacuum is applied for a specified time to the end of the column in order to inject a-sample in contact with the front of the column. An injection time of 5 s was used and gave - a on-column injectionvolume of about 3 nL. In order to obtain reproducible results, the column was flushed every 3-4 runs using the following procedure: iniect 30 uL of 0.1 M NaOH and flush 15 min later with- 30 L~each of water and then buffer. Output from the electropherograph was analyzed by two methods. In the manual method the output was received by a chart recorder. Migration times and peak heights were measured by hand using a ruler. In the automated method the output was sent to a IBM-compatiblepersonal computer via a data acquisition card and collected and analyzed using ChemResearch soilware (ISCO, Inc.) Results An equimolar mixture of 15 amino acids and the internal standaid were derivatized and separated by CE to give the electropherogram shown in Figure 2a. This f i m e represents ;typical student run rath;?r than the bestkn. ~ i c e l lent resolution of most amino acids was achieved in less than 16 min. An unidentified peak (U) was present in all CE electrophemgraphs whether standard mixtures or protein samples were analyzed. U is formed during the NDA reaction because it was not present in runs of blanks containing all reagents except NDA. Leucine (L) and phenylalanine (F) are not resolved and arginine (R) overlaps somewhat with lvsine (K). It was noted that if trvDtoohan is included in the mix it appears as a well resolved peak just in front of the LIF peak. - In Figure 2b is givenAtheelectropherogram for the NDA derivatized amino acids present in a sample of cytochrome c that had been hydrolyzed for 18h. By comparing Figures 2a and 2b, it is apparent that the resolution evident in runs on standard mixtures is reproducible when using protein hydrolyzates. Only a few small peaks, such as that migrating near the 15 min mark, are present in the electropherogram of the protein hydmlyzate. The output from the electropherograph that is used to generate figures such as those in Figure 2 also is used to determine the amino acid composition of a protein by either manual or automated methods. In the inexpensive but tedious manual method peak heights on both the standard mixture (such as Fig. 2a) and the unhown protein (such as Fig. 2b) are measured with a ruler. Peak areas cannot be determined manually because peak widths are too small to be measured accurately in a typical electro~heroeram.In the more costlv but simoler automated method, output is sent to a computer for data acquisition and analvsis usine ~ackaeedsoftware (ChemResearch). This softbare allows peak-areas to be determined easily and accurately using baseline drawing routines. Using either method, the standard run permits a response factor for each amino acid to be determined. Analy-
". .
Table 1. Amino Acid Composition of Cytochrome C
(Hoursof Hydroly~is)~
AminOAcid
H ~ CAreaC Ht.
Ser (S) Thr (T)
1.0
1.3
7.8
His (H)
2.8
G ~ (G) Y
it.^
Area
Ht.
Area
0.9
12
1.0
1.3
1
8.3
7.3
8.2
7.5
8.3
8
2.3
2.6
3.1
2.7
2.9
3
13.5
14
11.0
13.8
11.2
Ala (A)
5.3
6.2
5.4
6.7
5.4
6.5
6
Glu (E)
11.9
12.4
9.6
11.5
11.9
12.0
12
Tvr (Y)
3.7
3.8
3.8
4.4
3.8
4.1
4
ASP (Dl
7.1
7.9
6.6
6.9
7.1
7.4
8
Val (V)e
3.0
3.0
3.0
3.0
3.0
3.0
3
Met (M) Ile (I)
1.9
1.8
1.7
2.3
1.8
2.1
2
6.0
6.4
5.7
7.0
5.9
6.7
6
LeuIPhe (UF) 8.9
9.8
8.8
11.3
8.9
10.6
10
1.8
2.1
2.0
2.1
1.9
2
Arg
(4
11.4 13.2
2.1
'Protein was hydrolyzed in 6M HCi at 105" C for lhetimeindicated. Separation conditions are given in Figure 2. Each value represents the average of two
'Average' ms ine average va ue ollne 18 and 24 n nydro yses obta nea ,s ng manLa y rneasJlea pea* heights wn e Area' va Jeswere oora ned ,$.no areas nreorateo ov cornomr &'~it"is the literaturevalues for-each aminoacid asgiven in reference 9. Val was set at 3.0 and ail other values calculated relative to it. See text for C-nl' va des were
b
i
i
li
16
Time (min.) Figure 2.EleclropherogramsofNDAoerivatized am noacid mixlures. condlt~ons:as descr bed in -Experimenra "; column lengtn, 61 cm (36cm lo detector);appled voltage, 20.0kV; current. 33 uA: one-letter symbols are used for each amino acid (see Table 1 .):K= lysine: X = internal standanl,a-aminoadipicacid);U = unidentified peak. A. Stocksolutionof 15 aminoacids and IS. 6. Cytochrome c hydrolyzate (18h). Separation
sis of six replicate determinations of the response factors for the 14 amino acids listed in Table 1 using the automated method was performed. The average relative standard deviation in the mean response factor was only 4%, comparing quite well to a similar study using GC-MS (5). Thus, the determination of response factors is repmducible. Response factors are used along with corresponding peak heights (or areas) from the peaks identified in the electrophemgram of the unknown protein (such as Fig. 2b) in order to give relative amounts of each amino acid in the unknown hydrolyzate. These are called the "initial results". Then these results are used to determine the number of each type of amino acid residue present in the intact protein, theiiinal results", by the follnwing process. One scans the "initial results" to find which amino acid is uresent in lowest amount. In cvtochromec, this is serine ( ~ jwhich , is the smallest p e a k i n ~ i ~ u2b. r e Because there cannot be less than one residue of a oarticular amino acid per molecule of protein, the amino acid in lowest amount is likely to be present as one residue in the protein. One wuld then scale all of the other amino acids relative to this amino acid to detembine the "fmal results" but this is likely
. .
detaib:
to generate significant error because it is difficult to determine accurately the relative amount of a small peak relative to the internal standard. Therefore, one scans the "initial results" further to find another amino acid that is present in approximately twice or three times the amount of the amino acid in the smallest amount. In cytochrome c, valiie (V) is spotted easily as being present in about three times the amount that serine is. One then sets the relative amount of this amino acid to the appropriate whole number, such as two or three, and scales all other amino acids to this amino acid to determine the "final results". In cytochrome c, the amount of valine (V) is set a t three and all other amounts of amino acids are calculated relative to this value. This procedure for determining amino add composition was aoolicd tn 18 h and 24 h hvdrolvzates of cvtochrome c " " as indiiated in Table 1. From an inspection o"f the "Average" results, one can see that the manual method compares favorably with the automated method. Both methods seem to give a compositionclose to that reported in the literature (9). Also. there does not seem to be much difference between the h and the 24 h hydmlyzutes. Lysine is not reooncd since the NDAderivativc ofthis diderivntized amino acid seems to be relatively unstable. Data from analysis of a 28-h hydrolysis of cytochrome c indicated a sipnificant loss in a number of amino acids, notablv asuartica"d (D), glutamic acid ,El,serine (S,, and threonine'r~,. Asignificant problem in the practice of CE has been variations in migration times for a given analyte from one run to the next. The periodic flushing of the column given in the procedure has helped overcome this problem. An example of the relatively small deviation seen between runs is Volume 71 Number 7 July 1994
611
Table 2. Single Analysis of a Hldrozylate of Lysozyme (LYSO.) Composition
Mig. Time (min) Amino Acid Ser
Lyso. 8.67
Std. 8.68
Percent M.T. Exp . ~ev.~
Litc
4.1
9.3
10
the carboxylic acid and the amide in the intact protein. Although the lysine content can be determined, the lability of its NUAderivative makes the analysisof this amino acid a difficult task, but a semi-auantitative analvsis can be easily made. ~Gally,proline $ not a primary &nine and does not react with the NDAto give a detectable analvte. Although leucine and ph&yialanine were not separated in this analysis, experiments indicated that the use of a longer colu~& and higher voltages give some resolution. However, analysis times also would increase thus l i m i t i i the utility in a student laboratory. Along with the general advantages of CE mentioned in the introduction, this particular experimental procedure has the following additional benefits:
Thr
9.11
9.12
4.1
5.1
7
His
10.00
10.04
4.4
1.1
1
G~Y
10.68
10.71
4.2
-
-
xd
11.06
11.06
0.0
11.4
12
Ala
11.36
11.39
-0.3
5.5
5
Glu
11.70
11.71
0.0
3.0
3
Tyrs
12.37
12.42
-0.4
15.1
21
Asp
12.59
12.61
4.2
5.3
6
Val
13.63
13.68
4.4
1.2
2
Met ile
14.53
14.59
4.4
5.9
6
15.41
15.46
- 0
11.5
12
Ease
LeuIPhe 16.16
16.24
-05
11.6
11
Arg
17.73
4.5
11.4
11
Derivatization of samples using digital micropipets is a useful technique that is learned quickly The derivatization reaction is fast, and the appearance of the yellowgreen color of the NDA derivatives allows students to ascertain quickly whether the reaction is proceeding or not. Operation of the CE instrument is straightforward, much simpler than either GC or HPLC. If an error is made in attempting to run a sample, the run can be stopped immediately, the column flushed with fresh buffer in seconds, and another run quickly started. This is an especially important feature in a lab where students are just learning to use the instrument. Both the speed and ease of the overall lab, including data analysis, can be increased by using the ChernResearch software for automated analysis. Indeed, the use of such software could extend the utility of the laboratory so that introductory level labs could perform an analysis of an amino acid mixture.
17.65
'Protein was hydrolyzed in 6 M HCI at t05"Cfor20 h. Separationconditions: as described in "Experimental': wlumn length. 74 cm (53cm to detector);applied voltage. 24 kV; wrrent, 35 pA. The Percent Migration Tlme Deviation = ((M.T.L" a - MTw)lM.T.sm) x inn% "Lit'isthe literaturevaluesfor each amino acid as given in refersnce 9. d~ = the internal standard. 4 y r Composition was set at 3.0 and all other values calculated dative to it. See text for details.
given in Table 2. The migration times from a single run of the amino acid derivatives ina hydrolyzate oflysozyme are compared to those in a standard mix. The percent migration time deviation is not more than 0.5%. Also given in Table 2 is the composition of lysozyme as determined from this single analysis. Like eytochrome c, the experimental values compare quite well to those reported in the literature. Both the split-flow and vacuum injection methods work well in student hands. The vacuum injection method seemed to give a more reproducible amount injected oncolumn but this is not a concern because an internal standard is employed in these experiments. The vacuum injection method is easier and faster to use and also consumes a lot less sample (a few nL versus a few
a).
Discussion It has been shown that a relatively simple and inexpensive CE system can be used by undergraduate students to determine the amino acid composition of proteins such as cytochrome c and lysozyme. Good agreement between experimental and literature values for 14 amino acids was obta~ned.Amounts of six other common amino acids found in proteins were not dctfrmlncd Tryptophan is completely destroved while cvsteinc is uartiallv demaded bv acid hvdroly&. The hy&olysis afso converts the am"ides, gl;tamine and asparagine, to the carboxylic acids, glutamic acid and aspartic acid, respectively, and this change is reflected in the compositional analysis of the protein. That is, the composition of glutamic acid and aspartic acid determined experimentally reflect the total amounts of both A
612
Journal of Chemical Education
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Speed
Though the hydrolysis times are long, most of the time is spent unattended by the student. Furthermore, the addition and mixing of sample, reaction buffer and reagents for sample derivatization requires only a few minutes. Reaction time is about 45 min total. Most importantly, electrophoresis requires only 16 to 18 min. Thus, 10 prepared samples can be analyzed in a 4-h lab period on one instrument.
Acknowledgment We are grateful to ISCO, Ine. (Lincoln,NE)for providing the CE equipment and soilware used in this work. We also are thankful for a Kathy D a m e Research Grant (Briar Cliff College) which provided a summer fellowship to the authors for the work. A presentation on this work was given in November, 1992: Daniel R. Buck and Paul L. Weber, "Capillary Electrophoresis: AFast and Simple Method for the Determination of Amino Acid Composition of Peptides and Proteins," 27th Midwest Regional Meeting of the American Chemical Society, November 6,1992. Literature Cited 1. Kuhr. W. G.: Momin.. C.A.Anol.Ckm. 199%€4.S 8 9 R 4 I R . 2. He& T L J. ~ h & ~ 19W. d ~ 67,964-966. . 3. Hurst, M.0.;C0bb.D. K J . C k m . Edue 19m,67,978. 4. Clapp, C. H.; Swan, J. 6.;Poechmann, J. L. J. Ckm. Edm. 189%6.9, A122-A126. 5. Hamann,C.S.;Myas,D.P;RiUte,K J.;Wirth,E.F.:Moe,O.A. J Chem.Edue 1891, m. ? --,d.-6. Sepaniak, M . A; Pawell. A.C.; Saaile.D. E;Co1e.R 0. In Coprlla~Eloe(mpham8ir: Theory and Roc"; Dmssman, P D., co1bum. J. C., Ed%;Aesdw* Re-: San Diego, 1992: Chapter6. 7. Luntc, S. M.: Wong, 0.S. Current Sepzrofions 1890,10, 1WZ6. 8. U d a , T;Mitchell, R.; Kitamura, F.; Metcslf,T; Kuwana, T.; N h o t o , A J. Chmmafogr 199%5933,265-274. 9. Dayhoff, M. 0.AUos o f h t e i n S q u e n e and Sfmctum; National Biomdioal Research Foundation: Weshir@m, 1972:Vol. 5, pp D-14.0-138,
