grams of shredded coconut. The mixture was extracted with 3 X 150 ml. of water. The extract was dried by azeotropic distillation with pyridine and acetylated as described above. Gas chromatography followed b y the appropriate calculation revealed that the coconut meat contained 0.5 % sorbitol. By elimination of the internal standard it was shown that mannitol, if present, is in such a lorn concentration that it does not interfere with the accuracy. I n another experiment, additional sorbitol was added and the determination repeated. The results are recorded in Table T’. The Chromatography must be performed the same day as the acetylation because products appear which interfere with the mannitol hexacetate peak introduced as the internal standard.
LITERATURE CITED
Table
V.
Run Controla Controla
Determination of Sorbitol in Shredded Coconut
Sorbitol added Sorbitol Difference (5) found (ci) 0 00 0 00 1 00 1 00
0 50 0 50
1 50 0 00 1 52 -0 02 Control experiments were performed on fresh meat from different coconuts. (5
(1) Esposito, G. G., Smann, 11. H., AYAL.CHEM.33, 1854 (1961). (2) Haahti, E. 0. A4.,\-anden Heuval, 11.J. A , , Homing, E. C., d n a l . Bzochenz.
2,344-52 (1961).
(3) Homing. E. C., Moscatelli, E. -I., Sweeley, C. C., C h e m . I n d . ( L o n d o n ) 1959, 751. ( 4 ) Lew, B. 1Y. Kolfram, 11.L., Goepp, R. M.,J . A n i . ?hem. Soc. 6 8 , 144953 (1946). ( 5 ) Sjovall, J., Meloni, C R , Turner, I). *4.> J . Lzpid Res. 2 , 31’7-20 (1961,. (6) The Pharmacopeia of the United States of bmerica, Sixteenth Revision, pp. 692-3, hlack Printing Co , Easton,
P a , 1960.
ACKNOWLEDGMENT
( 7 ) 1-anden Heuval, JY. J. ;i,Homing, E. C Bzochem. Bzophys Res. Communs. ~
TT’e are indehted to Robert De Valeria
who contributed to various phases of the work and to Franklin Baker Coconut, General Foods Corp., for the generous supply of coconut.
4,399-403 (1961). (8) Zill, L. P., Khrm, \-, S.,Cheneae, G. ll,,J . A m . Chem. Soc. 75, 1339-42 (1953). RECEIYEDfor review May 7, 1962. Arcepted August 15, 1962.
Chromatographic Separation of Peptides on Ion Exchange Resins Separation of Peptides from Enzymatic Hydrolyzates of the a, and 7 Chains of Human Hemoglobins
p,
W. A. SCHROEDER, RICHARD T. JONES, JEAN CORMICK, and KATHLEEN McCALLA California lnsfifufe of Technology, Pasadena, Cc /if.
b Isolation of components in a mixture is most effectively accomplished by great alteration of conditions from step to step in the separation. This principle has been applied to the separation of complex mixtures of peptides in protein hydrolyzates. Excellent results have been obtained by chromatographing first on the cation exchanger Dowex-50 and then rechromatographing on the anion exchanger Dowex-1. Volatile developers were employed throughout. Experimental procedures are presented, and results are given of their application to the separation of peptides from human hemoglobins A and F.
r
r
HE 5UCCLSSFUL DETERMIXATIOK O f
the amino acid sequence in a protrin depends much upon the adequacy with JT hich the peptides, however obtained, may be separated. Unless the amount of protein available is small and p a p u chromatographic methods must 1~ used, the separation of peptides by column chromatography in amounts wfficient for complete characterization is clearly the method of choice. Although peptides may be adequately separated in many inPtances (17) by 1570 *
ANALYTICAL CHEMISTRY
sodium citrate or sodium acetate buffers of the type that are used in the analytical determination of amino acids by ion exchange chromatography ( I I ) , the product from pooled fractions is essentially only buffer salts that are contaminated with the peptide. The separation of the desired peptide from the buffer salts has been achieved in various ways. If the peptide is dinitrophenylated in the presence of the salts, it may usually be extracted from the solution (17). This procedure suffers from the disadvantage that the free peptide is no longer available for the determination of structure by step15 ise degradation. Other procedures have also been used. For example, peptides have been separated (19) by means of ammonium formate or acetate buffers which were then removed by wblimation ( 5 ) . A q an alternatile, the peptides have been separated \{ ith sodium-containing buffers which w r e converted to ammonium salts by passage through a column in the ammonium form. The ammonium salts were then sublimed (6). .Ilthough effective, these procedures are laborious and time consuming. The recent application of volatile orqanic developers has been a most sig-
nificant step in the separation of peptides on an ion exchange chromatogram, Vanecek et. al. (go), Margoliash and Smith ( I O ) , and Kimmel et al. (8) have described the separation of peptides by means of Do~vex-50 v i t h pyridineacetic acid developers, \Thereas Rudloff and Braunitzer (14) have used columns of Domex-1 with pyridine-collidineacetic acid developers. The separation of peptides from hydrolyzates of CY, p , and y chains of human hemoglobins A and F has been achieved by the use of both Dowex-1 and Doirex-50. I n the earliest experiments, the peptides were separated first on Dowex-50 and further purified by paper electrophoresis or paper chromatography. The purification by paper methods, in general, was unsatisfactory and has been superseded by rechromatography on Don-ex-1 ; paper methods always resulted in losses of 50 to 75% of the material and usually gave a product that still contained impurities. The use of Dowe.t-50 and Dowex-1 in succession (the reverse would probably be equally effective) takes advantage of the fact that vastly different conditions prevail in the two types of chromatography because one is a cation and the other an anion ex-
changer. Usually, peptides that are coincident on one are well separated on the other. This paper presents a detailed description of the procedures that have b w n employed in these laboratories and describes the results of the application of the procedures. The significance of the results in terms of the structure of' the CY, p, and y chains of human hemoglobins A and F will be published elsewhere. EXPERIMENTAL
Preparation of Buffers. T h e composition of t h e buffers and the quantity of t h e reagents used t o prepare them are given in Table I. T h e p H 3.1 buffer is 0 . 2 5 pyridine a n d t h e p H 5.0 buffer is 21- in pyridine. T h e p H of different preparations is 1 0 . 0 5 p H units of t h a t given. The buffers of p H 8.0, 8.3, and 9.3 are identical with or based on some buffers of Rudloff and Braunitzer (14). The p H is very sensitive to the amount of acetic acid which must be added carefullv t o give the desired value. Rudloff and Braunitzer (14) prepared their buffers with n-ater that had been degassed under V ~ C U I I ~ . Alctually, only the removal of carbon dioxide is necessary If carbon dioxide is absorbed by the basic developer, it is subsequently evolved as bubbles in the column as the pH of the developer becomes more acidic. It has been more convenient to remove carbon dioxide by passing nitrogen through the water before adding the reagents. Subsequent uptake of carbon dioxide can easily be prevented by attaching a soda lime tube to all vessels. Preparation of Columns of Dowex50 X 2. I n t h e course of these experiments. t h e procedure has been somewhat modified, b u t the description beloIv is t h a t presently in use for large scale columns. T h e procedure m a v be scaled u p or down n it hou t difficulty . The Don ex-50 X 2 had been obtained some years ago from the Dow Chemical c'o. The lot number was 3328-12, and the mesh size n-as given a? 200-400. A 5-pound portion was purified exactly as Moore and Stein describe ( 1 1 ) . The wet resin in the sodium form nould not pass a 200-mesh sieve but did pass with difficulty through a 120mesh sieve. It was stored in the sodium form. Before a column n a s packed, a n amount required for a column 3.5 x 100 em. in dimension was washed successively on a Buchner funnel with 5 liters of water, 5 liters of X KaOH, 5 liters of water, 3 liters of 2 5 HC1, 5 liters of water, 4 liters of 2 5 pyridine (distilled), and 3 liters of p H 3.1 buffer (Table I). The resin was then suspended in sufficient p H 3.1 buffer to give a ratio of two volumes of supernatant buffer t o one volume of resin, The column was packed a t room temperature under 10 t o 15 cm. of mercury
pressure as Moore and Stein (11) describe. About 1 liter of p H 3.1 buffer was passed through a column, 3.5 X 100 cm., before the sample was applied. Chromatographic Procedures with Columns of Dowex-50 X 2. Details of t h e hydrolytic procedures that produced t h e mixture of peptides will be published elsewhere. A t the completion of hydrolysis t h e p H of t h e sample was taken t o 6.5 with H C l and a n y insoluble material mas centrifuged off. T o t h e supernatant liquid was added a volume of 1N pyridine-acetic acid buffer at p H 3.5 sufficient t o bring the concentration of pyridine in the final solution t o 0.2N. Finally, 6 N HC1 was added to reduce the p H to 2 t o 2.5. The volume of sample after these adjustments has varied betn-een 30 and 75 ml. This variation has had no obvious influence on the subsequent course of the chromatogram. The hydrolyzate usually contained the peptides from 15 to 30 pmoles of protein (0.4 to 1.0 g.). With smaller amounts, the size of the column should be decreased. It map v-ell be possible to increase the load. Before the sample was added, the temperature of the column was raised to 38 O C. for a t least 1 hour and was maintained there throughout the chromatogram. The sample was carefully added and forced in with air at 10 em. of mercury pressure. =Ifter the sample had been rinsed in with a few milliliters of pH 3.1 buffer, the tube above the column was filled with the same buffer (about 200 ml.). Development was made with a rather flat convex gradient in pH and pyridine concentration. To produce the gradient, by the principle of Bock and Ling (3) as shown in their Figure 6, three containers of equal diameter were connected in series. h volume of 4.3 liters of p H 3.1 buffer n-as placed in the first container and an equal volume of pH 5.0 buffer in both the second and third containers, Developer flon-ed from the second and third containers into the first which was niagneticallv Ftirred and then to the column. The developer was pumi3ed through the column with a Milton Roy Minipump (Milton Roy Co., Philadelphia. Pa.) at 120 ml. per hour as long as the pressure did not rise above 30 pounds. The column shrinks to
Table 1.
PH
Reagents, ml. Pvridineb Glacial acetic acid. 0-Picdined
2,4,6-Collidinee .~7-ethylmorpholinel
about two thirds of its length during the chromatogram; the pressure tends t o rise, and the flow rate may need t o be reduced to keep the pressure below 30 pounds. I n earlier experiments, a flow rate of 60 ml. per hour was used, but more recent chromatograms have been made at twice this rate without deleteriously influencing the separations. The effluent from the column was collected in 10-ml. fractions. Although further discussion of this point will be made below, it should be mentioned t h a t in its present application i t \vas adequate to discontinue development with the gradient after 1000 fractions had been collected, and t o replace the developer with 2 N NaOH. Any very strongly absorbed material may thus be removed and detected. After a chromatogram had been completed, the resin was removed from the column and regenerated as previously described before the column was repoured for another chromatogram. Preparation of Columns of Dowex1 X 2. Chromatography on a large column of Domex-50, in general, reduced a compIex mixture of peptides t o a series of very simple mixtures. T h e separation of such simpler mixtures m a p be achieved on smaller columns of Don-ex-1 a t a considerably higher loading. Accordingly, columns of Dowex-l h a r e been only 1 x 100 cm. in dimension. Don-ex-1 as AG 1 X 2 in the chloride form was obtained from Bio-Rad Laboratories, Richmond, Calif. The dry mesh size was listed ae 200 to 400 and the wet size as 80 t o 200 (Control S o . 5306-151. One pound was suspended in 1.5 liters of water six times with settling for 20 minutes to remove fines. It was then washed on a Buchner funnel rl-ith 2 liters of water at 60" C., and a t room temperature 1%-ith1 liter of carbonate-free 0 . 5 S S a O H , 3 liters of water, 0.5 liters of 1 S HCl, and 3 liters of water. It was not sized further and was stored wet as the chloride. Before a column was packed, 100 ml. of the wet resin was placed on a Buchner funnel, and the following solvents were allowed to flow through under gravity: 200 ml. of water, 200 ml. carbonate-free 0 . 5 4 S a O H , 500 ml. of water, 200 ml. of I S HOdc, 200 ml. of water, and 500 ml. of buffer of the desired pH (pH 8.0, 8.3, or 9.31. It n-as finally slurried
Composition of Aqueous Buffers (4 Liters)
3.1
64.5 1114
5.0 645
573
8.0 47 CaO 5
8.3n
40
1 5
9.3
30
0 4to2
113 40
50
Essentially the huffer described by Rudloff and Braunitzer ( 1 4 ) . b Baker and Adamson, purified, redistilled. Buffer prepared from distilled RIallinckrodt analytical reagent pyridine has discolored on standing. c Du Pont reagent grade. d Matheson Coleman and Bell, practical grade, redistilled. e Matheson Coleman and Bell. f Eastman, pracbical, redistilled. a
VOL. 34, NO. 12, NOVEMBER 1962
1571
with buffer of such volume that the supernatant buffer was twice the volume of settled resin. It has been more difficult t o obtain satisfactory columns of Dowex-1 than of Domex-50. The resin is stickier and tends to trap bubbles of air. The following procedure is adequate : Buffer was added to a chromatographic tube and allowed to pass through t o fill the interstices of the sintered disk and the stem immediately below. A 10-cm. layer of buffer was allowed to remain above the disk but the flow of buffer was stopped. Sufficient slurry a t 38" C. to form about a 15-em. length of column was added, and the re,'.in was allowed to settle under gravity. During the first part of the settling, the upper part of the tube was evacuated to aid the release of trapped bubbles of air. After the resin had settled, excess buffer (except for a 10-em. layer above the resin) m s removed and the procedure was repeated until the desired length of column was attained. The column should be poured a t 38" C., the temperature of use. Finally, the column was equilibrated with 400 to 500 ml. of the desired buffer. The flow rate during this equilibration is unimportant. -kt the end of the equilibration, a small amount of yellow material that accumulated at the top was removed before the sample was applied. Chromatographic Procedures with Columns of Dowex-1 X 2. T h e appropriate sample was dissolred in 3 ml. of the desired buffer and adjusted with 0.2N SaOH to a p H about onehalf unit higher than the p H of t h e developer. T h e sample was allowed to percolate into t h e column under gravity and rinsed in with small portions of buffer. Finally, t h e chromatographic tube above t h e column was filled with developer and the chromatogram was maintained at 38'
C.
The choice of developer is dependent to a degree upon the charge of the peptides to be separated; the criteria for choice will be discussed below. Although the basic procedure for the use of Dowex-1 is that of Rudloff and Braunitzer (14), a different sequence of developers has been found to be more satisfactory. The following procedure has been used with the three types of buffer. The top of the chromatographic tube was connected through polyethylene tubing to a magnetically stirred constant volume mixer of 135-ml. volume. The mixer was connected through a 6-inch length of polyethylene tubing to a reservoir. (Tygon tubing is unsatisfactory with high concentrations of acetic acid.) Mixer and reservoir were attached to a single rod and could be moved as a unit to control the flow rate through the column b y the hydrostatic head. Tubing, mixer, and reservoir were filled with buffer, and the flow rate was adjusted to 15 ml. per hour. The fraction size was maintained a t 1.5 ml. throughout the chromatogram although the flow rate was altered from time to time. Development was then made a t 15 ml. per hour until 60 fractions had been collected. ilt this
1572
ANALYTICAL CHEMISTRY
HEATING BAT TWO
2
I.
-
S11osl1c Dbluenl
-
Polyethylene
4 rnrn c o i l
034
DISCARD
Figure 1.
System for ninhydrin analyses with the Technicon AufoAnalyzer
point, the buffer in the reservoir was removed and replaced with 0 . 1 s HOAc, and the flow rate was reduced to 9 ml. per hour. The solution in the reservoir was changed t o 0.5N HOAc a t fraction No. 160 (flow rate to 20 ml. per hour), to IN HOAc at fraction No. 260 (flow rate to 9 ml. per hour), and to 2 5 HOAc at fraction KO.360 (9 ml. per hour). The chromatogram was generally stopped after 500 fractions. If the change of 0.1N HOAc is made in the late afternoon, the suggested flow rates permit succeeding changes to be made a t convenient times in the morning or afternoon. The flow rate may be maintained throughout at 15 to 20 nil. per hour (or perhaps more). rlfter the chromatogram had been completed, the solvent m-as replaced with the desired buffer, and the column was re-equilibrated by passing through B total of 500 ml. a t any convenient rate. Prior t o the addition of the next sample, a small amount of yellow material that had accumulated at the top was removed. These columns may be used repeatedly without repacking. Examination of Efffuent Fractions. T h e organic bases in the developers do not interfere with t h e use of the ninhydrin reagent. Therefore, in order to determine the progress of the chromatogram, t h e effluent has been evamined with ninhydrin reagent. I n the first chromatograms, the ninhydrin procedure of Moore and Stein (12) was applied to 0.5-ml. and 0.2-ml. portions, respectively, from alternate fractions from the Dowex-50 and Dowex-1 chromatograms. However, more recently, a Technicon AutoAnalyzer (Technicon Corp , Chauncey, N. Y.) has been used to carry out the determinations automatically. Figure 1 outlines the system that was used: The niaterials and sizes of the tubes in the proportioning pump are given as well as other data pertinent to the system. The diluent was peroxide-free methyl Cellosolve and water in the ratio 1:1 by volume. The ninhydrin reagent was
that of Moore and Stein (18). Inasmuch as only qualitative information was derived from this procedure, the reagent has been used satisfactorily over periods of two weeks. Sampling was done at 40 per hour. For convenience in handling and to avoid loss of material, special test tubes have been made which fit into the sampler plate and, by the use of adapters, into the fraction collectors. These have been used with the Dowex-1 chromatograms. Alternate fractions may then be readily transferred to the sampler for sampling. Pooling of Efffuent Fractions. Fractions shown t o contain ninhydrin positive material were pooled. F o r the most part, fractions belonging t o the interzone between adjacent peaks have not been discarded. Rather, adjacent fractions in a n interzone have been added to t h e appropriate peak. There has been little evidence of appreciable mutual contamination of adjacent peaks. The pooled fractions were evaporated almost t o dryness in a rotary evaporator a t 40" C., transferred to a test tube, and evaporated to dryness a t 40" C. in a stream of filtered air (Koby Air Purifier, Koby Corp., Boston, Mass.). Further Examination of the Peptides. The peptides in t h e pooled fractions from the Dowex-50 chromatograms were examined b y unidimensional paper electrophoresis and b y paper chromatography. T h e electrophoresis was made on Whatman 3hIM paper a t pH 6.4 in t h e pyridineacetic acid buffer of Ingram ( 7 ) at 40 t o 45 volts per cm. for 45 minutes. The ascending paper chromatograms were developed with 7 :7 :6 isoamyl alcohol-pyridine-water. In recent experiments, these means have been used only to determine the charge and, t o a degree, the homogeneity of the material in a given peak. From this information, a better choice could be made of the developer for rechromatography on Dowex-1. .ifter isolation from the Dowex-1,
t
Figure 2. Separation of Peptides in a tryptic hydrolyzate human hemoglobin A on Dowex-50 Hydrolyzate was made at p H 8 and 40" C. for
the determination of the structure of the peptide was begun. Further description of this phase of the work will he published elsewhere. RESULTS
I n Figures 2 to 6 is shown the separation of the soluble peptides that result from the tryptic digestion of henioglobins A and FII (1) and of the CY*,P A , and yF chains of these hemoglobins. Because hemoglobin A contains CY and p chains, the tryptic peptides of both types of chain are present in the chromatogram depicted in Figure 2 where the absorbance a t 5T0 mp after reaction with ninhydrin is plotted against the fraction number. The clashed line in any figure indicates the p H of the effluent. The separation of the chains themselves was made by the
Table
Peak No. in figs. 2, 3J 1 2
3 4 5
5a 6 7
8 9
10
11 12
13 14
15 16 17 18
II.
Tryptic peptidea BT-3 pT-5
aT-1 pT-5 pT- 13 aT-S aT-1 I pT-8 aT-9 aT-9 (oxid j aT-2 CYT-1.2 BT-14
RT-6 pT-9 pT-1 aT-5 pT-15 CYT-4
pT-2
CYT-10 pT-S,S(?) CXT-7 pT-7 aT-3
PT-4
Charge*
-_ __
Hydrolytic conditions as in Figure
procedure of Wilson and Smith (21). The separation of peptides from tryptic digests of the individual chains is presented in Figures 3 and 4. The numbers of the peaks in Figures 3 and 4 have been assigned t o correspond to identical peaks in Figure 2. Hemoglobin FII contains both a and y chains, and the peptides from both chains are present in the chromatogram of Figure 5 . The separation of the peptides from the y chains is given in Figure 6. The numbers of the peaks of Figure 6 have been assigned to correspond with peaks of Figure 5 . The chromatogram t h a t is represented b y Figure 5 followed in detail the procedure as given in the experimental section. I n the chromatograms of Figures 2-4 and 6 a slightly modified procedure was used: A t fraction 900, p H 5.0 buffer was removed from the
+00 +0 ++0
++
+ 0 0
1 2 3 4
5
6r
A 9
10 11
I
I
-
++ + ++t + I
+ aT-14 pT-l4,15 20 ++A 21 aT-6 22 aT-7,8 pT-7,S See text for explanation of nomenclature. At neutral pH.
Tryptic
peptidea
5, 6
0
__
++ +++ +++
*
90 minutes (see text)
Peak 50. in figs.
12 13
11 15 16 li
18 19
20 21
rT.-3 :.T-9
aT-1 aT-11 aT-8 yT-8
aT-9 (oxid.) yT-15 (oxid.) CYT-9 -;T-15 aT-2 aT-l,2
Chargeb
-0 0 0
+
L
0 4-
0 -T-
-... c
9
rT-G
-;T-10 aT-5 rT-S,R -/T-16
4
0 i
+
t -
. CYT-4 rT-2 aT-10
++
aT-7 yT-7 aT-3 yT-4
+-c
aT-14
yT-1,2 aT-6 aT-i 8 yT-7,S
i
Figure 3. Separation of peptides in a tryptic hydrolyzate of a* chains on Dowex-50
Correlation of Peak Numbers and Identity of Tryptic Peptides
19
(1
Of
i
+A L
L -L
0 J-+ +++ +++
2
second and third containers of the gradient device and was replaced in the second container only with a volume of 2iL' pyridine equivalent t o that of the buffer in the mixer, while at fraction 1000, the developer was changed to 2 S pyridine, and a t fraction 1100 to 2 5 NaOH. At approximately fraction 1200, the front of NaOH emerged from the column with a concomitant rise in pH. This modification and similar ones do not have any special advantage. It is not germane here to discuss the determination of the sequence of the peptides that have been isolated in this way. However, a correlation of the content of the peaks iTith the structure of the peptide is recorded in Table 11. The nomenclature for the peptides is that published by Baglioni ( 2 ) in which all possible tryptic peptides are numbered in order from the IY-terminus of the chain. The ordering of the tryptic peptides in these chains is based on the investigations of several groups (4, 9, 18), and the sequence of the individual peptides is given in these references. The procedure has also been used satisfactorily for the isolation of peptides from a chymotryptic hydrolyzate of the y chains: The resulting chromatogram has been published elsewhere (16). Dowex-1 has been used to purify many peptides that have been isolated from the chromatograms presented in Figures 2 t o 6 or other chromatograms from other kinds of hydrolysates. T o present all of these chromatograms is clearly impractical. Representative chromatograms have, therefore, been chosen t o illustrate the results. Figures 7 , 8, and 9 typify the separations that result from the use of Doweu-1 with the three types of developer. The legend of each figure gives the source of the material that was chromatographed. Although each chromatogram has been continued to a total of 500 fractions with changes to 1.4; and 2.47 HOAc as described, only the peaks depicted were observed. Indeed, only in occasional instances has any peptide emerged after the change t o Z X HOAc. (If a change is made t o glacial acetic acid, the flow rate slows markedly. The column may have to be repoured.) VOL. 34, NO. 12, NOVEMBER 1962
1573
,F Figure 5. Separation of peptides in a tryptic hydrolyzate of human fetal hemoglobin (Frr) on Dowex-50 Hydrolytic conditions os in Figure 2
Figure 4. Separation of peptides in a tryptic hydrolyzate of P A chains on Dowex-50 Hydrolytic conditions as in Figure 2
When fraction 500 has been reached, the p H in all cases had decreased to about 2.2. During this investigation, two ninhydrin procedures have been used to detect the presence of peptides. Although the effect has not been studied in detail, i t is apparent that the intensity of color that is produced b y a given peptide may be rather different in the two procedures. When the usual procedure of Moore and Stein (12) is used, the reaction mixture is heated in a relatively small volume and then diluted. However, in the procedure with the Technicon AutoAnalyzer, the dilution is made prior to the heating. I n some instances, the intensity of color produced by the second procedure is less. DISCUSSION
Rudloff and Braunitzer (14) rejected Dowex-50 as an ion exchange medium for separating peptides because some peptides mere strongly adsorbed and therefore difficult to develop through the column. I n the present investigation, this difficulty has not been encountered. All soluble peptides regardless of their amino acid composition, sequence, or charge have emerged from the column by the point a t which the p H of the developer is about 4.7 and the pyridine concentration is about 1.gAV. This statement is based upon two facts: All soluble tryptic peptides from hemoglobin that may be detected by the so-called “fingerprint” procedure of Ingram (7) have been detected in the chromatogram, and the unnumbered peak a t the very end of the chromatogram is minor in comparison to the sum of the others and yet contains all that is mashed from the column with 2N SaOH. Modifications of the gradient
have been ineffective in sharpening the peaks or speeding up the emergence of zones. Experience suggests that little is t o be gained by continuing the gradient beyond 900 fractions. If a t that point the developer is replaced by 2N NaOH, about 100 fractions more will be collected before the front of S a O H reaches the bottom of the column. With peptides from other proteins, modification of the described procedure may be necessary. The gradient that has been used on the Dowex-50 chromatograms is based on preliminary experiments in which sodium-containing buffers of sodium ion concentration and p H equivalent to the pyridine-acetic acid buffers were used. The gradients for the most part have been linear or slightly convex; these alterations have had relatively little influence on the quality of the separations. The effect of a rather rapidly changing gradient, such as would be produced by a relatively small constant volume mixing chamber, has not been investigated. As Rudloff and Braunitzer (14) point out, one expects negatively charged peptides (where the charge is that a t neutral pH) t o emerge most rapidly from a column of Dowex-50 and to be followed by neutral and positively charged peptides, whereas the order of emergence from Dowe-i-1 would be the reverse. So simple a relationship between charge
and the order of emergence of the peptides from Dowex-50 does not exist. Examination of Table I1 clearly shows that charge is a n important but not necessarily the determining factor. However, until many more data are available, an attempt to correlate structure and chromatographic behavior in detail is likely to meet with small success. Chromatography on Dorvex-50 is capable of very fine distinctions; for example, the separation of val-thr-leu and thr-Val-leu from a chymotryptic hydrolyzate of aF chains has been observed. I n earlier investigations with gelatin ( l 7 ) , separation of such pairs as ala-gly and gly-ala as well as gly-glu and glu-gly was achieved without difficulty. The order of emergence on Dowex-1 appears to be far more influenced by the charge than that on Dowex-50. The basic peptides tend to emerge rapidly to be followed by the neutral and acidic peptides. If development is made with the p H 8.3 buffer, a very basic peptide will emerge almost with front of developer. -4s a result, a mixture of basic peptides sometimes is not successfully separated. The separation can usually be much improved by the use of the p H 9.3 buffer which retards the movement considerably as shown, for example. in Figure 9. The separation of neutral peptides on Dowex-1 may be difficult. This is apparent also from the results of Rudloff
c
A 0
ala-Val-his1 Ithr-pro-ala-Val-his
I
g t
6 PH
4
01
I
,
I
I
Fraction No
k p H 8 O buffer
Figure 6.
Separation of peptides in a tryptic hydrolyzate of
y chains Hydrolytic conditions as in Figure 2
1574
ANALYTICAL CHEMISTRY
I
I
100
50
FO1N
HOAc
I
1
I50
+05N
HOAc
Figure 7. Separation of a peptide fraction on Dowex-1 beginning with p H 8.0 buffer Peptide fraction derived from a chymotryptic digest of oxidized aF chains and emerged about fraction 310 from a Dowex-50 column
gl y-lys-lys-Val-ala-asp-ala~leu
08OI c 0
e 06P 0 4
I
I
1
F r o ~ I ~ oNO n
t P H 8 3 bu!!er
SO
lo3
bO1N
HOAC
IS0
200
r 0 5 N HOAc
PI0
FN HOAC
Figure 8. Separation of a peptide fraction on Dowex-1 beginning with p H 8.3 buffer Peptide fraction derived from a 24-hour tryptic digest of carbamidomethyl y chains and emerged from a Dowex-50 column at a position equivalent to p e a k 12 of Figure 6
and Braunitzer (14). When the p H 8.3 buffer is used, a sharp break in p H froin about 7 to 5.7 occurs regardless of the way in which the change t o higher concentrations of HOAc is made (Figure 8). At this point, a mixture of neutral peptides is likely to emerge. The p H 8.0 buffer eliminates the rapid change of p H through this range; Figure 7 is a n example of its usefulness. The separation of acidic peptides is little influenced b y the p H of the starting buffer. A knowledge of the charge of the peptides to be separated as determined b y paper electrophoresis at p H 6.4 aids greatly in choosing the appropriate conditions for rechromatographing on Dowex-1. I n the Dowes-1 procedure, the peptides are eluted by constantly increasing the concentration of acetic acid. Although they have used a gradient, Rudloff and Braunitzer (14) have changed consecutively from 0.1N t o 2N t o glacial HOdc. I n our experience these rather abrupt changes have lessened the separation of the neutral peptides, For this reason, the sequence from 0.114’ to 0.LV t o lA* to 2-lr HOAc has been devised. I n occasional instances, the phenomenon of “double zoning” has been observed in chromatograms on Dowex-1. This behavior in which a n apparently homogeneous material forms two zones 011 the chromatogram has been observed n-ith simpler compounds (15). The variety of ionizable groups in peptides might lead to double zoning by the separation of molecules that are differently charged. The phenomenon has not been observed so frequently that its appearance can be correlated definitely with the structure of the peptide, It is not, for example, restricted to peptides which contain aspartic acid and which, therefore, might undergo a n 01 to /3 arrangement (13) or to those which contain histidine whose charge n-ould be much influenced a t pH’s near 6.
04-
02-
I Fraction hlo
I
I
I
50
100
150
k - P H 9 3 buffer
Folk
HOAc
F O S N HOAc
Figure 9. Separation of a peptide fraction on Dowex-1 beginning with pH 9.3 buffer Peptide fraction derived from a chymotryptic digest of oxidized aF chains and emerged about fraction 5 4 0 from a Dowex-50 column
If the purification of a compound necessitates rechromatography, the greatest chance of success clearly requires the use of vastly different conditions. However, if a peptide is isolated from one ion exchanger and rechromatographed on the same, the conditions may be changed t o a limited extent only. The use of trvo greatly different ion exchangers as described in this paper has resulted in the effective isolation of many peptides in high purity. I n many instances, adjacent peaks that were not 1%-ellseparated have been found on further examination t o have little mutual contamination. Whether Dowex-50 or Dowes-1 is used first for the separation of the complex mixture into simpler mixtures is probably unimportant. Because a column of Dowex-50 shrinks during chromatography and must be repacked before the next chromatogram whereas a column of Dowex-1 does not and need only be re-equilibrated, considerable time and effort are saved by using first Dowex-50 and then Dowes-1. ACKNOWLEDGMENT
Parts of this investigation have had the assistance of J. Roger Shelton and iT7illiam Fenninger. The composition and use of the pyridine-acetic acid developers on Dowes-50 were suggested b y Joe R . Kimmel of the University of Utah. LITERATURE CITED
(1) Allen, D. W.,Schroeder, IT. A., Balog, J., J . 24m.Chem. SOC.80, 1628
(1958’1. (2) Baglioni, C., Biochim. Biophys. Acta 48, 392 (1961). (3) Bock, R. RI., Ling, N. S., ASAL. CHEM.26, 1543 (1954).
(4) Braunitzer, G., Gehring-lluller, R., Hilschmann, S.,Hilse, K., Hobom, G., Rudloff, V., Wittmann-Liebold, B., Z. physiol. Chem. 325, 283 (1961). (5) Hirs, C. H. W.. Moore. S.. Stein. W.H.’, J . Biol. Chem. 195,’669’(1952): (6) Ibid.., 219., 623 11956). (7) Ingram, V. hi., Biochinz. Biophys. Acta 28, 539 (1958). (8) Kimmel, J. R., Kato, G. K., Paiva, A. C. M., Smith, E. L., J . B i d . Chem. (in press). (9) Konigsberg, IT., Guidotti, G., Hill, R. J., Ibid., 236, PC55 (1961). (10) Margoliaah, E., Smith, E. 1L., A‘ature 192, 1121 (1961). (11) Moore, S., Stein, W. H., J . Biol. Chem. 192, 663 (1951). (12) Ibid.. 211. 907 (1954). (13) Naughton; M.A., Sanger, F., Hartley, B. S., Sha--, D. C., Biochem. J . 77, 149 (1960). (14) Rudloff, I-., Braunitzer, G., 2. physzol. Chem. 323, 129 (1961). (15) Schroeder, W. A.. Ann. h’. Y . Acad. Sci. 49, 204 (1948). (16) Schroeder, ‘A7. 4., Jones, R. T., Shelton, J. R., Shelton, J. B., Cormick, J., McCalla, K., Proc. S a t . Acad. Sci. 47, 811 (1961). (17) Schroeder, W. A., Kay, L. >I., LeGette, J., Honnen, L., Green, F. C., J . A m . Chem. SOC.76, 3556 (1954). (18) Schroeder, W. A., Shelton, J. R., Shelton, J. B., Cormick, J., Proc. A-at. Acad. Sci. 48, 284 (1962). (19) Thompson, A. R., Bzochem. J . 61, 253 (1955). (20) 1-anecek, J., hleloun, B., Kostka, V., Keil, B., Sorm, F., Collection Czech. Chem. Commun. 25,2358 (1960). (21) Wilson, S., Smith, D. B., Can. J . Biochem. Physiol. 37, 405 (1959). RECEIVEDfor review June 11, 1962. Accepted August 30, 1962. Contribution S o . 2857 from the Division of Chemistry and Chemical Engineering, California Institute of Technology. Presented in part at the 141st meeting of the American Chemical Society, Washington, D. C., March 1962, a t the symposium honoring L. Zechmeister as recipient of the ACS Award in Chromatography and Electrophoresis. This investigation has been supported in part by a grant (H-2558) from the Kational Institutes of Health, United States Public Health Service.
VOL. 34, NO. 12, NOVEMBER 1962
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