ANALYTICAL
BIOCHEMISTRY
120, 198-203 (1982)
Analytical
ion Exchange
WOJCIECH
ARDELT
Chromatography
AND
MICHAEL
LASKOWSKI,
of Proteins JR.
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907 Received September I I, 1981 A low-pressure system for analytical ion exchange chromatography of proteins is described. Elution of proteins is performed by salt gradient and protein concentration is monitored at 206 nm. Protein mixtures consisting of microgram amounts of each component can be quantitatively resolved in 40 min. Quantitation of results is done by integration of the peak areas using a digital recorder with a data processing system. The method was successfully used for separation of proteins different by 1 unit charge only.
Separation of proteins differing from one another by small charge differences have typically been carried out by either ion exchange or disc gel electrophoretic methods. It is generally true that ion exchange chromatography is far better than gel electrophoresis as a preparative technique. However, disc gel electrophoresis is regarded as a superior analytical technique because of (a) higher sensitivity, and (b) multiple running of samples. Quantitative disc gel electrophoresis has been extensively used in our laboratory and in many other laboratories to measure the conversion of one protein species into another different by 1 unit charge (e.g., conversion of virgin into modified proteinase inhibitors). This worked exceedingly well for the relatively large soybean trypsin inhibitor (Kunitz) which exhibits good electrophoretic characteristics on polyacrylamide gels ( 1,2). However, more recently we have found that small protein proteinase inhibitors such as pancreatic secretory trypsin inhibitors (Kazal) (3,4), pancreatic trypsin inhibitor (Kunitz), and avian ovomucoid domains (unpublished) stack, fix, and stain poorly in gels. This makes quantitation of results extremely difficult. Therefore, we have decided to explore the possible analytical application of ion exchange chromatography as an alternative technique. In order 0003-2697/82/030198-06$02.00/O Copyright 0 1982 by Academic Press, Inc. AU rights of reproduction in any form reserved.
to increase sensitivity and to decrease the amount of material required, we decided to monitor protein concentration at 206 nm. However, at this wavelength even a sodium chloride gradient produces a very significant background which interferes with quantitation of the results. We did not completely eliminate this background but we did manage to make it highly reproducible. We record this background alone on a digital recorder and subtract it from the digitally recorded run to obtain a clean and highly reproducible chromatogram. We can readily follow the conversion of ovomucoid third domains into their modified forms (with reactive site peptide bond hydrolyzed) by anionic exchange chromatography at pH 9.0 using microgram quantities of protein per assay. The end-to-end run is about 40 min and the quantitative accuracy is much better than that of disc electrophoresis for our system. MATERIALS AND METHODS Biochemicals. Bovine trypsin and a-chymotrypsin were obtained from Worthington Biochemical Corporation (Freehold, N. J.). Aspergillus alkaline proteinase (aspergillopeptidase B) and Staphyloccocus aureus proteinase V8 were generous gifts from Drs. G. Kalnitsky and G. Drapeau, respectively. 198
ANALYTICAL
ION EXCHANGE
CHROMATOGRAPHY
The chromogenic trypsin burst titrant, p-nitrophenyl p’-guanidobenzoate (GdnBzONp)’ was purchased from Nutritional Biochemical Corporation (Cleveland, Ohio). The fluorogenic burst titrant of trypsin 4methylumbelliferyl p-guanidobenzoate (5) was synthesized in this laboratory by P. Fankhauser, using a method similar to that of Chase and Shaw (6) for GdnBzONp. The chymotrypsin burst titrant 4-methylumbelliferyl-p-trimethyl ammonium cinnamate chloride (MUTMAC) was a product of Sigma Chemical Company (St. Louis, MO.). N- Succinyl- glycyl- glycyl- L- phenylalanine p-nitroanilide (Sue-Gly-Gly-Phe pNA) was given to us by Dr. S. Blumberg and DEAESepharose CL-6B was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden).
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lyzed (for sequence see Ref. ( IO))-was obtained by treatment of TKY III with cu-chymotrypsin at low pH followed by isolation of the product on DEAE-Sepharose CL-6B (manuscript in preparation). Determination of the concentrations of TKY ZZZsolutions. This was performed by
titration of the inhibitor solutions with standardized solution of cY-chymotrypsin using Sue-Gly-Gly-Phe pNA as a substrate. Standardization of cu-chymotrypsin was in turn carried out as described by Estell et al. (11) except that MUTMAC was used as a burst titrant. Difference spectrum. Difference spectrum of TKY III* vs TKY III was recorded in 0.05 M ammonium chloride buffer (pH 9.0) using Cary 118 double-beam recording specPreparation of turkey ovomucoid third trophotometer. domain (TKY ZZZ). Turkey ovomucoid was Analytical chromatography system. Anprepared by a modified procedure of Linealytical glass columns (9 X 250 mm) as well weaver and Murray (7) as described by Bo- as a septum injector were products of Altex gard et al. (8). Carbohydrate-free third do- Scientific Inc. (Berkeley, Calif.). Each colmain was generated by limited proteolysis umn was equipped with one regular bed supwith staphyloccocal proteinase (9,10) and port and a plunger to obtain 9 X 80-mm bed isolated by molecular sieving chromatogradimensions. The valves (three- and four-way phy (8,lO). Final purification was accomslide ones) were obtained from Durrum plished by DEAE-Sepharose CL-6B chro- Chemical Corporation (Palo Alto, Calif.). matography (not shown) using O-O. 1 M linear Three-channel peristaltic pumps (P-3) of sodium chloride gradient in 0.04 M TrisPharmacia Fine Chemicals (Uppsala, SweHCl buffer, pH 8.8. den) were used. The monitor employed was The ovomucoid third domain preparations the 2138 Uvicord S (LKB-Produkter A. B., from other avian species (Japanese quail and Bromma, Sweden) single-beam uv monitor Gambel’s quail, ruffed grouse, and goose) equipped with a standard low-pressure flowwere prepared by the above method by Drs. through cell (pathlength, 2.5 mm; volume, W. C. Bogard, M. Wieczorek, and I. Kato 0.07 ml) and a 206-nm interference filter. of this laboratory, respectively. The recorder was the 8120 Recorder with Preparation of turkey ovomucoid third’ Data Processing and Storage manufactured domain, modified (TKY ZZZ*). The modified by Bascom-Turner Instruments (Newton, form of the inhibitor-with the -Leu( 18)Mass.). Glu( 19)-reactive site peptide bond hydroThe system we developed is presented in Fig. 1. It consists of two subsystems: one ’ Abbreviations used: GdnBzONp, p-nitrophenyl p’- (right side of the figure), operated by pump guanidobenzoate; MUTMAC, 4-methylumbelliferyl-p1, is used for chromatographic runs and the trimethyl ammonium cinnamate chloride; Suc-Glyother (left side of the figure), operated by Gly-Phe pNA, N-succinyl-glycyl-glycyl-L-phenylalapump 2, serves for regeneration of the colnine p-nitroanilide; TKY III, turkey ovomucoid third umns. Both subsystems work simultadomain; TKY III*, turkey ovomucoid third domain, modified. neously; chromatography is carried out in
200
ARDELT
AND LASKOWSKI
* PUMP
2
E
5
I N
:
LIMITING
STARTING
BUFFER
BUFFER c3
PROCESSOR
WASTE
FIG. I. Schematic representation of the system for analytical chromatography text for details.
one of the two columns while the other column is regenerated. All solutions used are previously filtered through a 0.22-pm Millipore filter. The columns are filled with DEAE-Sepharose CL-6B equilibrated in 0.02 M ammonium chloride buffer, pH 9.0. The samples (5-30 pg of protein in l-100 ~1) are introduced with a Hamilton syringe through the septum injector. The column is developed at 55 ml/h flow rate by a linear sodium chloride gradient (O-O.1 M) generated by a three-channel pump according to the instructions of the pump manufacturer. Twenty milliliters of the starting (equilibrating) buffer is used to make the gradient with 0.1 M sodium chloride made in the equilibrating buffer used as a limiting buffer. The elution is monitored at 206 nm (absorbance range 0.05) and the pattern is recorded on a Bascorn-Turner computerized recorder. After the end of a run (about 42 min), the gradient-forming device and the injector are washed (2-3 min) with the equilibrating buffer by directing the stream to waste with valve 1. The used column is then connected to the regeneration subsystem and the re-
of proteins. See the
generated column is connected to the operating subsystem by switching valves 2 and 3. Before the next sample injection, the injector is connected back to the column with valve 1. The used column is regenerated by passing 0.5 M sodium chloride made in the equilibrating buffer (5 mitt, 90 ml/h) and washed (after switching valve 4) with the starting buffer until a run on the other column is completed. This long and fast washing regime is essential. With shorter or less exhaustive washing, the baseline of a subsequent run was not reproducible. RESULTS AND DISCUSSION
To test and illustrate the system developed, a set of four proteins was selected in such a manner that each subsequent protein differed from the former by one negative charge. The proteins were ovomucoid third domains (carbohydrate-free) of different avian species. Ovomucoid domains, which are Kazal-type protein proteinase inhibitors, have been extensively isolated and sequenced in our laboratory. Third domains can be prepared from entire ovomucoid (which consist
ANALYTICAL
ION
EXCHANGE
CHROMATOGRAPHY
of three domains) by S. aureus V8 proteinase cleavage of the connecting peptide between second and third domain (9,lO). They consist of 54-56 amino acid residues accounting for a molecular weight of 6000, and are highly homologous species to species. Japanese quail, Gambel’s quail, ruffed grouse, and goose ovomucoid third domains were chosen for the experiment presented in Fig. 2. Their sequences were published previously (lo), except for that of ruffed grouse third domain which was determined recently (M. Wieczorek and M. Laskowski, Jr., unpublished result). The last three proteins of the set differed from the first one-Japanese ovomucoid third domain-by one, two, and three negative charges, respectively, as a result of the following replacements: Lys(l8) - Leu (Gambel’s quail); Lys
t TIME
(minutes)
FIG. 2. Analytical DEAE-Sepharose chromatography of a mixture (about 10 pg each) of ovomucoid third domains derived from four different avian species. The peaks (l-4) correspond to Japanese quail, Gambel’s quail, ruffed grouse, and goose ovomucoid third domains, respectively. Each protein differed from its neighbor on the chromatogram by one negative charge. Chromatography conditions are given under Materials and Methods. (A) The apparent chromatographic pattern; (B) the baseline; (C) The pattern after baseline subtraction (A - B); (D) integrated pattern C.
OF PROTEINS
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(18) + Met and Lys(55) - Thr (ruffed grouse); Lys( 18) - Val, Arg(21) - Met, and Gly(32) - Asp (goose). The four inhibitors could be well resolved in our system (Fig. 2). However, the pattern was distorted by a strong background due to the sodium chloride gradient used for elution. This background was digitally recorded in a separate run and stored in the memory of the Bascorn-Turner recorder. It could be precisely subtracted from the pattern by means of a data processing system of the recorder. The resolution obtained was not as good as that of preparative chromatography (not shown) in which a “down to the baseline” separation could be achieved, but it was still quite adequate for quantitative determination of the concentration ratios of the resolved proteins. This could be done by integration of the peak areas using the computerized recorder. The ratios were 1.00:0.92:0.98:0.90, respectively. It must be stressed, however, that the elution times of third domains differing in sequence but bearing the same net charge were not always the same. They sometimes differed up to 20%. We intended to use the method for measuring the conversion of TKY III into its modified form-TKY III* (with the reactive peptide bond hydrolyzed) catalyzed by some proteinases. It was clear that at pH 9.0, TKY III* must differ from TKY III by one negative charge derived from the newly formed carboxyl end group which is largely dissociated, while the newly formed amino terminal is uncharged at this pH value. Therefore, the two forms of the inhibitor should be resolved in our system. It was also expected that the absorbance index at 206 nm of TKY III* may be different from that of TKY III, and therefore, appropriate corrections of the quantitative results might be necessary. It was found that during hydrolysis of the reactive-site peptide bond of TKY III by aspergillopeptidase B (manuscript in preparation), absorbance of the inhibitor at 277 nm (maximum of the spectrum) was not changed even when 85-90s
202
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AND
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conversion was achieved. It was thus evident 1 that the absorbance index at 277 nm of TKY III* was the same as that found of TKY III: A 19b,lcm= 8.4. Using this value to match precisely the solution concentrations, a difference spectrum of TKY III* vs TKY III was recorded. Only a small negative difference spectrum (not shown) was detected in the region 200-230 nm. The values of absorC bance index at 206 nm for TKY III and TKY III* were 340 and 329, respectively. \ D The difference between these values is too large to be due only to one missing peptide E bond in TKY III*. A slight conformational A change during TKY III - TKY III* conversion is therefore probable. The system was also independently calibrated by running standard mixtures of TKY III and TKY III* prepared from their solutions previously matched according to the absorption at 277 nm. The results are FIG. 3. Analytical DEAE-Sepharosechromatography given in Fig. 3. The patterns were integrated of the standard mixtures of turkey ovomucoid third doas described before. From the integrals (not main virgin and modified. The lines present the patterns conditions shown in the figure) apparent values of a after baseline subtraction. Chromatography fraction of TKY III* were calculated. They are given under Materials and Methods. (A) 100% TKY III; (B) 80% TKY III, 20% TKY III*; (C) 65% TKY appeared lower than those expected from III, 35% TKY III*; (D) 50% TKY III, 50% TKY III*; compositions of the standard mixtures by a (E) 35% TKY III, 65% TKY III*; (F) 20% TKY III, factor of 1.05 + 0.01. This number is slightly 80% TKY III*; (G) 100% TKY III*. higher than the factor 1.033 calculated diIt would make a resolution of proteins with rectly from the values of absorbance index at 206 nm determined for TKY III and TKY one charge difference impossible. Moreover, III*. This discrepancy may be attributed to the higher salt gradient necessary for elution a possible difference in light bandpass be- would increase baseline distortion which, in tween the Cary 118 spectrophotometer used turn, would force a change of absorption for measuring the absorbance indexes and range of the monitor at the expense of sensitivity of the system. If necessary, it is posthe uv monitor employed in our system. The sible to use a cationic exchanger instead of correction factor 1.05 was used in further experiments for multiplying the results, given the anionic exchanger DEAE-Sepharose CLas fractions of TKY III*, to obtain reliable 6B . The best choice would be CM-Sepharose CL-6B because of the same crosslinked values. The method presented in this paper ap- agarose support is used. Excellent mechanpeared very useful in measuring TKY ical properties of this support allow high flow III - TKY III* conversion in many experrates as well as regeneration of the exchanimental systems (manuscript in preparation). ger without column repackings. It can be used for analytical resolution of When only a few samples are to be run (up to six per day), the method given in this other proteins (e.g., following a limiting propaper is faster than disc gel electrophoresis teolysis in general). However, strong binding of proteins to the exchanger must be avoided. because time-consuming steps such as poly-
ANALYTICAL
ION EXCHANGE
CHROMATOGRAPHY
acrylamide gel preparation, staining, and destaining are avoided. In some cases destaining must be done by diffusion (3,4). This greatly prolongs the time of the complete experiment. In such cases, the difference between our method and disc gel electrophoresis is much more pronounced. When a large number of runs is needed, the method presented is slower than electrophoresis. However, efforts are presently being made in our laboratory to automate the system to make it more time efficient. Results obtained from the ion exchange method are more direct and more quantitative than those of disc gel electrophoresis. The method presented in this paper is convenient for low-molecular-weight proteins which do not stack and/or stain well in the gels. It is particularly good for proteins with low absorptivity at 280 nm (e.g., ovomucoid domains) which must be applied in large amounts when other analytical techniques (e.g., disc gel electrophoresis with direct uv scanning) are used. It is, however, worth mentioning that when sensitivity of the process is not of great importance, e.g., a larger amount of material is available, the system eventually can be used without the expensive Bascom-Turner instrument. At the absorbance range 1-2, any recorder can be employed providing 20-40 times more protein is applied and integration of the peak areas is performed.
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ACKNOWLEDGMENTS This research was supported by grants from the Division of General Medical Sciences, National Institutes of Health (GMl0831). and from the National Science Foundation (PCM77-17554). We are grateful to Professors S. Blumberg, G. P. Drapeau, and G. Kalnitsky for gifts of materials used in this research.
REFERENCES 1. Niekamp, C. W., Hixson, H. F., Jr., and Laskowski, M., Jr. (1969) Biochemistry 8, 16-22. 2. Mattis, J. A., and Laskowski, M., Jr. (1973) Biochemistry 12, 2239-2245. 3. Sealock, R. W., and Laskowski, M., Jr. (1973) Biochemistry 12, 3 139-3 146. 4. Sealock, R. W. (1972) Ph.D. Thesis, Purdue University, W. Lafayette, Ind. 5. Jameson, G. W., Robert, D. V., Adams, R. W., Kyle, W. S. A., and Elmore, D. T. (1973) Biochem. J. 131, 107-117. 6. Chase, T., Jr., and Shaw, E. (1967) Biochem. Biophys. Res. Commun. 29, 508-514. 7. Lineweaver, H., and Murray, C. W. (1947) J. Biol. Chem. 171, 565-581. 8. Bogard, W. C., Jr., Kato, I., and Laskowski, M., Jr. (1980) J. Biol. Chem. 255, 6569-6574. 9. Kato, I., Kohr, W. J., and Laskowski, M., Jr. (1977) Fed. Proc. 36, 764. IO. Kato, I., Kohr, W. J., and Laskowski, M., Jr. ( 1978) in Regulatory Proteolytic Enzymes and Their Inhibitors (Magnusson, S., Ottesen, M., Foltman, B., Dano, K., and Neurath, H., eds.), Vol. 47, pp. 197-206, Pergamon, Oxford. 11. Estell, D. A., Wilson, K. A., and Laskowski, M., Jr. (1980) Biochemistry 19, 131-137.