Lipoprotein Staining Following Polyacrylamide Disc Electrophoresis of Chick Serum

SALICYLIC ACID AND SALICYLATE

tylsalicylic acid and sodium salicylate used in this experiment and Dr. Tao Wong and Jimmy A. Wilson for their laboratory assistance. REFERENCES Campbell, B., 1948. Inhibition of anaphylactic shock by acetylsalicylic acid. Science, 108: 478-479. Carlo, P. E., N. M. Cambosos, G. E. Feeney and P. K. Smith, 19SS. Plasma levels after the oral administration of acetylsalicylic acid and N-acetyl-p-aminophenol in different forms to human subjects. J. Am. Pharm. Assoc. Si. Ed. 44: 396-399. Chirigos, M. A., and S. Undenfriend, 1959. A simple fluoremetric procedure for determining salicylic acid in biologic tissues. J. Lab. Clin. Med. 54: 769-772. Collier, H. O. J., and G. Shorley, 1960. Analgesic antipyretic drugs as antagonists of bradykinin. J. Brit. Pharmacol. IS : 601-610. Collier, H. O. J., 1963a. Aspirin. Sci. American, 209: 96-108. Collier, H. O. J., 1963b. Antagonism by aspirin and like-acting drugs of kinins and SRS-A in guinea-pig lung. I n : Salicylates, Edit, by A. St. J. Dixon, M. J. H. Smith, B. K. Martin and P. H. N. Wood, Boston. Collier, H. O. J., A. R. Hammond and B. Whiteley, 1963. Antianaphylactic action of acetylsalicylate in guinea pig lung. Nature, 200: 176-178. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 1 1 : 1-42. Glick, B., 1962. Some physiological effects of ace-

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tylsalicylic acid and sodium salicylate in the chicken. Ohio J. Sci. 62(1): 13-17. Glick, B., 1963. Influence of acetylsalicylic acid on the body temperature of heat stressed chickens. Nature, 200: 603. Glick, B., and K. Sato, 1964. The bursa of Fabricius, acetylsalicylic acid, and anaphylactic shock. Poultry Sci. 43 : 1322. Hutchins, M. O., W. S. Newcomer and R. H. Thayer, 1962. Effects of sodium salicylate on thyroid function of heat-stressed chickens. Poultry Sci. 4 1 : 1807-1815. Makinodan, T., H. R. Wolfe, M. Goodman and R. Ruth, 1952. Precipitin production in chickens. VII. The relation between circulating antibody and anaphylactic shock. J. Immunology, 68: 219-227. Morgan, G. W., Jr., 1966. The effect of acetylsalicylic acid and salicylates on anaphylaxis and plasma salicylate levels in the chicken. M.S. Thesis, Miss. State Univ., State College, Miss. Shorley, P. G., and H. O. J. Collier, 1960. Direct comparison of the synthetic nonapeptide with trypsin bradykinin. Nature, 188: 999-1000. Sims, D., 1966. Moorman modification of the Chirigos and Undenfriend method, (developed by David Sims, unpublished communication). Quincy, 111. Smith, W., and J. H. Humphrey, 1949. The effect of sodium salicylate upon hypersensitivity reactions. Brit. J. Exptl. Path. 30: 560-571. Steele, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Company, Inc., New York. Weigle, W. O., C. G. Cochrane and F. J. Dixon, 1960. Anaphylactogenic properties of soluble antigen antibody complexes in the guinea pig and rabbit. J. Immunol. 85: 469-477.

Lipoprotein Staining Following Polyacrylamide Disc Electrophoresis of Chick Serum L. PROSKY, R. G. O'DELL, D. A. LIBBY AND D. F. FLICK Division of Nutrition, Bureau of Science, Food and Drug Administration, U. S. Department of Health, Education, and Welfare, Washington, D.C. 20204 (Received for publication May 20, 1967)

ISC GEL electrophoresis has provided a technique for rapidly resolving whole serum into more than twenty protein fractions (Davis and Ornstein, 1959). Our

D

investigations of the serum protein changes of the chick during the first days after hatching (Libby et al., 1966) require identification and routine determination of these

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L. PROSKY, G. R. O'DELL, D. A. LIBBY AND D. F. FLICK

the standard separating gel3 containing 7% acrylamide. The gel was subjected to electrophoresis at pH 9.5 for 25 min. at 5 ma. per gel. Tracking dye was omitted in those gels which were to be stained for lipoprotein because of interference with the final color. The lipid stains were prepared by adding 100 mg. of either Sudan green,1 oil red 0,1 Sudan orange1 or Bleu BZL1 to each of four Erlenmeyer flasks containing 10 ml. of methanol and 90 ml. of 8.3% acetic acid. The final concentration of acetic acid was 7.5%. The vessels were stoppered and placed over a steam bath. After 30 min., the solutions were filtered and the clear colored filtrates were collected. The disc gels, after electrophoresis, were placed in the dye solutions at room temperature and allowed to develop overnight. The gels were rinsed three times in 7.5% acetic acid before analysis. Amidoschwarz 10 B 1 and Aniline Blue Black1 were both used as protein stains iniMATERIALS AND METHODS tially; however, only Aniline Blue Black All dyes were Chroma-Gesellschaft was used for later studies since both gave stains.1 2,4-Dinitroso-l,3-naphthalenediol2 identical results. Glycoproteins were identiwas used for the iron determination of fied as red bands when the acrylamide gel transferrin. Albumin was isolated and puri- was treated with periodic acid and then exfied from chick serum by ammonium sulfate posed to Schiff Reagent. Transferrin was fractionation methods (Gornall et al., identified as a single light green band which 1949). developed 15 minutes after treatment with For these studies, we used the standard 2,4-dmitroso-l,3-naphthalenediol and hydisc polyacrylamide electrophoresis proce- droquinone. This band persisted for less dure3 with the Canalco Model 12 ap- than 15 minutes after reaching maximum paratus and Model 300 power supply. Five intensity. Before densitometer analysis of 17,1. of chick serum or plasma, containing ap- the gels which had been stained with genproximately 100 pig. of protein, was added to eral protein stains, it was necessary to remove the unbound dye. This was accom1 Purchased from Roboz Surgical Instrument plished by placing the gel in the electrophoCo., Washington, D.C. resis apparatus for 30 min. at 12.5 ma. per 2 Purchased from Canal Industrial Corp., Rockgel and using 7.5% acetic acid in both ville, Md. Prepared according to bulletin "Deterbaths. mination of Iron in Gels", available from Canal In several cases, for absolute correlation Industrial Corp. 3 Bulletin "Disc Polyacrylamide Electrophoresis between the specific band stain and its Procedure", Canal Industrial Corp., Rockville, Md. counterpart protein stain, the disc gels were proteins. The major problems arising in the evaluation of the lipoprotein fractions have been caused by the inadequacy of analytical ultracentrifugation and moving boundary electrophoretic techniques (Narayan et al., 1965). Attempts to resolve and identify these fractions by varying gel pore size and staining techniques have usually given unsatisfactory results and are complicated to perform (Raymond et al., 1966). Furthermore, prestaining the lipid fractions may alter the charge on the protein and consequently, its migration in the electrical field. This alteration makes it difficult to compare gel fractions. The present work describes lipoprotein staining techniques performed on polyacrylamide disc gels after whole serum electrophoresis. Attempts are made to correlate these fractions in serum and plasma samples run concurrently and then differentially stained for protein, transferrin, and glycoprotein.

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LIPOPROTEIN STAINING

A

B

C

FIG. 2. Disc gel electrophoretic separation of chick serum followed by staining with (A) Sudan Green (B) Oil Red 0 and (C) Bleu BZL and their respective densitometric tracings. FIG. 1. (a) Paper electrophoretic separation of chick plasma protein and densitometric trace.

Paper electrophoretic separations of the same chick sera were analyzed in the Densicord and Analytrol after staining with alcoholic bromphenol blue. Protein was determined by the Lowry colorimetric method (Lowry et al., 1951). RESULTS AND DISCUSSION

(b) Disc gel electrophoretic separation of the same chick plasma protein and densitometric trace. A =: Albumin, T = Transferrin. See methods section for complete description.

frozen in li Freon" and sectioned longitudinally with a scalpel before staining. The gels were analyzed in the Photovolt Densicord4 densitometer with an accompanying integrator. Two micro slits were employed and the ratio of gel scan drive to recording paper speed was maintained at 1:5 to improve resolution of the stained fractions. 4

A. H. Thomas Co., Philadelphia, Pa.

Resolution of protein mixtures is improved when molecular sieving (using adjustable gel pore size) and electrophoresis are employed simultaneously (Smithies, 1959). Fig. la and lb show the high resolution of chick plasma protein achieved. The plasma from one experimental chick yielded seven protein components by paper electrophoresis (Fig. la) and 15 specific fractions when the polyacrylamide gel was used (Fig. lb). The photograph (Fig. lb) clearly shows, in the central portion of the gel, four distinct protein bands to the right of the band identified as transferrin. This indicates that with the possible exception of the albumin fraction, all of the fractions shown on paper are actually protein mixtures, and that paper electrophoretic data may be of limited value. Starch gel electrophoresis of avian plasma proteins yielded at most nine components (Merriman and Darcel, 1964),

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L. PEOSKY, G. R. O'DELL, D. A. LIBBY AND D. F. FLICK

1, f

Fie. 3. Disc gel electrophoretic separation of chick serum followed by "Freon" freezing of gel and splitting gel in half. One half was stained for protein and the other for lipid using Sudan Orange. The accompanying densitometric trace is that of the lipoprotein fractions.

again indicating incomplete resolution of protein fractions. Fig. 2 shows gels which were stained for lipoproteins after the electrophoresis of chick sera. Specific lipid dyes were preferentially bound by the lipid-containing proteins. Sudan green, oil red 0 and Bleu BZL gave almost identical patterns, and as the densitometric tracings indicate, showed three lipid fractions. The slowest moving li-

Fic. 4. Disc gel electrophoretic separation of serum (S) and plasma (P) from the same chick. The densitometric tracing were identical with the exception of the diminished fibrinogen (F) fraction in serum.

poprotein, probably a single protein fraction, is the sharp band near the top of the gel and is probably a ^-lipoprotein. The two diffuse lipid staining components are mixtures of from two to four proteins. To determine the exact position of the lipoprotein bands, the gels were frozen in "Freon" after electrophoresis and then cut longitudinally. One half was stained for protein with Aniline Blue Black, and the other half was stained for lipid with Sudan Orange. The half gels were then placed side by side to determine the protein corresponding to the particular lipid staining fraction. The comparison is shown in Fig. 3. The smaller of the diffuse lipoprotein bands moves more slowly than transferrin; the larger band moves with a greater velocity. This larger, diffuse component corresponds to the region of the four discrete bands in Fig. 1. For identification of lipoprotein fractions in studies of lipid metabolism and transport, it is often important to stain the lipid component rather than the protein. Changes in the lipid component of the lipoprotein may be quite different from changes in the protein components. Albumin, the major protein component in plasma, was identified by using a purified chick albumin preparation. Fig. 4 shows disc gels and their respective densitometric traces after fractionation of serum and plasma from the same chick. Fibrinogen was identified as the only protein present in the plasma mixture which was considerably lower in the serum preparation. This is clearly demonstrated in the magnified photograph of the upper portion of the gel and by the smaller fibrinogen peak in the densitometer recording. A photograph of a gel which had been split longitudinally is shown in Fig. 5. One half of the gel was treated with the 2,4-dinitroso-l,3-naphthalenediol reagent for the iron-staining reaction. The transferrin was identified as a sharp, light green band which developed rapidly and disap-

LIPOPROTEIN

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STAINING

while the other two migrate to positions not as far from the origin as transferrin. ACKNOWLEDGEMENTS

The authors wish to thank Mrs. R. M. Branch for her technical assistance and Mr. James C. Fritz for his excellent assistance with the photographs. SUMMARY

A procedure is described for the staining of lipoprotein fractions after separation of chick plasma proteins in 7% acrylamide disc gels by electrophoresis. This method eliminates prestaining techniques which complicate the procedure as well as interfere with the migration of proteins. Several lipid stains were employed and showed similar staining properties. The best stains were Oil Red 0, Sudan Orange. Bleu BZL and Sudan Green. REFERENCES Fir,. S. Disc gel separation of chick serum followed by splitting of the gel and staining one half for transferrin and the other for protein. Another gel was stained for glycoprotein (G) and showed two distinct bands.

peared in a short time. The other half of the gel was stained for protein. Transferrin was seen as the fine protein band at the midpoint of the gel, between the origin and the albumin fraction. Fig. 5 also shows another gel which had been stained for glycoproteins. These appeared as two distinct red bands. Other components which may give a positive reaction with the Schiff reagent are either trapped in the stacking gel because of their low molecular weight or remain at the interphase of the stacking and separating gel because of their low net charge. Two distinct glycoprotein bands are always found in the sera and as many as four were sometimes visible. The two most common ones migrate short distances into the gel

Davis, B. J., and L. Ornstein, 1959. A new high resolution electrophoresis method. Presented at The Society for the Study of Blood at the New York Academy of Medicine. Gornall, A. G., C. J. Bardawill and M. M. David, 1949. Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177: 751. Libby, D., R. G. O'Dell and D. F. Flick, 1966. Daily plasma globulin levels in the first week posthatch of the S. C. White Leghorn cockerel. Poultry Sci. 45 : 1420. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265. Merriman, M., and C. Le Q. Darcel, 1964. Confirmation of plasma protein changes in avian erythroblastosis. Canad. J. Biochem. 42: 293. Narayan, K. A., S. Narayan and F. A. Kummerow, 1965. Disc electrophoresis human serum lipoproteins. Nature, 205: 246. Raymond, S., J. L. Miles and J. C. J. Lee, 1966. Lipoprotein patterns in acrylamide gel electrophoresis. Science, 151: 356. Smithies, O , 1959. Zone electrophoresis in starch gels and its application of studies of serum proteins. Advan. Protein Chem. 14: 65.