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Hydrazinoacridine staining of proteins and glycoproteins in polyacrylamide gels
ANALYTICAL
BIOCHEMISTRY
T&428-435
Hydrazinoacridine Glycoproteins
(1977)
Staining of Proteins and in Polyacrylamide Gels
STEVEN D. CARSON Department
of Human Biological Chemistry The University of Texas Medical
and Genetics, Division of Human Branch, Galveston, Texas 77550
Genetics,
Received July 6, 1976; accepted November 15, 1976 The histochemical method for staining polyaldehydes in tissue sections with p-hydrazinoacridine has been adapted for use in polyacrylamide gels. While staining of histological preparations was reported to be specific for polyaldehydes and independent of bisulfite, both glycoproteins (p chain of haptoglobin) and nonglycoproteins (lysozyme and (Ychain of haptoglobin) were stained following periodate oxidation, and satisfactory results were highly dependent on the presence of bisulfite. Hydrazinoacridine staining of periodate-treated gels produced an extremely sensitive fluorescent labeling of the haptoglobin /3 chain and also stained haptoglobin a! chain and lysozyme. The proteins could be visualized under visible light as yellow bands which were scanned spectrophotometrically at 440 nm. The /3 chain of haptoglobin could be subjectively distinguished from the nonglycoproteins both by differential intensity of staining with hydrazinoacridine and Coomassie brilliant blue and by the yellow nature of the fluorescence. The sensitivity of hydrazinoacridine staining of the p chain of haptoglobin compared favorably to that of the commonly used periodic acid-Schiff staining procedures and provided the advantage that nonglycoproteins in complex mixtures could be localized in the gels.
Many methods have been presented for detecting proteins and carbohydrates in polyacrylamide gels after electrophoresis (1). Following the work of de St. Groth et al. (2), Coomassie brilliant blue has become a common reagent for general protein staining, and modifications of the periodic acid-Schiff (PAS) method are usually employed for detecting glycoproteins (3,4). Stoward studied histological staining of periodateengendered aldehydes with fluorescent Schiff-type reagents (5) and by hydrazone formation with salicylhydrazide (6). Weinblatt et al. (7) have recently extended the use of hydrazone formation for fluorescent staining of aldehydes in histological preparations usingp-hydrazinoacridine (HA). These authors reported that staining was independent of bisulfite in the reaction and provided evidence that the reaction was specific for aldehydes. With regard to the histochemical systems reported by Stoward (5,6), the conditions of staining periodate-engendered aldehydes with HA reported by Weinblatt et al. (7) were adapted for use in polyacrylamide gels. Similar to Stoward’s findings with other stains (5), HA staining was most intense 428 Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN WJO3-2697
HYDRAZINOACRIDINE
STAINING
IN GELS
429
for glycoproteins but was not specific for proteins with carbohydrate moieties. Furthermore, satisfactory results were found to be highly dependent on the presence of bisulfite. Therefore, HA was investigated for sensitive staining of glycoprotein as well as nonglycoprotein after completion of electrophoresis. Fluorescent staining procedures for polyacrylamide gel electrophoresis have utilized dansyl chloride (8), fluorescamine (9), ando-phthalaldehyde (10) with which the proteins are first reacted and subsequently electrophoresed in the presence of sodium dodecyl sulfate (SDS). The most familiar fluorescent stain used for detecting proteins after electrophoresis is anilinonaphthalene sulfonate (ANS) which stains proteins with less sensitivity than does Coomassie brilliant blue (1). This paper demonstrates the use of p-hydrazinoacridine for staining polyacrylamide gels after electrophoresis, with attention directed toward subjective identification of proteins with carbohydrate moieties. MATERIALS
AND METHODS
Polyacrylamide gel electrophoresis was conducted in 5 x 70-mm cylindrical gels in either SDS-urea as described by Swank and Munkres (1 I) or in acid-urea as described by Panyim and Chalkley (12). The SDS-urea gels were 10% acrylamide with a ratio of N,N’-methylene-bisacrylamide to acrylamide of 1:29. The proteins to be electrophoresed were dissolved at 1 mg/ml in the appropriate solution and applied to the gels in amounts from 5 to 50 pg. The (Y and /3 chains of human haptoglobin (Hpc~ and Hpfl) were a gift from Dr. Alex Kurosky, and egg white lysozyme was from Sigma. Acrylamide was from Bio-Rad. Analytical isoelectric focusing of human serum was adapted from the methods described by Awdeh et al. (13) and Vesterberg (14). The gel was poured between Column Coat (Canalco)-treated glass plates into dimensions of 85 x 150 x 1 mm. The gel contained 5% acrylamide with a ratio of acrylamide to N,N’-methylene-bis-acrylamide of 34.4: 1. The gel contained 4 M urea and 1.4% Ampholine (LKB), pH 3.5- 10. Electrode buffers were 5% ethylene diamine (Fisher) at the cathode and 5% H,PO, (Fisher) at the anode. Samples were applied in 20-~1 aliquots on Whatmann 3 MM paper. The gel was run for 4 hr with a final potential of 600 V. Cylindrical gels were stained with either Coomassie brilliant blue R (Sigma) as described by Carson et al. (15), with PAS as described by Glossmann and Neville (4), or with p-hydrazinoacridine (Eastman) as described here. The gels stained with Coomassie were destained by diffusion in 10% isopropanol- 10% acetic acid (Mallinckrodt) and scanned at 590 nm on a Gilford spectrophotometer. The isoelectric focusing gel was stained with p-hydrazinoacridine, PAS, or a combination dye containing 0.07% Coomassie brilliant blue R, 0.07% fast green (Allied), and 0.07% red
430
STEVEN
D. CARSON
W (Canalco) in 10% trichloroacetic acid (TCA; Mallinckrodt), 10% isopropanol, and 10% acetic acid. This stain has been shown to have sensitivity equivalent to Coomassie blue alone for most proteins and increased sensitivity for the small peptide bacitracin (unpublished data). The stain remains stable with no alteration of the individual components as determined by thin-layer chromatography. For staining with p-hydrazinoacridine, gels were fixed overnight in 12.5% TCA. The gels were then washed in water for 5 min and subjected to 0.5% periodic acid (Ames) for 30 min followed by a 5-min water wash. The p-hydrazinoacridine was prepared by dissolving 20 mg of HA in 20 ml of 95% ethanol. The dye was diluted to 100 ml with distilled water to which 5 ml of 2 N HCl (Fisher) was added. No more than 5 min prior to staining, 1 g of potassium metabisulfite (Fisher) was added with mixing. The gels were stained for 20 min or until the transient brown coloration was gone and were then placed in 1% HCl-20% ethanol for 15 min. For development of fluorescent bands, the gels were soaked in a solution containing 12.5% TCA and 1% sulfosalicylic acid (Mallinckrodt). Bands could be detected after 5 min by illumination with long-wave ultraviolet light. Gels were scanned at 440 nm. Several modifications of the staining method were investigated. The time of periodate oxidation was varied from 10 min to 1 hr under ambient conditions and in the dark at 0°C without altering the results. Oxidation in dimethyl sulfoxide (Fisher) did not improve the differential staining of protein and glycoprotein. The 1% potassium metabisulfite employed was found to produce better staining than 0.5% bisulfite, even though a precipitate appeared after 5 min of exposure to the HA solution. RESULTS
The p chain of human haptoglobin (HP/~) contains 19.4% carbohydrate (16) and was chosen to compare with the nonglycoproteins, lysozyme and the (Ychain of haptoglobin (Hpo). Staining with HA as described resulted in fluorescent labeling of all three proteins, with the fluorescence of HpP most intense. Furthermore, the HI>@ fluorescence was yellow, whereas lysozyme and Hpcl! fluoresced light green. No fluorescence was observed prior to placing the gels in TCA, and 1% sulfosalicylic acid intensified the fluorescence. No staining was evident without prior periodate oxidation of the proteins. Although some staining of all three proteins occurred in the absence of potassium metabisulfite, the bands were too weak for successful photography. Only in the presence of bisulfite was staining satisfactory, producing the yellow fluorescence noted for HP/?. The stain produced a notable fluorescence after 5 min in the TCA-sulfosalicylic acid solution, which reached maximum intensity after 24 hr. The stained proteins could also be seen under normal lighting as yellow bands and were scanned with good results at 440 nm. After 24 hr, the fluorescent and visible bands slowly
HYDRAZINOACRIDINE
STAINING
IN GELS
431
lost their intense yellow color and acquired a deep orange color. This orange band, although less intense than the yellow, remained stable after several weeks in the TCA-sulfosalicylic acid solution, Staining results were similar for all of the gel systems employed, SDS-urea polyacrylamide gels were run with 5 to 50 pg of HpP, Hpcr, and lysozyme and stained with HA, Coomassie brilliant blue R, and PAS (Figs. 1 and 2). Figure 1 shows that, with HA, even 5 pg of each protein was
a b
c d
A
E3
C
D
FIG. 1. SDS-urea polyacrylamide gels stained with HA and photographed under long-wave ultraviolet light. Gels contained: (A) 5, (B) 10, (C) 20, and (D) 30 pg each of Hp& Hpa, and lysozyme; (E) 30 pg of HpP and 2Opg of lysozyme; and(F) 50 pg each of HpP and Hpcy.Bands are: (a) HpP contaminant, (b) HpP, (c) Hpa. and (d) lysozyme.
432
STEVEN
D. CARSON
a b
c d
A
6
CD
E
F
G
FIG. 2. SDS-urea polyacrylamide gels stained with (A-C) Coomassie brilliant blue R and (D-G) PAS. Gels contained equal amounts of HP/~, Hpa, and lysozyme: (A and E) 10 Fg, (B and F) 20 pg, (C and G) 30 pg. and(D) 5 pg. Bands are (a) Hp@ contaminant, (b) HP/~, (c) Hpcu, and (d) lysozyme.
easily detected under long-wave ultraviolet light. Figure 2 shows similar polyacrylamide gels stained with Coomassie and PAS. When the gels containing 10 pg of Hpfi, Hpa, and lysozyme stained with Coomassie or HA were scanned in the visible range and the peak heights were compared, the HA staining was found to be about 80% as sensitive as Coomassie for HpP. Coomassie staining of lysozyme was threefold more intense than HA staining when compared in the visible spectrum. Inspection of Figs. 1 and 2 reveals that HA staining was more sensitive for demonstration of Hpfl than PAS (especially notable at the 5-pg level) and comparable to Coomassie. The HA stained both lysozyme and Hpc~ to a lesser extent than did Coomassie. and PAS stained neither. FIG. 3. Polyacrylamide gel isoelectric serum. Gel sections were stained with described in Materials and Methods; (B) (D) HA photographed under long-wave
focusing, pH 3.5 to 10, of 20-4 aliquots of humar (A) Coomassie brilliant blue-fast green-red W a: PAS; (C) HA photographed under visible light; am ultraviolet light.
HYDRAZINOACRIDINE
STAINING
IN GELS
433
--“-.r-”,-----.., .,!’ a
434
STEVEN
D. CARSON
Aliquots of whole serum (20 ~1) were subjected to isoelectric focusing in polyacrylamide gel. The gel was sectioned and stained with Coomassiefast green-red W, PAS, or HA as described in Materials and Methods. Figure 3 is a photograph of stained gel sections. After HA staining (Fig. 3D), bands a (predominantly y-globulin), b, f, and g showed the yellow fluorescence characteristic of glycoproteins, whereas bands c and e displayed blue-gray fluorescence, and band d fluoresced white. Human serum albumin (Schwarz/Mann) was shown to focus at positions corresponding to bands c and e. PAS staining (Fig. 3B) confirmed the glycoprotein nature of bands b, f, and g, but was not sufficiently sensitive to stain the carbohydrate moieties of the y-globulins. Staining of the gels with the compound protein stain (Fig. 3A) revealed that HA staining was comparably sensitive for proteins b, d, and g, but of decreased sensitivity for bands a, c, and f. Since the glycoproteins could be recognized by the yellow fluorescence, HA staining was preferable to PAS with regard to both sensitivity and localization of the glycoproteins among other bands stained with the general stain. DISCUSSION
p-Hydrazinoacridine has been demonstrated as a fluorescent stain suitable for detecting glycoproteins in polyacrylamide gels after electrophoresis. The yellow fluorescence of the glycoproteins allowed their identification among other protein bands which fluoresced less intensely and at wavelengths other than yellow. Comparison of HA staining with Coomassie brilliant blue R (15) and PAS (4) staining of known amounts of HP/~, Hpcy, and lysozyme demonstrated that HA was more sensitive for detecting HpP 119.4% carbohydrate (16)] than PAS (Figs. 1 and 2). The visible yellow bands produced with HA were scanned at 440 nm and shown to be of a sensitivity comparable to that of Coomassie for Hpfl and approximately one-third as sensitive as Coomassie for the nonglycoprotein, lysozyme. As used here for staining polyacrylamide gels, the behavior of HA was similar to fluorescent histological stains described by Stoward (5). A weak green fluorescence was obtained when bisulfite was omitted from the reactions, which may have been simple hydrazone formation as discussed by Weinblatt et al. (7). However, such hydrazone formation must not be specific for aldehydes derived from the oxidation of carbohydrates as evidenced by fluorescence of Hpcw and lysozyme. In the presence of potassium metabisulfite, the intensity of fluorescence was greatly enhanced. Furthermore, HpP (Fig. 1) and the PAS-positive bands from isoelectric focusing (Fig. 3) acquired the yellow fluorescence found to be characteristic of glycoproteins in this study. Since these characteristics of HA are similar to those of the Schiff-type reagents studied by Stoward (5), these stains may share even more properties. Stoward found ambiguity in
HYDRAZINOACRIDINE
STAINING
IN GELS
435
distinguishing some glycoprotein tissue constituents from nonglycoprotein structures on the basis of fluorescent color alone. He also noted that the fluorescent Schiff-type reagents he studied did not stain some PAS-positive mucosubstances. Although neither of these problems was encountered when isoelectrically focused serum was stained with HA, the possible occurrence of anomalous staining has not been eliminated and should be considered in the evaluation of protein staining. As presented here,p-hydrazinoacridine is a sensitive fluorescent reagent suitable for staining polyacrylamide gels after electrophoresis. When glycoproteins were present, they fluoresced more intensely than nonglycoproteins and could be distinguished by the yellow color of the fluorescence. Compared to PAS staining, HA provided advantages of both sensitivity and localization of nonglycoproteins within the gels. ACKNOWLEDGMENTS I thank Dr. Alex Kurosky for the haptoglobin preparations and suggestions and Dr. B. H. Bowman and Dr. R. J. Klebe for reviewing the manuscript. This work was supported, in part, by a grant from the Robert A. Welch Foundation H-378.
REFERENCES 1. Maurer, H. R. (1971) Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis, pp. 72-78. Walter de Gruyter, New York. 2. de St. Groth, S. F., Webster. R. G., and Datyner. A. (1963)Biochim. Biophys. Acra 71, 377. 3. Zacharius, R. M., Zell, T. E., Morrison, J. H., and Woodlock, J. J. (1969)Anal. Biochem. 30, 148. 4. Glossmann, H., and Neville, D. M., Jr. (197l)J. Biol. Chem. 246, 6339. 5. Stoward, P. J. (1967) J. Roy. Microsc. Sot. 87, 237. 6. Stoward, P. J. (1967) J. Roy. Microsc. Sot. 87, 247. 7. Weinblatt, F. M.. Shannon. W. A., Jr., and Seligman, A. M. (1975) Histochemisrry 41, 353. 8. Talbot, D. N., and Yphantis, D. A. (197l)Anal. Biochem. 44, 216. 9. Eng, P. R., and Parkes, C. 0. (1974) Anal. Biochem. 59, 323. 10. Weidekamm. E., Wallach, D. F. H., and Fluckiger, R. (1973) Anal. Biochem. 54, 102. 11. Swank, R. T., and Munkres, K. D. (1971) Anal. Biuchem. 39, 462. 12. Panyim, S., and Chalkley, R. (1969) Arch. Biochem. Biophys. 130, 337. 13. Awdeh, Z. L., Williamson, A. R., and Askonas, B. A. (1968) Nature (London) 219,66. 14. Vesterberg, 0. (1973) Sci. Tools 20, 22. 15. Carson, S. D., Harper. B. L., Bamett, D. R. Kurosky, A., Lankford, B. J., and Bowman, B. H. (1976) Tex. Rep. Biol. Med. 34, 209. 16. Black, J. A., Chan, G. F. Q., Hew. C. L., and Dixon. G. H. (1970) Gun. J. Biochem. 48. 123.
BIOCHEMISTRY
T&428-435
Hydrazinoacridine Glycoproteins
(1977)
Staining of Proteins and in Polyacrylamide Gels
STEVEN D. CARSON Department
of Human Biological Chemistry The University of Texas Medical
and Genetics, Division of Human Branch, Galveston, Texas 77550
Genetics,
Received July 6, 1976; accepted November 15, 1976 The histochemical method for staining polyaldehydes in tissue sections with p-hydrazinoacridine has been adapted for use in polyacrylamide gels. While staining of histological preparations was reported to be specific for polyaldehydes and independent of bisulfite, both glycoproteins (p chain of haptoglobin) and nonglycoproteins (lysozyme and (Ychain of haptoglobin) were stained following periodate oxidation, and satisfactory results were highly dependent on the presence of bisulfite. Hydrazinoacridine staining of periodate-treated gels produced an extremely sensitive fluorescent labeling of the haptoglobin /3 chain and also stained haptoglobin a! chain and lysozyme. The proteins could be visualized under visible light as yellow bands which were scanned spectrophotometrically at 440 nm. The /3 chain of haptoglobin could be subjectively distinguished from the nonglycoproteins both by differential intensity of staining with hydrazinoacridine and Coomassie brilliant blue and by the yellow nature of the fluorescence. The sensitivity of hydrazinoacridine staining of the p chain of haptoglobin compared favorably to that of the commonly used periodic acid-Schiff staining procedures and provided the advantage that nonglycoproteins in complex mixtures could be localized in the gels.
Many methods have been presented for detecting proteins and carbohydrates in polyacrylamide gels after electrophoresis (1). Following the work of de St. Groth et al. (2), Coomassie brilliant blue has become a common reagent for general protein staining, and modifications of the periodic acid-Schiff (PAS) method are usually employed for detecting glycoproteins (3,4). Stoward studied histological staining of periodateengendered aldehydes with fluorescent Schiff-type reagents (5) and by hydrazone formation with salicylhydrazide (6). Weinblatt et al. (7) have recently extended the use of hydrazone formation for fluorescent staining of aldehydes in histological preparations usingp-hydrazinoacridine (HA). These authors reported that staining was independent of bisulfite in the reaction and provided evidence that the reaction was specific for aldehydes. With regard to the histochemical systems reported by Stoward (5,6), the conditions of staining periodate-engendered aldehydes with HA reported by Weinblatt et al. (7) were adapted for use in polyacrylamide gels. Similar to Stoward’s findings with other stains (5), HA staining was most intense 428 Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN WJO3-2697
HYDRAZINOACRIDINE
STAINING
IN GELS
429
for glycoproteins but was not specific for proteins with carbohydrate moieties. Furthermore, satisfactory results were found to be highly dependent on the presence of bisulfite. Therefore, HA was investigated for sensitive staining of glycoprotein as well as nonglycoprotein after completion of electrophoresis. Fluorescent staining procedures for polyacrylamide gel electrophoresis have utilized dansyl chloride (8), fluorescamine (9), ando-phthalaldehyde (10) with which the proteins are first reacted and subsequently electrophoresed in the presence of sodium dodecyl sulfate (SDS). The most familiar fluorescent stain used for detecting proteins after electrophoresis is anilinonaphthalene sulfonate (ANS) which stains proteins with less sensitivity than does Coomassie brilliant blue (1). This paper demonstrates the use of p-hydrazinoacridine for staining polyacrylamide gels after electrophoresis, with attention directed toward subjective identification of proteins with carbohydrate moieties. MATERIALS
AND METHODS
Polyacrylamide gel electrophoresis was conducted in 5 x 70-mm cylindrical gels in either SDS-urea as described by Swank and Munkres (1 I) or in acid-urea as described by Panyim and Chalkley (12). The SDS-urea gels were 10% acrylamide with a ratio of N,N’-methylene-bisacrylamide to acrylamide of 1:29. The proteins to be electrophoresed were dissolved at 1 mg/ml in the appropriate solution and applied to the gels in amounts from 5 to 50 pg. The (Y and /3 chains of human haptoglobin (Hpc~ and Hpfl) were a gift from Dr. Alex Kurosky, and egg white lysozyme was from Sigma. Acrylamide was from Bio-Rad. Analytical isoelectric focusing of human serum was adapted from the methods described by Awdeh et al. (13) and Vesterberg (14). The gel was poured between Column Coat (Canalco)-treated glass plates into dimensions of 85 x 150 x 1 mm. The gel contained 5% acrylamide with a ratio of acrylamide to N,N’-methylene-bis-acrylamide of 34.4: 1. The gel contained 4 M urea and 1.4% Ampholine (LKB), pH 3.5- 10. Electrode buffers were 5% ethylene diamine (Fisher) at the cathode and 5% H,PO, (Fisher) at the anode. Samples were applied in 20-~1 aliquots on Whatmann 3 MM paper. The gel was run for 4 hr with a final potential of 600 V. Cylindrical gels were stained with either Coomassie brilliant blue R (Sigma) as described by Carson et al. (15), with PAS as described by Glossmann and Neville (4), or with p-hydrazinoacridine (Eastman) as described here. The gels stained with Coomassie were destained by diffusion in 10% isopropanol- 10% acetic acid (Mallinckrodt) and scanned at 590 nm on a Gilford spectrophotometer. The isoelectric focusing gel was stained with p-hydrazinoacridine, PAS, or a combination dye containing 0.07% Coomassie brilliant blue R, 0.07% fast green (Allied), and 0.07% red
430
STEVEN
D. CARSON
W (Canalco) in 10% trichloroacetic acid (TCA; Mallinckrodt), 10% isopropanol, and 10% acetic acid. This stain has been shown to have sensitivity equivalent to Coomassie blue alone for most proteins and increased sensitivity for the small peptide bacitracin (unpublished data). The stain remains stable with no alteration of the individual components as determined by thin-layer chromatography. For staining with p-hydrazinoacridine, gels were fixed overnight in 12.5% TCA. The gels were then washed in water for 5 min and subjected to 0.5% periodic acid (Ames) for 30 min followed by a 5-min water wash. The p-hydrazinoacridine was prepared by dissolving 20 mg of HA in 20 ml of 95% ethanol. The dye was diluted to 100 ml with distilled water to which 5 ml of 2 N HCl (Fisher) was added. No more than 5 min prior to staining, 1 g of potassium metabisulfite (Fisher) was added with mixing. The gels were stained for 20 min or until the transient brown coloration was gone and were then placed in 1% HCl-20% ethanol for 15 min. For development of fluorescent bands, the gels were soaked in a solution containing 12.5% TCA and 1% sulfosalicylic acid (Mallinckrodt). Bands could be detected after 5 min by illumination with long-wave ultraviolet light. Gels were scanned at 440 nm. Several modifications of the staining method were investigated. The time of periodate oxidation was varied from 10 min to 1 hr under ambient conditions and in the dark at 0°C without altering the results. Oxidation in dimethyl sulfoxide (Fisher) did not improve the differential staining of protein and glycoprotein. The 1% potassium metabisulfite employed was found to produce better staining than 0.5% bisulfite, even though a precipitate appeared after 5 min of exposure to the HA solution. RESULTS
The p chain of human haptoglobin (HP/~) contains 19.4% carbohydrate (16) and was chosen to compare with the nonglycoproteins, lysozyme and the (Ychain of haptoglobin (Hpo). Staining with HA as described resulted in fluorescent labeling of all three proteins, with the fluorescence of HpP most intense. Furthermore, the HI>@ fluorescence was yellow, whereas lysozyme and Hpcl! fluoresced light green. No fluorescence was observed prior to placing the gels in TCA, and 1% sulfosalicylic acid intensified the fluorescence. No staining was evident without prior periodate oxidation of the proteins. Although some staining of all three proteins occurred in the absence of potassium metabisulfite, the bands were too weak for successful photography. Only in the presence of bisulfite was staining satisfactory, producing the yellow fluorescence noted for HP/?. The stain produced a notable fluorescence after 5 min in the TCA-sulfosalicylic acid solution, which reached maximum intensity after 24 hr. The stained proteins could also be seen under normal lighting as yellow bands and were scanned with good results at 440 nm. After 24 hr, the fluorescent and visible bands slowly
HYDRAZINOACRIDINE
STAINING
IN GELS
431
lost their intense yellow color and acquired a deep orange color. This orange band, although less intense than the yellow, remained stable after several weeks in the TCA-sulfosalicylic acid solution, Staining results were similar for all of the gel systems employed, SDS-urea polyacrylamide gels were run with 5 to 50 pg of HpP, Hpcr, and lysozyme and stained with HA, Coomassie brilliant blue R, and PAS (Figs. 1 and 2). Figure 1 shows that, with HA, even 5 pg of each protein was
a b
c d
A
E3
C
D
FIG. 1. SDS-urea polyacrylamide gels stained with HA and photographed under long-wave ultraviolet light. Gels contained: (A) 5, (B) 10, (C) 20, and (D) 30 pg each of Hp& Hpa, and lysozyme; (E) 30 pg of HpP and 2Opg of lysozyme; and(F) 50 pg each of HpP and Hpcy.Bands are: (a) HpP contaminant, (b) HpP, (c) Hpa. and (d) lysozyme.
432
STEVEN
D. CARSON
a b
c d
A
6
CD
E
F
G
FIG. 2. SDS-urea polyacrylamide gels stained with (A-C) Coomassie brilliant blue R and (D-G) PAS. Gels contained equal amounts of HP/~, Hpa, and lysozyme: (A and E) 10 Fg, (B and F) 20 pg, (C and G) 30 pg. and(D) 5 pg. Bands are (a) Hp@ contaminant, (b) HP/~, (c) Hpcu, and (d) lysozyme.
easily detected under long-wave ultraviolet light. Figure 2 shows similar polyacrylamide gels stained with Coomassie and PAS. When the gels containing 10 pg of Hpfi, Hpa, and lysozyme stained with Coomassie or HA were scanned in the visible range and the peak heights were compared, the HA staining was found to be about 80% as sensitive as Coomassie for HpP. Coomassie staining of lysozyme was threefold more intense than HA staining when compared in the visible spectrum. Inspection of Figs. 1 and 2 reveals that HA staining was more sensitive for demonstration of Hpfl than PAS (especially notable at the 5-pg level) and comparable to Coomassie. The HA stained both lysozyme and Hpc~ to a lesser extent than did Coomassie. and PAS stained neither. FIG. 3. Polyacrylamide gel isoelectric serum. Gel sections were stained with described in Materials and Methods; (B) (D) HA photographed under long-wave
focusing, pH 3.5 to 10, of 20-4 aliquots of humar (A) Coomassie brilliant blue-fast green-red W a: PAS; (C) HA photographed under visible light; am ultraviolet light.
HYDRAZINOACRIDINE
STAINING
IN GELS
433
--“-.r-”,-----.., .,!’ a
434
STEVEN
D. CARSON
Aliquots of whole serum (20 ~1) were subjected to isoelectric focusing in polyacrylamide gel. The gel was sectioned and stained with Coomassiefast green-red W, PAS, or HA as described in Materials and Methods. Figure 3 is a photograph of stained gel sections. After HA staining (Fig. 3D), bands a (predominantly y-globulin), b, f, and g showed the yellow fluorescence characteristic of glycoproteins, whereas bands c and e displayed blue-gray fluorescence, and band d fluoresced white. Human serum albumin (Schwarz/Mann) was shown to focus at positions corresponding to bands c and e. PAS staining (Fig. 3B) confirmed the glycoprotein nature of bands b, f, and g, but was not sufficiently sensitive to stain the carbohydrate moieties of the y-globulins. Staining of the gels with the compound protein stain (Fig. 3A) revealed that HA staining was comparably sensitive for proteins b, d, and g, but of decreased sensitivity for bands a, c, and f. Since the glycoproteins could be recognized by the yellow fluorescence, HA staining was preferable to PAS with regard to both sensitivity and localization of the glycoproteins among other bands stained with the general stain. DISCUSSION
p-Hydrazinoacridine has been demonstrated as a fluorescent stain suitable for detecting glycoproteins in polyacrylamide gels after electrophoresis. The yellow fluorescence of the glycoproteins allowed their identification among other protein bands which fluoresced less intensely and at wavelengths other than yellow. Comparison of HA staining with Coomassie brilliant blue R (15) and PAS (4) staining of known amounts of HP/~, Hpcy, and lysozyme demonstrated that HA was more sensitive for detecting HpP 119.4% carbohydrate (16)] than PAS (Figs. 1 and 2). The visible yellow bands produced with HA were scanned at 440 nm and shown to be of a sensitivity comparable to that of Coomassie for Hpfl and approximately one-third as sensitive as Coomassie for the nonglycoprotein, lysozyme. As used here for staining polyacrylamide gels, the behavior of HA was similar to fluorescent histological stains described by Stoward (5). A weak green fluorescence was obtained when bisulfite was omitted from the reactions, which may have been simple hydrazone formation as discussed by Weinblatt et al. (7). However, such hydrazone formation must not be specific for aldehydes derived from the oxidation of carbohydrates as evidenced by fluorescence of Hpcw and lysozyme. In the presence of potassium metabisulfite, the intensity of fluorescence was greatly enhanced. Furthermore, HpP (Fig. 1) and the PAS-positive bands from isoelectric focusing (Fig. 3) acquired the yellow fluorescence found to be characteristic of glycoproteins in this study. Since these characteristics of HA are similar to those of the Schiff-type reagents studied by Stoward (5), these stains may share even more properties. Stoward found ambiguity in
HYDRAZINOACRIDINE
STAINING
IN GELS
435
distinguishing some glycoprotein tissue constituents from nonglycoprotein structures on the basis of fluorescent color alone. He also noted that the fluorescent Schiff-type reagents he studied did not stain some PAS-positive mucosubstances. Although neither of these problems was encountered when isoelectrically focused serum was stained with HA, the possible occurrence of anomalous staining has not been eliminated and should be considered in the evaluation of protein staining. As presented here,p-hydrazinoacridine is a sensitive fluorescent reagent suitable for staining polyacrylamide gels after electrophoresis. When glycoproteins were present, they fluoresced more intensely than nonglycoproteins and could be distinguished by the yellow color of the fluorescence. Compared to PAS staining, HA provided advantages of both sensitivity and localization of nonglycoproteins within the gels. ACKNOWLEDGMENTS I thank Dr. Alex Kurosky for the haptoglobin preparations and suggestions and Dr. B. H. Bowman and Dr. R. J. Klebe for reviewing the manuscript. This work was supported, in part, by a grant from the Robert A. Welch Foundation H-378.
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