Anal. Chem. 1995, 67, 2539-2547
The Absence of Chiral Recognition Ability in Ovomucoid: Ovoglycoprotein=BondedHPLC Stationary Phases for Chiral Recognition Jun Haginaka,* Chikako Seyama, and Naoko Kanasugi
Faculty of Pharmaceutical Sciences, Mukogawa Women's University, I 1-68, Koshien Kyuban-cho, Nishinomiya 663, Japan
Commercial chicken ovomucoid (OMCHI) and OMCHI, isolated by precipitation of egg whites with organic solvents, both of which as crude products, were hclionated by anion- and cation-exchange chromatogmphy. The obtained four fractions were characterized by reversedphase chromatography, N-terminal sequencing, matrixassisted laser desorption ionization time-of-ftight(MALDITOE) mass spectrometry,determination of sugar contents, and trypsin-inhibitory activities. Three fractions were OMCHI variants ditrering in carbohydrate composition, especially in sialic acid content, and the other draction was tentatively termed ovoglycoprotein (OGCHI). The OMCHI and OGCHI are ditrerent in physicochemical and biochemical properties: average molecular weight, 26 00027 700 for OMCHI variants and 29 700 for OGCHI; N-terminal amino acid, Ala for OMCHI and Thr for OGCHI; and trypsin-inhibitory activity, positive for OMCHI and negative for OGCHI. These OMCHI variants and OGCHI were bound to aminopropyl silica gels to evaluate chiral recognition ability. OMCHI is reported to have chiral recognition ability (Miwa, T.; et al. Chem. Pharm. Bull. 1987, 35, 682-686). However, neither OMCHI variant had appreciable chiral recognition ability, while the OGCHI had excellent chiral recognition properties as compared to those of the OMCHI reported previously. This reveals that the chiral recognition ability of the OMCHI reported previously comes fkom the OGCHI, which is present in crude OMCHI as an impurity. Protein-bonded stationary phases including albumins such as bovine serum albumin @SA)' and human serum albumin,2 mucoids such as al-acid glycoprotein3 and ovom~coid,~ and enzymes such as cellulase5have been developed for separation of enantiomeric forms. These protein-bonded columns are now commercially available. Among those, al-acid glycoprotein and ovomucoid-bonded columns can separate a wide range of weakly acidic, weakly basic, and neutral racemates.6 Recently, Kirkland et a1.7 compared commercially available mucoid columns based (1) Allenmark, S. J. Liq. Chromatogr. 1986,9,425-442. (2) Domenici, E.; Bertucci, C.; Salvadori, P.; Felix, G.; Cahagne, I.; Montellier S.; Wainer, I. W. Chromatographia 1990,29,170-176. (3) Hermansson, J. J. Chromatogr. 1983,269,71-80. (4) Miwa, T.; Ichikawa, M.: Tsuno, M.; Hattori, T.; Miyakawa, T.; Kayano, M.; Miyake, Y. Chem. Pharm. Bull. 1987,35, 682-686. (5) Erlandsson, P.; Marle, I.; Hansson, L.:Isaksson, R; Petterson, C.; Petterson, G. J. Am. Chem. SOC.1990,112,4573-4574. (6) Allenmark, S. Chromatographic Enantioseparation. Methods and Applications, 2nd ed.; Ellis Horwood; New York, 1991; pp 129-142. (7) Kirkland, K. M.; Neilson, K. L.; McCombs, D. A.J. Chromatogr, 1991,545, 43-58.
0003-2700/95/0367-2539$9.00/0 0 1995 American Chemical Society
on al-acid glycoprotein and ovomucoid. They confirmed that the latter column showed generally higher enantioselectivity and column efficiency and better column ruggedness. With regard to the ruggedness of the ovomucoid-bonded column, it was thought that native ovomucoid was inherently stable as reported by many in~estigators.8~~ Also, ovomucoid did not denature irreversibly even when subjected to 100 "C for 1 h,8 or to high concentrations of organic solvents such as ethanol and acetone during isolation.8J0 The advantages of protein-bonded stationary phases generally include the use of a mobile phase like reversed-phase HPLC, enantioselectivity for a wide range of compounds, and direct analysis without derivatization. The disadvantages have included low capacity, lack of column ruggedness, and limited understanding of the chiral recognition mechanism. Different pathways may be accessible for overcoming the disadvantages of protein-bonded stationary phases. Protein modification includes chemical modification of side chains of amino acidsll and using biosynthetic analogues by changing the DNA sequences.12 We modified commercial chicken ovomucoid (OMCHI)-bonded materials with glutaraldehyde, formaldehyde, glycerylaldehyde, and acetic anhydride.13J4 The obtained materials showed better column ruggedness for repetitive injections of samples than the unmodified 1nateria1s.l~ A protein fragment or protein domain is prepared by chemical or enzymatic ~leavage15-'~or can be prepared by overexpression with genetic technology and peptide synthesis. In principle, if a chiral binding site exists on one domain or one fragment and if it acts independently of the other domains or fragments, one should be able to make chiral phase columns with a domain or fragment. It could be of higher capacity because only the active protein mass would be used. It is also possible to understand the chiral recognition site (s) of protein columns by investigating whether independent chiral binding sites existed on each domain or ~
~~
~
(8) Fredericq, E.; Deutsch, H. F. J. Biol. Chem. 1949,181, 499-510. (9) Das, B. K; Aganval, S. IC;Khan, M. Y. Biochim. Biophys. Acta 1991,1076, 343-350. (10) Lineweaver, H.; Murray, C. W.J Biol. Chem. 1947,171, 565-581. (11) Hirs, C. H.W., Timasheff, S. N., Eds. Methods in Enymology; Academic Press; New York, 1977; Vol. 47. (12) Goeddel, D. W. Methods in Enzymology; Academic Press: New York, 1987: Vol. 185, pp 3-7. (13) Haginaka, J.; Seyama, Ch.; Yasuda, H.; Fujima, H.; Wada, H.J.Chromatogr, 1992,592,301-307. (14) Haginaka, J.; Murashima, T.; Seyama, Ch.; Fujima, H.; Wada, H. J. Chromatog. 1993,631,183-190. (15) King, T. P. Arch. Biochem. Biophys. 1973,156,509-520. (16) Geisow, M. J.; Beaven, G. H. Biochem. J. 1977,161.619-625. (17) Laskowski, M., Jr.; Kato, I.;Ardelt, W.; Cook, J.; Denton, A; Empie, M. W.; Kohr, W. J.; Park, S. J.; Parks, K; Shatzley, B. L.; Schoenberger, 0. L.; Tashiro, M.; Vichot, G.; Whatley, H. E.; Wieczorek, A; Wieczorek, M. Biochemisty 1987,26, 202-221.
Analytical Chemistry, Vol. 67, No. 15, August 1, 1995 2539
fragment or not. Erlandsson and NilssonI8 and Anderson et al.19 made a column with BSA fragments, whose molecular weights are -38 000. However, the BSA fragment-bonded columns prepared by Anderson et all9 had lower capacity and poorer stability than the intact BSA-bonded columns. Recently, we modified isolation and binding methods of BSA fragments, whose molecular weights are -35 000.20 The obtained BSA fragmentbonded columns gave higher enantioselectivity and higher capacity than the intact BSA column because of higher density of chiral recognition site($. On the other hand, various turkey ovomucoid (OMTKY)21and OMCHI domains,?? which exist as three tandem, independent domain^,?^ were isolated, purified, and characterized. Columns were made with purified OMTKY and OMCHI domains to test the chiral recognition properties. The third of the OMTKY and OMCHI domains was found to be enantioselective to at least two classes of compounds, benzodiazepines and 2-arylpropionic acid derivatives. The chral recognition mechanism of the third domain was elucidated by using NMR measurements, molecular modeling, and computational chemistry. However, either the first or second domain of the OMTKY, a combination of the first and second domains of the OMTKY,21or the second domain of the OMCHP had no appreciable chiral recognition ability. These results may suggest that three domains should work in concert for chiral recognition of various solutes, because columns made with the whole, intact O M T W 4as well as OMCH125-28can resolve a wide range of weakly acidic, weakly basic, and neutral racemates. If a chiral binding site on one domain acts independently of the other domains, other proteins, with excellent chiral recognition ability, might be present in OMTKY and OMCHI proteins. Previously, Davis et aLZ9reported that commercial OMCHI proteins as well as the OMCHI isolated by precipitation with acetone contained other egg white proteins such as lysozyme, ovoinhibitor, conalbumin, ovalbumin and flavoprotein as impurities. The presence of some of those proteins was shown by other investigator^.^^,^^ These reports reveal that commercial OMCHI and OMCHI isolated by precipitation with organic solvents are crude. Also, our preliminary result suggests the presence of an unknown protein portion, -10% estimated from peak area in the commercial OMCHI and in the OMTKY and OMCHI isolated by precipitation with organic solvents.24 The unknown protein portion could be ascribable to the chiral recognition ability of the crude OMCHI and OMTKY. (18) Erlandsson, P.; Nilsson, S. J. Chromatogr. 1989,482,35-51. (19) Anderson, S.: Allenmark, S.; Erlandsson, P.; Nilsson. S. J. Chromatogr. 1990,498,81-91. (20) Haginaka, J.; Kanasugi, N. J. Chromafogr. 1995,694,71-80. (21) Pinkerton, T. C.; Howe, W. J.; Ulrich. E. L.; Comiskey, J. P.; Haginaka, J.; Murashima, T.; Walkenhorst, W. F.; Westler, W. M.; Markley, J. L. Anal. Chem. 1995,67,2354-2367. (22) Haginaka, J.; Seyama, Ch., unpublished results. (23) Kato, I.; Schrode, J.; Kohr, W. J.; Laskowski, M., Jr. Biochemistry 1987, 26, 193-201. (24) Haginaka, J.; Seyama. Ch.; Murashima, T. J. Chromatogr., A, in press. (25) Haginaka, J.; JVakai, J.; Yasuda, H.; Takahashi. IC;Katagi,T. Chromatographia 1990,29, 587-592. (26) Iredale. J.; Aubry, A.-F.; Wainer, I. W. Chromatographia 1991,31,329334. (27) Okamoto, M.; Nakazawa, H. J Chromatogr. 1990,504,445. (28) Kirkland, IC M.; Neilson, IC L.; McCombs, D. A,; DeStefano, J. J. LC-GC 1992,10, 322-342. (29) Davis, J. G.; Mapes, C. J.; Donovan, J. W. Biochemistry 1971,10,39-42. (30) Donovan, J. W. Biochemistry 1967,6, 3918-3927. (31) Feeney, R E.; Stevens, F. C.; Osuga, D. T.J Bid. Chem. 1963,238,565581.
2540
Analytical Chemistry, Vol. 67,No. 15, August I , 1995
In this study, we focused on commercial OMCHI proteins because the OMCHI-bonded column, Ultron E S O V V I ,is~ ~made with the proteins and used for chral resolution of a wide range of compounds.25-28 This paper deals with the isolation of OMCHI variants differing in carbohydrate composition and an unknown protein portion from the commercial OMCHI proteins and characterization of these proteins by reversed-phase chromatog raphy, N-terminal sequencing, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, determination of sugar contents and trypsin-inhibitory activities. Also, columns were made with these proteins in order to clanfy whether an OMCHI protein has chiral recognition ability or not and where the chiral recognition ability of the commercial OMCHI comes from. EXPERIMENTAL SECTION Reagents and Materials. Ibuprofen, ketoprofen, chlorpheniramine maleate, and hexobarbital were kindly donated by Kaken Pharmaceutical Co. (Tokyo, Japan), Chugai Pharmaceutical Co. (Tokyo, Japan), Essex Nippon (Osaka, Japan), and Teikoku Chemicals (Osaka, Japan). Benzoin, alprenolol, and propranolol were purchased from Sigma Chemicals (St. Louis, MO). Ovomucoid proteins from chicken egg whites (OMCHI) were purchased from Eisai (Tokyo, Japan). DEAE Sepharose CLGB, Sephadex G25 (fine), and SP Sepharose FF were purchased from Pharmacia Biotech (Tokyo, Japan). Trypsin, N-benzoylargininePnitroanilide (BAPNA) and NF-disuccinimidyl carbonate @SC) were purchased from S i a Chemical Co. HPLC grade ethanol was obtained from Wako Pure Chemical Industries (Osaka, Japan). Aminopropyl silica gels (Ultron-NHz,5pm diameter, l 2 @ l pore size) used are kindly donated by Shinwa Chemical Industries (Kyoto, Japan). Other solvents and reagents were used without further purification. Water purified with a Nanopure I1 unit (Barnstead, Boston, MA) was used for the preparation of the eluent and the sample solution. Isolation of OMCHI from Egg Whites. OMCHI was precipitated with ethanol and acetone, respectively, according to procedures modified slightly from those of Kat0 et al?3 and Forsythe and Foster.33 The obtained OMCHI was further purified by column chromatography using a m o d ~ e dform of the method reported by Kat0 et al.23 The obtained OMCHI and commercial OMCHI is termed crude OMCHI. Ion-Exchange Chromatography of Crude OMCHI. Two grams of crude OMCHI was applied to a DEAE Sepharose C L 6B column (5 x 12 cm) that was equilibrated with 40 mM Tris buffer (PH 7.1) applying a 50, 100, and 200 mM NaCl stepwise change using an average flow rate of 100 mWh. The eluant was monitored at 280 nm with a Model AC-500 spectrophotometric monitor (Atto, Tokyo, Japan). The separation was performed at 4 "C. Three fractions were collected and lyophilized. The lyophilized samples were desalted with a Sephadex G25 (fine) column (5 x 20 cm) using 15 mM NHdHC03 as the buffer with an average flow rate of 120 mWh. The eluate was collected and lyophilized. The isolated fraction were termed fractions 1,2, and 3 4. The third fraction, fraction 3 4, was further isolated by SP Sepharose FF column (5 x 12 cm) that was equilibrated with 10
+
+
(32) An Ultron ESOVM column made with the crude OMCHI from Eisai (Tokyo, Japan) is now available from Shinwa Chemical Industries (Kyoto, Japan). (33) Forsythe, R H.; Foster, J. F. J. Bid. Chem. 1950,184,385-392.
3+4 I
0
I
4
I
8
Time (h) Figure 1. Chromatogram of crude OMCHI (commercial OMCHI) proteins on a preparative anion-exchange column: column, DEAE Sepharose CL-6B column (5 x 12 cm); eluent, 0.04 M Tris buffer (pH 7.1) applying 50, 100, and 200 mM NaCl stepwise change; average flow rate, 100 mUh; detection, 280 nm.
1
8 I Fraction 3
6
Fraction 4
4
I
8
Time (h) mM CH3COONH4 (PH 4.6) applying a liner gradient to 500 mM CH3COONH4 (PH 4.6) for 4.5 h at an average flow rate of 100 mL/h, and then the eluent was changed to 700 mM CH3COONHd (PH 4.6). The separation was performed at 4 "C. Detection was carried out at 280 nm. Two fractions were collected and lyophilized. The lyophilized samples were desalted with a Sephadex G25 (fine) column as described above, and the eluate was collected and lyophilized. The isolated fractions were termed fractions 3 and 4. N-Terminal Sequencing. A 7Gpg sample of each protein was reconstituted with 50 pL of water. A 5pL portion of the dilution was spotted on a solid support for N-terminal sequencing analysis using an AB1 473A protein sequencer (Applied Biosystems Division, Perkin Elmer Japan, Tokyo, Japan). MALDI-TOF Mass Spectrometry. The mass spectrometer used was a Vision 2000 reflector-type TOF instrument (Finnigan MAT, Tokyo, Japan) equipped with an N2 laser operating at a wavelength of 337 nm with a pulse duration of 3 ns. The laser beam diameter at the sample surface was 70 pm; laser irradiances were in the low lo6 W/cm2 range, close to the threshold for obtainiig ions. The ions generated were accelerated to a potential of 5 kV in the ion source and postaccelerated to a potential of 20 kV for detection with a secondary ion multiplier. The MALDITOF spectra represent the accumulation of 20-25 single laser shots. They were calibrated externally by a standard sample (Subtilisin Cudsberg, molecular weight of 27 288) that was placed on the same target. The matrix used was 2,5dihydroxybenzoic acid, dissolved in a 2:l mixture of 0.1% aqueous TFA and acetonitrile at a concentration of 50 mM. Samples were dissolved in a water at a concentration of M. A 0.5pL portion of the sample solution was mixed with an equal volume of the matrix solution on the target, resulting in a used sample amount of 500 fmol. After deposition on the stainless steel target, the sample was air-dried and introduced into the mass spectrometer.
Figure 2. Chromatogram of fraction 3 + 4 on a preparative anionexchange column. Column, SP Sepharose FF column (5 x 12 cm); eluent, 0.01 M CH3COONH4 (pH 4.6) applying a liner gradient to 0.5 M CH3COONH4 (pH 4.6) for 4.5 h and then applying a stepwise change to 0.7 M CH3COONH4 (pH 4.6); average flow rate, 100 mU h; detection, 280 nm.
Determination of Sugar Contents. The contents of sugars, hexoses, glucosamine, and sialic acid in each fraction were determined. Determination of hexose was based on the method of Hartley and J e v o n ~ .The ~ ~ sample was hydrolyzed in 4 M HCl at 100 "C for 3 h; determination of glucosamine was based on the method of Cessi and P i l i e g ~ .After ~ ~ hydrolysis in 0.05 M H2S04 at 80 "C for 1 h, determination of sialic acid was based on the method of A t n i i ~ f f . ~ ~ Trypsin Inhibitory Activities. Trypsin solution was prepared by dissolving 1 mg of trypsin in 0.001 M HCl and 0.02 M CaC12. The substrate was BAPNA. A solution of 50 mg of BAPNA in 50 mL of distilled water was prepared over a warm water bath. A 1WpL aliquot of trypsin solution, 1.9 mL of 0.1 M Tris-HC1 containing 0.02 M CaClz (PH 7.8), and 1.0 mL of substrate solution were added. The absorbance at 405 nm was measured as a function of time to determine the rate of absorbance increase, using a Model 228 spectrophotometer (Hitachi, Tokyo, Japan), To test for the inhibitory activity in each fraction, 100 pL of the solution was incubated at room temperature for 5 s with 100 pL of trypsin solution and 1.8 mL of 0.1 M Tris-HC1 containing 0.02 M CaCl2 (PH 7.8) . Afterward, 1.0 mL of substrate solution was added. Again,the change in absorbance was monitored with time. If the change in absorbance with time for each protein was significantly less than that of a substrate control, then inhibitory activity for trypsin was indicated. (34) Hartley, F. K; Jevons, F. R Biochem. 1. 1962,84, 134-139. (35) Cessi, C.; Piliego, F. Biochem. /. 1960,77,508-510. (36)Aminoff, D. Biochem. 1. 1961,81, 384-392.
Analytical ChetnMry, Vol. 67, No. 15, August 1, 1995
2541
Preparationof Protein-BondedMaterials. Five grams of aminopropyl silica gels was slurried in 100 mL of acetonitrile and reacted with 5 g of DSC at 30 "C for 20 h. The obtained DSCactivated aminopropyl silica gels were washed with acetonitrile, water, and methanol. The obtained materials were dried in vacuo at 40 "C for overnight. Each protein was bound to the DSCactivated aminopropyl silica gels as described below. A 900-mg sample of the DSC-activated silica gels was slumed in 10 mL of 20 mM phosphate buffer (PH 6.8). A 300-mg sample of each protein dissolved in 5 mL of the same buffer was added slowly to the mixture at room temperature for 1 h by adjusting the pH to 6.6; the resultant mixture was further stirred for 15 h at 30 "C. The reaction mixture was washed with water and reacted with aminoethanol-water (1:50) adjusted to pH 6.6 with hydrochloric acid at room temperature for 1 h. The reaction mixtures were filtered and washed with water and 5%ethanol. The obtained materials were packed into a 2.0 mm i.d. x 100 mm stainless steel column by the slurry packing method.35 The slurry and packing solvents were 5% ethanol. Chromatography. For chiral resolution of racemic solutes on the protein-bonded columns, the HPLC system used was composed of an LC-SA pump, an SPD-6A spectrophotometer, a Reodyne 7125 injector with a 5-pL loop, and a C-R6A integrator (all from Shimadzu, Kyoto, Japan). The flow rate was maintained at 0.2 mL/min. Detection was performed at 220 or 254 nm. Capacity factors ( k ) , enantioseparation factor (a),and resolution (RJ of racemates were calculated. The asymmetry factor (q)was calculated as reported p r e v i ~ u s l y .All ~ ~ separations were carried out at 25 "C using a water bath. The eluents are prepared by using phosphoric acid-sodium dihydrogen phosphate or sodium dihydrogen phosphate-disodium hydrogen phosphate and ethanol. The eluents used are specified in the figure and table legends. For reversed-phase chromatography of the isolated protein, the same HPLC system as described above was used except that two pumps were used for gradient elution. The eluents used are as follows: (eluent A) HaO-CH3CN (80:20, v/v) containing 0.1% trifluoroacetic acid (?F'A); (eluent B) HzO-CH~CN (20230, v/v) containing 0.1%TFA linear gradient from 0%eluent B at 0 min to 100%eluent B at 90 min. The column used was Cosmosil5C18 AR (4.6 mm i.d. x 250 mm) (Nacalai Tesque, Kyoto, Japan). Detection was carried out at 280 nm. The flow rate was 1.0 mL/ min. All separations were performed at 30 "C. Sample Preparation. A known amount of a racemic solute was dissolved in methanol or water, and the solution was diluted with the eluent to the desired concentration. A 5pL aliquot of the sample solution was loaded onto a column. The loaded amount was 0.04-0.1 pg. RESULTS AND DISCUSSION
Ion-ExchangeChromatographyof Crude OMCHI. Figure 1 shows a chromatogram of commercial OMCHI proteins follow-
ing preparative anion-exchange chromatography. Three fractions, 1, 2, and 3 + 4, were isolated. The fraction 3 4 was further isolated by preparative cationexchange chromatography, as shown in Figure 2. The isolated fraction was termed fractions 3 and 4. The OMCHI proteins, isolated from egg whites by precipitation with ethanol or acetone showed almost the same pattern as the commercial OMCHI following anion- and cation-exchange chromatograuhy.
+
(37) Snyder, L. R.; Kirkland, J. J. Introduction to M o d e m Liquid Chromatography, 2nd ed.; John Wiley & Sons: New York, 1979.
2542 Analytical Chemistry, Vol. 67, No. 15, August 1, 7995
0
2 0 Time (mill)
o
m
0
Time (min)
20
0
20
bin)
T i c (min)
o
m Time ( m i 3
Figure 3. Chromatogram of crude OMCHI (commercial OMCHI) (A) and fractions 1 (B), 2 (C), 3 (D),and 4 (E) on a reversed-phase column: column, Cosmosil 5C18-AR (4.6 mm i.d. x 250 mm); eluents, eluent A, HzO-CH~CN (80:20, v/v) including 0.1% TFA; eluent 6 , HzO-CH~CN (20:80,v/v) including 0.1% TFA; linear gradient from 0% eluent Bat 0 min to 100% eluent B at 90 min; flowrate, 1.O mumin; detection, 280 nm; sensitivity, 0.004 AUFS for (AD) and 0.032 AUFS for (E); loaded amount, 200 pg. Table 1. Carbohydrate Composition of Fractions 1-4.
concn (dl00E) glucosamine hexose
_ _ _ _ _ _ _ ~ ~ ~
fraction 1 2 3 4 a
16.4 16.6 14.5 15.1
5.75 4.65 7.23 7.26
sialic acid 0.13 0.40 1.50 2.82
All values were averages of six replicates.
Characterization of Fractions 1-4. The isolated fractions were characterized by reversed-phase chromatography, N-terminal sequencing, MALDI-TOF mass spectrometry, determination of sugar contents, and trypsin-inhibitory activities. Figure 3, parts A-E, shows chromatograms of commercial OMCHI and fractions 1-4, respectively, by reversed-phase chromatography. It is found that the commercial OMCHI contains one major protein portion at a retention time of 15 min and two minor protein portions at retention times of 20 and 30 min. Taking into account the chromatographic behaviors of fractions 1-4, fractions 1-3 could be OMCHI, and fraction 4 might be other protein portion (s). The N-terminal sequences of the first 15 amino acids in fractions 1-3 are A-E-V-D-X-SR-F-P-X-A-T-D-K-E,where X is a cystine or glycosylated amino acid residue. These amino acid sequences are in good agreement with those of OMCHI reported by Kat0 et aLZ3 On the other hand, the N-terminal sequence of the first 15 amino acids in fraction 4 is T-E-SP-X-SA-P-LV-P-AD-M-D, where X is a cystine or glycosylated amino acid residue. The second sequences of fractions 1-4 were not observed. Fractions 1-3 had inhibitory activities toward trypsin, but fraction 4 had no inhibitory activity. It was reported that OMCHI had
,
P -
a -
-
IO
2M’
I
I
I I”
O
H u t In**]
I w
XGm
C
I
I
I
1
6”
I
I 90000
P
IW
+a BO
m W
P
a IO 1p
IO 0
I
I
I
/I
w -
I 0
Mu. IW
I
m
M’
I
I
100)
lSmO
I
,w
I
I
I
llpm
5300)
7lmO
Figure 4. MALDI-TOF mass spectra of fractions 1-4. Mass spectra A-D correspond to fractions 1-4, respectively. For other conditions, see Experimental Section.
inhibitory activities toward trypsin.’O These results suggest that fractions 1-3 could be OMCHI and fraction 4 could be an unknown protein portion.
Table 1 shows the glucosamine, hexose, and sialic acid contents of fractions 1-4. There are slight differences in hexose content among fractions 1-3 and large d3erences in sialic acid Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
2543
A
B
C
D
E
N
4
I
E
‘8
2
sf
T
IC
(min)
Time (min)
Time (min)
I
Time (min)
Figure 5. Chiral resolution of benzoin on columns made with crude OMCHI (commercial OMCHI) (A), OMCHI-A (B), OMCHI-B (C), OMCHI-C (D), and OGCHI (E): eluent, 20 mM phosphate buffer (pH 5.0)-ethanol 9 O : l O (v/v); flow rate, 0.2 mUmin; detection: 220 nm; loaded amount, 100 ng.
content. Fraction 4 had the highest sialic acid content among all the fractions. It was reported that OMCHI variants differing in carbohydrate composition were separated into two components by electrophoresis3*and into three major and two minor components by cation-exchange c h r o m a t ~ g r a p h y .Also, ~ ~ ~ ~these ~ variants separated by cation-exchange chromatography had a slight variation in the content of glucosamine, galactose, and hexose and a large variation in the content of sialic acid. These results reveal that fractions 1-3 are the OMCHI variants differing in carbohydrate contents and that the sialic acid content is larger in the order of fractions 1, 2, and 3. Fraction 4 has a higher sialic acid content than the OMCHI. Fractions 1 and 2 are the major components of the OMCHI variants, as shown in Figures 1 and 2. Figure 4, parts A-D, shows MALDI-TOF mass spectra of fractions 1-4, respectively. The molecular weights averaged from MZt, Mt, and 2M+ ions or M2+,M+ and 2M+ and 3Mt ions are as follows; 27 747 f 170 for fraction 1, 26 023 f 25 for fraction 2, 26 478 f 100 for fraction 3, and 29 731 f 325 for fraction 4. It is clear that all the fractions are glycosylated and contains s i g d c a n t microheterogeneity in the carbohydrate moiety, especially significant in fraction 3. There are many reports for the average molecular weight of the OMCHI, which ranges from 27 OOO to 32 600.4O It is found from the MALDI-TOF mass spectrum, which can determine the molecular weight of a protein with an accuracy of fO.Ol%,41that the averaged molecular weight of the OMCHI is -27 000. Fraction 4 is a minor component in egg whites. We have searched the literature and data bases of proteins in egg whites to determine what fraction 4 was. Recently, we found two reports by Kettere1-,4~3*~ which suggest that fraction 4 might be tentatively named “ovoglycoprotein”. The physical properties of ovoglycoprotein reported by Ketterer are as follows: glucosamine, hexose, and sialic acid contents are 13.8, 13.6, and 3.0%, respectively; N-terminal amino acid is threonine; average molecular weight is 24 400; and ovoglycoprotein has no trypsin inhibitory activity. He mentioned that ovomucoid and ovoglycoprotein were coprecipitated from egg whites by organic solvents such as ethanol or 2544 Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
acetone. The two proteins could be separated by ion-exchange chromatography or electrophoresis. There is good agreement on the glucosamine and sialic acid contents between his and our data, while the content of hexose in fraction 4 is slightly lower than the ovoglycoprotein reported by Ketterer. The N-terminal amino acid is the same between the two glycoproteins isolated by Ketterer and us. The only discrepancy between his and our data is the average molecular weight. It is concluded that fractions 1-3 should be the OMCHI variants, especially because of the differing sialic acid content and that fraction 4 could be the ovoglycoprotein isolated by Ketterer. We have also tentatively termed fraction 4 ovoglycoprotein (OGCHI). We are now currently determining the amino acid sequences of the OGCHI. In the following section, fractions 1-4 are abbreviated OMCHI-A, OMCHI-B, OMCHI-C, and OGCHI, respectively. It is well-knownz3that the OMCHI has polymorphism in the amino acid sequences, which means the major form with 186 amino acid residues and the minor form with Va1134Ser135 deleted, as well as microheterogeneity in the carbohydrate moiety. It is plausible that the OGCHI as well as the OMCHI might have microheterogineity and polymorphism. However, we do not know whether the split of the OGCHI peak in Figure 3 is due to the microheterogeneity or polymorphism of the OGCHI or due to both. Chiral Recognition Ability of OMCHI Variants and OGCHI. Next, we covalently bound the OMCHI variants and OGCHI to porous silicas and packed the bonded-phase materials into stainless steel columns to evaluate the chiral recognition properties. For comparison, columns were also made with the commercial OMCHI, which includes -10% OGCHI by weight. Figure 5, parts A-E, shows the chiral resolution of benzoin, a neutral compound, on the columns made with crude OMCHI (commercial OMCHD, OMCHI-A, OMCHLB, OMCHI-C, and OGCHI, respectively. Figures 6 and 7, parts A-E, show the chiral resolution of chlorpheniramine and ketoprofen on the same columns as in Figure 5. These results reveal that OMCHI-A and OMCHI-B have no chiral recognition properties for benzoin, chlorpheniramine,
B
C
Table 3. Comparison of Capacity Factor (ki'), Enantloselectivity(a),and Resolution (Rs)of Various Solutes on Columns Made with Commerclal OMCHI and Isolated OGCHP
m
r!
W
N
P
'B .-E 5 c
~:
2
P
~
P
OMCHI
.m
g
E
3
h
h
i
column
8
'2 E E c v
v
h
i
kl'
a
R,
kl'
a
R,
benzoin hexobarbital alprenolol propranolol chlorpheniramine ibuprofen ketoprofen
2.50 0.35 2.53 7.49 1.03 4.05 7.69
2.71 1.00 1.12 1.12 2.05 1.18 1.11
6.06
11.4 1.52 15.9 42.6 5.42 9.03 23.5
3.18 1.29 1.13 1.18 2.27 1.39 1.20
10.1 0.83 0.84 0.78 5.89 2.58 1.97
0.31 0.44 3.09 0.88 0.82
All values were averages of three replicates. HPLC Conditions: column, 2.0 mm i.d. x 100 mm; eluent, 20 mM phosphate buffer @H 5.1)-ethanol 9010 (v/v); column temperature, 25 "C; flow rate, 0.2 mL/min; detection, 220 nm.
L
%me (min)
OGCHI
compound
c (min)
1
Time (min)
:(min)
Time (min)
Figure 6. Chiral resolution of chlorpheniramine on columns made with crude OMCHI (commercial OMCHI) (A), OMCHI-A (E), OMCHI-B (C), OMCHI-C (D), and OGCHI (E). Asterisk indicates maleic acid. Other conditions as in Figure 5. Table 2. Chlral Resolution of Benzoln, Chlorpheniramine, and Ketoprofen on Columns Made with OMCHI-C and a Mixture of OMCHI-A and OGCHP
OMCHI-C
column OMCHI-A 1.0%OGCHI
+
compound
kl'
a
kl'
a
benzoin chlorpheniramine ketoprofen
1.54 1.00 7.64
1.86 1.60 1.10
1.73 0.69 8.22
2.06 1.88 1.00
Table 4. Influence of Loading Amount of Benzoin on the Capacity Factor ( K ) , Peak Symmetry ( q ) , Enantloselectivity (a),and Resolution (Rs)of Columns Made with Commercial OMCHI and Isolated OGCHP
injected amt (nmol) 0.5 1.0
+
2.5
a
5.0
OMCHI-A 1.5%OGCHI kl'
2.59 1.16 10.46
2.45 2.24 1.21
All values were averages of three replicates. HPLC Conditions: column, 2.0 mm i.d. x 100 mm; eluent, 20 mM phosphate buffer (PH 5.1)-ethanol955 (v/v); column temperature, 25 "C; flow rate, 0.2 mL/ min; detection, 220 or 254 nm.
10 20
packing
kl'
kz'
ql
72
a
Rs
OMCHI OGCHI OMCHI OGCHI OMCHI OGCHI OMCHI OGCHI OMCHI OGCHI OMCHI OGCHI
2.20 9.66 2.10 9.42 1.95 8.96 1.80 8.58 1.65 8.12 1.55 7.57
5.54 30.5 5.19 29.7 4.63 28.1 4.25 26.6 3.79 24.9 3.25 22.7
0.69 0.67 0.97 0.73 1.24 0.77 0.96 0.83 0.93 1.13
1.77 0.59 2.19 0.81 4.79 0.85 4.37 0.92 2.39 1.20
1.68
1.65
2.52 3.16 2.47 3.15 2.38 3.13 2.36 3.10 2.29 3.07 2.09 3.00
4.77 9.03 4.15 7.86 2.67 4.69 1.81 3.69 1.13 2.82 0.70 2.04
a
and ketoprofen but that OMCHI-C has slight chiral recognition for benzoin and chlorpheniramine. These results suggest that the chiral recognition property of the column made with commercial OMCHI does not come from the OMCHI, but mainly comes from the OGCHI. As shown in Figure 8.4, OMCHI-C contains -1% OGCHI, estimated from the areas of peaks at retention times of 15 and 29 min. Note that the peak at a retention time of 31 min is different from OGCHI and that it is mainly an unknown protein. We examined whether the OMCHI-C free from contaminated OGCHI had chiral recognition ability or not. It is impossible to purify the OMCHI-C free from the OGCHI even by preparative HPLC. Therefore, 1.0 and 1.5% OGCHI, respectively, by weight were added to the OMCHI-A, which has no chiral recognition properties for benzoin, chlorpheniramine, and ketoprofen, as shown in Figure 8B,C. The contents of the OGCHI in Figures 8B,C were estimated to be 0.9 and 1.6%, respectively, from the areas of peaks at retention times of 15 and 29 min. The columns were made with OMCHI-C, OMCHI-A + 1.0% OGCHI, and OMCHI-A + 1.5% OGCHI and their chiral recognition properties were compared. Table 2 shows the comparison of the retentive and enantioselective properties of benzoin, chlorpheniramine, and ketoprofen on the
a All values were averages of three replicates. The ki and kz' are the capacity factors of the first and second eluted benzoin enantiomers, respectively. The qland 72 are the asymmetry factors of the first and second eluted benzoin enantiomers, respectively. HPLC conditions: column, 2.0 mm i.d. x 100 mm; eluent, 20 mM phosphate buffer @H 5.1)-ethanol 9O:lO (v/v); column temperature, 25 "C; flow rate, 0.2 mL/min; detection, 220 nm.
three columns. The enantioselective property of the OMCHI-C column is very similar to the OMCHI-A 1.0%OGCHI column, while the retention properties are slightly different. The former retains basic compounds more than the latter and retains acidic compounds less. This is plausible because the OMCHI-C contains larger amounts of sialic acid than the OMCHI-A. Thus, we conclude that the chiral recognition ability of the OMCHI-C for benzoin, chlorpheniramine and ketoprofen comes from the contaminated OGCHI. Separation of large number of compounds was tested on the OMCHI-A and OMCHI-B columns. However, no chiral resolution was observed. Thus, it is concluded that the OMCHI has no appreciable chiral recognition ability and that the chiral recognition
+
(38) Melamed, M. D. Biochem. J. 1967,103, 805-810. (39) Beeley, J. G. Biochem. J. 1971,123, 399-405. (40) Waheed, A; Salahuddin, A Biochem. 1.1975,147, 139-144. (41) Beavis, R C.; Chait, B. T. Proc. Natl. Acad. Sci. U S A . 1990,87,68736877. (42) Ketterer, B. Life Sci. 1962,1, 163-165. (43) Ketterer, B. Biochem. J 1965,96,372-376.
Analytical Chemistty, Vol. 67,No. 15, August 1, 1995
2545
B
A
u
D
C
c
c eP
E:
e
E
9
Y
t?
a 0
h
+I
h
v
I
? h
11 Time (min)
Time (min)
Time (min)
Time (min)
I Time (min)
Figure 7. Chiral resolution of ketoprofen on columns made with crude OMCHI (commercial OMCHI) (A), OMCHI-A (B), OMCHI-B (C), OMCHI-C (D), and OGCHI (E). Other conditions as in Figure 5. A
w -i
n
A OMCHI-C
fl
n
OMCHI-A
r 20 Time (min)
- r
10
0
Time (min)
o
m
I
4
1
0
Time (min)
Figure 9. Chromatograms of benzoin at 20-nmol injection on columns made with crude OMCHI (commercial OMCHI) (A) and isolated OGCHI (9). HPLC conditions as in Figure 5.
-
0
20 Time (min)
v
0
20 Time (min)
Figure 8. Chromatogram of OMCHI-C and a mixture of OMCHI-A and OGCHI on a reversed-phase column: (A) OMCHI-C; (9) OMCHI-A including 1.O% OGCHI; (C) OMCHI-A including 1.5% OGCHI. Other conditions as in Figure 3.
ability of crude OMCHI reported previouslyl comes from the OGCHI, which is present in commercial or isolated OMCHI as an impurity. Comparison of Chird Recognition Properties of Columns Made with Commercial OMCHI and Isolated OGCHI. Table 3 shows a comparison of retention, enantioselectivity, and resolution of various racemic solutes on columns made with the crude OMCHI and isolated OGCHI. The OGCHI column gave longer retentions, higher enantioselectivity, and higher resolution for all solutes tested than the OMCHI column. The tendencies of retentive and enantioselective properties for both OMCHI and OGCHI columns are very similar. 2546
B
4
OMCHI-A
(I 0
2
Analytical Chemistry, Vol. 67,No. 15, August 1, 1995
Table 4 shows influence of loading amounts of benzoin on the capacity factor (k? ,peak symmetry (7), enantioselectivity (a), and resolution (R,) of the OMCHI and OGCHI columns. A slight decrease in a values was observed as an increase in the loading amounts for both the OMCHI and OGCHI columns, while a marked decrease in the R,values occurred. The 7 value of each benzoin enantiomer on the OMCHI column at 20-nmol injection was not calculated because of the overlapping. The 72 value at 0.5nmol injection on the OMCHI column was almost the same as that at 20-nmol injection on the OGCHI column. Figure 9 shows chromatograms of benzoin at 20-nmol injection on the OMCHI (As and OGCHI (E9 columns, respectively. These results reveal that the OGCHI column is of much higher capacity for benzoin. These results support the suggestion that the OMCHI gives no appreciable chiral recognition ability and that the OGCHI has excellent chiral recognition ability for a variety of compounds. CONCLUSION
Crude OMCHI was fractionated by anion- and cation-exchange chromatography. The obtained fractions were as follows: three
fractions that were OMCHI variants differing in carbohydrate composition, especially in sialic acid content and a fourth fraction tentatively termed OGCHI. It is found that the OMCHI gives no appreciable chiral recognition ability and that chiral recognition ability of the crude OMCHI reported previously comes from the OGCHI, which is present in the crude OMCHI as an impurity.
are due to D. K, Nishi, Takeda Chemical Industries, for N-terminal sequencing of the OGCHI. This work was partly supported by a Grand-in-Aid for Scientiilc Research (05671799 and 06672159) from the Ministry of Education, Science and Culture, Japan. The work was also supported in part by funds from UPLUpjohn LaboratoriesJapan & Asia Vsukuba, Ibaraki, Japan).
ACKNOWLEDCPMENT We thank Dr. H. Wada of Shinwa Chemical Industries (Kyoto, Japan) for kind donation of the silica materials. Also, we thank Dr. A Ingendoh of Fmnigan Mat (Tokyo, Japan) for measurement of the MALDI-TOF mass spectra of OMCHI and OGCHI. Thanks
Received for review January 17, 1995. Accepted May 17,
1 995. AC950055X Abstract published in Advance ACS Abstracts, July 1, 1995.
Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
2547
