ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
82,
78-82 (1959)
Conalbu~n: A Rapid, High-Yield Preparation from Egg White Robert C. Woodworth
and Arthur L. Schade
From the United States Department of Health, E~u~~t~~, and Weljare, Public Health Service, rational I~st~t~~s of Health, rational Institute oj Allergy and fnfectious Diseases, Bethesda, ~ar~~an~ Received October 1, 1958
The elegant use of cellulose anion exchange columns for the chromatographic separation of blood serum proteins by Sober et al. (I), led us to attempt an adaptation of their methods to the purification of conalbumin, the iron-binding protein of hens’ egg white (2, 3). Experience showed us that the cation exchange column is more suitable than the anion exchange column for this purpose.1 The advantages of the preparation herein described are simplicity, rapidity, avoidance of po~ntial~y denaturing substances, and high yields of very pure conalbumin suitable for many uses without the necessity for crystallization. EXPERIMENTAL
General Procedure Fresh egg white was treated with ammonium sulfate to remove mucin and the globulins and to crystallize the ovalbumin according to the procedure of S$rensen and Heyrup (5). The supernatant from and a single wash of the crystalline ovalbumin were combined, and the conalbumin was precipitated by increasing the ammonium sulfate to 62% of saturation. A solution of the crude conalbumin in a minimum volume of water was dialyzed to remove the salt; and the dialyzed solution, pH 4.7, was charged in a carboxymethylcel~~ose cation-exchange column equilibrated with 0.005 M potassium phosphate buffer, pH 6.8. This same buffer served to wash ovomucoid and remaining ovalbumin from the column. The conalbumin was then eluted with 0.05 M potassium phosphate buffer, pH 6.8.
Detailed Procedure with Monitoring
Analyses
Pre~4r~~~on of Crude Conalbu~in ~o~~t~on. In a typical preparation, two dozen day-old hens’ eggs yielded 807 ml. of egg white, which was estimated to contain 10 g. 1 During the course of this investigation an article appeared (4) describing the complete chromatographic separation of various egg-white proteins on cellulose cation-exchange columns. 78
CONALBUMIN
FROM
EGG
WHITE
79
conalbumin by determination of the total iron-binding capacity (TIBC) (6). Mucin and globulins precipitated upon addition of an equal volume of saturated ammonium sulfate. After addition of saturated ammonium sulfate to the filtrate to the point of permanent turbidity and subsequent adj~lstment of the pH to 4.7 with 0.2 rlr sulfuric acid, the ovalbumin crystallized (5). These additions were made slowly and with vigorous stirring. Centrifugation of the suspension of crystalline ovalbumin yielded 1450 ml. of clear yellow supernatant and a paste which was washed by suspending in 500 ml. of 50% saturated ammonium sulfate solution, pIl4.7, and recentrifuging, The resulting 625 ml. of clear supernat~ant was added to t,he original supernatant from the crystalline ovalbumin. The addition of 50 g. of solid ammonium sulfate to this combined solution resulted in a precipitate which was recovered by centrifugation, dissolved in 150 ml. of distilled water, and dialyzed against running tap water overnight and against. distilled water for 2.5 hr. The resulting light-yellow solution was filtered to remove a small flocculent, precipitate. The filtrate amounted to 340 ml., had a pH of 4.95, a specific conductance of 7.45 X IO+ mho/cm., an optical density at 280 mp with a l-cm. light path (c&&~.) of 50.0, and a TlBC of 35.6 pg. Fe/ml. indicating a total conalbumin content of 8 g. Analysis of a small dialyzed sample of the 1906mf. s~~pernatant from the last precipitation indicated a total conalbumin content of 2 g. We added 38 g. of solid ammonium sulfate to the cu. 1900-ml. supernatant. The resulting precipitate was recovered, dissolved in 30 ml. of distilled water, and dialyzed as usual. The dialyzed solution afber centrifllgation yielded a clear, light-yellow supernatant with a volume of 43 ml., a specific conductance of 1.06 X 10-4 mho/cm., and a TIBC of 55.5 sg. Fe/ml. (1.6 g. conalbumin). The total recovery of conalbumin to this point equalled 9.6 g. or 96ya of theory. Isolation of Pure ConaEbumin. A 60-cm. column with an inside diameter of 7.5 cm. was packed with 306 g. of carboxymethylcellulose (7) (lOO- to 250-mesh Solka-Floe SW-B containing carboxyl groups to the extent of 0.59 meq./g. and having a pK’ of 5.0 in water). The column was equilibrated with 0.005 M potassium phosphate, pH 6.8, and then charged with 339 ml. of the crude conalbumin solution from the major (S-g.) conalbumin precipitate above. The starting eluant was 0.005 M potassium phosphate buffer, pH 6.8. We collected approximately 20-ml. effluent fractions at a rate of 5-10 ml./min. As may be seen from Fig. 1, protein first appeared in the effluent at fraction 47 and, after rising to a maximum concentration at fraction 56, fell to a negligible value by fraction 75 and to zero by fraction 180. Continued elution with the above buffer removed no additional protein from the column. At fraction 175 the eluant was changed to 0.05 M potassium phosphate buffer, pH 6.8, and protein again appeared in the effluent by fraction 270. After rising to a maximum concentration at fraction 259, it fell to a negligible amount by fraction 330.* 2 The chromatography columns may be reused a number of times by simply washing with about four times the column hold-up of starting buffer and without the necessity for removing and reprocessing the packing. We have not determined the limit of this reuse. It should also be made clear that fraction-collecting equipment is not needed for this p~paration, but only occasional monitoring of the column effluent by reading the &~a in an ultraviolet spectrophotometer or by detection of protein with 10% trichloroacetic acid. Under the procedure described for this fractionation, the column hold-up (including the volume of crude conalbumin solution charged into the column) before the first protein fraction emerges from the column is approximately one-half the total volume of the packing. The hold-up for the conalbumin fraction (calculated from the point at which the 0.05 M potassium phosphate buffer
80
WOODWORTH
L
t
100
I
,
,l , , 175
AND
,
200 FRACTION
,
SCHADE
$1
300 NUMBER
400
500
FIG. 1. Chromatographic separation of conalbumin from crude conalbumin solution on carboxymethylcellulose. Eluant: 0.005 M potassium phosphate buffer, pH 6.8, to fraction 175; 0.05 M potassium phosphate buffer, pH 6.8, after fraction 175.
A pool of fractions 48-70 was dialyzed against running tap water overnight, then against distilled water for 3 hr. After dialysis the pool amounted to 600 ml., had a d:8cgm’ of 8.63 and TIBC of 0.64 pg. Fe/ml. (0.26 g. conalbumin), and was light yellow in color. Analysis by paper-strip electrophoresis showed this solution to contain ovomucoid and the three egg-white albumins. The solution was discarded. A pool of fractions 275-327 was dialyzed as above. The dialyzed solution, filtered to remove a small amount of suspended matter, gave 1115 ml. of filtrate which had a d :srn’ of 6.78, pH 6.8, specific conductance of 4.59 X 10m4mho/cm., and TIBC of 8.18 (bound iron = 0.08 pg. Fe/ml.; unsaturated iron-binding capacity = 8.10 pg. Fe/ml.; 6.3 g. conalbumin). A 568-ml. portion of this solution was exhaustively dialyzed against distilled water and lyophilized to yield 3.3 g. protein. The remaining 547 ml. solution was placed in a cylinder and stirred under a 5% carbon dioxide-95% nitrogen atmosphere. Into this stirred solution was slowly pipetted 52 mg. ferrous ammonium sulfate and a small amount (es. 2 mg.) of ascorbic acid dissolved in 25 ml. of distilled water. The salmon-pink color of the iron-conalbumin complex appeared immediately. Stirring was continued with the solution exposed to air for 3 hr. The final pH was 6.6. Spectral absorption maxima were d@’ = 9.20, d&?’ = 0.366; and = 0.265, giving d&dtes = 25.1 and dmldtw = 1.38. the absorption minimum was d$’ Crystallization of Iron-ConaZbumin. The iron-conalbumin was crystallized from 50 ml. of the above iron-conalbumin solution by the method of Warner and Weber (8), and the resulting l-mm. crystalline rosettes were used for seeding later iron-conalbumin solutions. A 450.ml. portion of the iron-conalbumin solution was concentrated by partial lyophilization to 61 ml., exhaustively dialyzed against distilled water, adjusted to pH 6.0 with 1 N acetic acid, and cooled to -2°C. No turbidity or opalescence appeared. After the solution was seeded with crystals of iron-conalbumin and is introduced into the column head) is approximately packing.
equal to the total volume of the
CO~~LBU~I~
FROM
EGG WHITE
81
allowed to stand at 0°C. overnight, a voluminous crystalline precipitate, composed of elongated rectangular plates, formed. Crystallization was complete only after the solution had remained at 0°C. for 5 days. The supernatant solution then had a 1 om. d 280 = of 41.2; and a solution of the precipitate, made to 7.6 ml. with water, had a d:8”O”’ of 95.6. The supernatant solution had spectral ratios d&dm = 25.4 and d&dm = 1.34; and the solution of the precipitate had spectral ratios dm/dm = 23.0 and dre/daw = 1.39. In the absence of salt, these spectral ratios are independent of pEI between 6.0 and 7.2. Iron-conalbumin crystallized spontaneously from the latter solution upon cooling to 0°C.
The conalbumin obtained by chromatography, and before crystallization, contained less than 0.~17~ lysozyme as determined by the krbidity decrease with time of a suspension of freshly harvested bacteria, f~~~crococcue l~sode~k~icus. Immunochemical studies of the conalbumin by the agar-plate precipitin test of Ouchterlony (9, 10) indicated that the only detectable protein contaminant was a very small amount of ovalbumin in the supernatant from the crystallization of ironconalbumin from water. When 0.1 ml. of this supernatant, containing 3 mg. conalbumin, was placed in the antigen well, 8 days was required for a barely visible ovalbumin precipitin line to appear, corresponding to an absolute amount of ovalbumin of 1 pg. or less. This line did not appear in 8 days when 0.1 ml. of a solution containing 2 mg. iron-conalbumin, once-crystallized from water, was placed in the antigen well. The conalbumin was tested against rabbit antisera to conalbumin, ovalbumin, ocomucoid, and lysozyme. No preeipitin lines corresponding to ovomueoid or lysozyme appeared in 8 days. Sedimentation analysis of chromatographed, noncrystalli~ed eonalbumin from a similar run showed a s~metrical schlieren pattern with no indication of eontaminating proteins. A solution of 93.9 mg. of this conalbumin (containing 3.1% moisture) dissolved in 5 ml. water, saturated with iron, and made to 10 mt. with 0.01 M potassium phosphate buffer, pH 7.9, had a d%?” of 13.55 and a d&?. of 0.585. Therefore, t,he extinct.ion coefficients for iron-saturated conalbumin are &?“’ = 1.49 ml./mg. I cm. = 0.645 ml./mg. This same solution had spectral ratios d&dnss = 23.1 and CP~:, and d&dm = 1.39. DISCUSSION
Rhodes, Azari, and Feeney (4)) using cellulose cation-exchange columns, have recently reported finding multiple maxima in the conalbumin effluent band. We have observed that a single maximum is obtained when freshly prepared crude conalbumin solutions are processed with a minimum of delay. However, it is our experience that multiple maxima may appear when variously aged crude conalbumin solutions are fractionated. The conalbumin prepared by our described method retains unaltered its ability to equilibrate with iron in a manner exhibited by this protein in fresh egg white. Cells of Staphylococcus aureus, in a medium containing either egg white or this purified conalbumin, take up iron in proportion to the square root of the ratio of iron-saturated to iron-free conalbumin, whereas other variously prepared conalbumins do not exhibit this clearcut equilibrium-controlled response (11).
82
WOODWORTH AND SCHADE ACKNOWLEDGMENTS
We are indebted to Dr. William R. Carroll of the National Institute of Arthritis and Metabolic Diseases for conducting the ultracentrifugal sedimentation analyses. The technical assistance of John R. Miller and Robert W. Reinhart is gratefully acknowledged. SUMMARY
A method is described for the rapid, high-yield isolation of hens’ egg conalbumin of immunologically demonstrated high purity and relatively low (1% or less of maximum) iron content. The use of potentially denaturing substances is avoided. REFERENCES 1. SOBER, H. A., GUTTER, F. J., WYCKOFF, M. M., AND PETERSON, E. A., J. Am. Chem. Sot. 78, 756 (1956). 2. SCHADE, A. L., AND CAROLINE, L., Science 100, 14 (1944). 3. SCHADE, A. L., RBINHART, R. W., AND LEVY, H., Arch. Biochem. 20, 170 (1949). 4. RHODES, M. B., AZARI, P. R., AND FEENEY, R. E., J. Biol. Chem. 230, 399 (1958). 5. SORENSEN, S. P. L., AND H~YRUP, M., Compt. rend. trav. lab. Carlsberg 12, 12 (1915-17). 6. SCHADE, A. L., OYAMA, J., REINHART, R. W., AND MILLER, 3. R., Proc. Sot. Exptl. Biol. Med. 87, 443 (1954). 7. PETERSON, E. A., AND SOBER, H. A., J. Am. Chem. Sot. 78,751 (1956). 8. WARNER, R. C., AND WEBER, I., J. Biol. Chem. 191, 173 (1951). 9. OUCHTERLONY, O., Arkiv Kemi, Mineral. Geol. 26, 1 (1949). 10. WILSON, M. W., AND PRINGLE, B. H., J. Immunol. 73, 232 (1954). 11. SCHADE, A. L., Abstr. Proc. Intern. Congr. Biochem., 4th Congr., Vienna, 1968,127.