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Electrophoresis of honey: Characterization of trace proteins from a complex biological matrix by silver staining
ANALYTICAL
BIOCHEMISTRY
167,30
I-303
( 1987)
Electrophoresis of Honey: Characterization of Trace Proteins from a Complex Biological Matrix by Silver Staining Biochemistry
Research
THOMAS MARSHALL AND KATHERINE M. WILLIAMS Laboratory, Biology Department, University of Ulster, ColeraineBTj2
IsA,
Northern
Ireland
Received June 29, 1987 The proteins of unconcentrated honey have been detected with the methylamine-incorporating silver stain (T. Marshall, 1984, Anal. Biochem. 136, 340-346) following sodium dodecyl sulfate-polyacrylamide gel electrophoresis or high-resolution two-dimensional electrophoresis. The former consistently reveals at least 19 protein bands in a variety of Australian honeys. Two-dimensional electrophoresis gave patterns of poor resolution but proved useful for further characterization of the major protein constituents. The protein patterns were unaffected by centrifugation of the Samples prior to preparation fOt’ electrophoresis. 0 1987 Academic POW. IN. KEY WORDS: honey; proteins; SDS-PAGE: two-dimensional electrophoresis: silver staining.
The production and composition of honey have been extensively reviewed (1). It contains approximately 0.2% protein (2), of bee and plant (pollen/nectar) origin (3-5), including bee cY-amylase (4) and constituents capable of clarifying apple juice (3). The protein content and distribution could prove useful for electrophoretic monitoring of the adulteration and misrepresentation of honey (5); studies are currently complicated by its high sugar and low protein content which necessitate extensive sample manipulation, e.g., ultrafiltration, column chromatography, concentration, and lyophilization (3-5). We demonstrate avoidance of these problems by ultrasensitive silver staining (67) which, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)’ of unconcentrated native honey, reveals an unprecedented number of protein constituents. We also demonstrate high-resolution two-dimensional (2-D) electrophoresis of honey for further characterization of these proteins, an application not previously reported. ’ Abbreviations used: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 2-D, two-dimensional; IEF, isoelectric focusing. 301
MATERIALS
AND METHODS
Sample preparation. A variety of commercially available (“Epicure”) Australian honeys were purchased locally. In each case, 1 g of sample was diluted with 1 ml of ultrapure water (MilhQ, Millipore, UK), thoroughly mixed, centrifuged (Microfuge), and mixed with an equal volume of sampledenaturing solution (0.0625 M Tris-HCl, pH 6.8, containing 2% (w/v) SDS, 5% (v/v) 2mercaptoethanol, and 20% (w/v) glycerol) prior to heating at 95°C for 5 min. Duplicate samples were prepared without centrifugation. SDS-PAGE (8). The samples (l-2.5 ~1 SDS mixture) were loaded in agarose wells precast on the upper surface of 4-20% (w/v) polyacrylamide gradient gels (75 X 75 X 3 mm) and electrophoresed (without a stacking gel) at 50 mA/gel for 1 h in 0.025 M Tris containing 0.2 M glycine and 0.1% (w/v) SDS (9, IO). 2-D electrophoresis (11). Isoelectric focusing (IEF, first dimension) in 4% (w/v) polyacrylamide gel cylinders (65 X 3 mm) containing 9 M urea, 0.5 or 2% (w/v) Nonidet P-40 (NP-40), and 2% (w/v) Ampholine (pH range 2.5-4, 3.5-10; 2:9; v/v) was followed, 0003-2697187
$3.00
Copyright 0 1987 by Academic F’req Inc. All rights of reproduction in any form reserved.
302
MARSHALL
AND WILLIAMS
FIG. I. Silver-stained SDS-PAGE protein patterns of 300 pg (lanes 1-8) or 750 ~g (lanes 9-12) of strawberry clover (lanes 1, 2, 9) sunflower (lanes 3,4. 10). coolibah (lanes 5, 6, 1I), and orange blossom (lanes 7, 8. 12) Australian honey. Samples centrifuged prior to SDS denaturation correspond to lanes 2,4,6, and 8; the respective noncentrifuged samples correspond to lanes I, 3, 5, and 7 (and 9-12 at higher protein loads). The major constituents are lettered A-S and an additional sunflower component is marked with an asterisk. M, indicates relative molecular weight X 1O-3
distinctive bands (denoted A-S) were reproducibly detected following SDS-PAGE (Fig. 1). These proteins were further characterized by 2-D electrophoresis-the respective constituents being tentatively identified by their prominence and M, position (Fig. 2). The major constituents, in order of prominence, comprised G (Mr 56,000; pZ 4.8-7.7), F (M, 68,000; pZ 5.2-6), S (Mr 10,500; pZ 5.1), N (A& 23,000; pZ 4.8-6.1) H (Mr 45,000), E (Ad, 79,000; pZ 7.2), C (A4r 12 1,000; pZ 5.2-5.8), and D (M, 102,000; pZ 5.2-5.8). Thus while S and E were quite homogeneous and C and D were diffuse over a narrow pZ range, the other major constituents were highly heterogeneous and apparently comprised of poorly resolved multiple strings of spots. Component G was particularly complex consisting of at least two parallel strings of prominent spots and a minor string of lower M,. Very high-molecular-weight components A (M, 500,000) and B (Mr 350,000) were also detected in all samples (Fig. 1). A low-molecular-weight component (M,
with or without IEF gel equilibration, by SDS-PAGE as described above ( 12,13). Silver staining (6,7). The electrophoresis gels were soaked for 48 h in 50% methanol, 10% acetic acid, and the proteins were detected using the overstain/destain strategy of the methylamine-incorporating silver stain ( 14). RESULTS
Representative silver-stained SDS-PAGE and 2-D electrophoresis patterns of honey are shown in Figs. 1 and 2, respectively. Both methods gave highly reproducible patterns which were remarkably similar for each of the honey types analyzed. Centrifugation of the samples prior to SDS denaturation did not significantly affect the protein patterns (Fig. 1). In all samples analyzed 19 clearly
FIG. 2. Silver-stained 2-D electrophoresis pattern of 3 mg of orange blossom Australian honey. The major constituents have been tentatively identified according to prominence and M, with respect to Fig. 1. The anode of the IEF gel is to the left and electrophoresis performed is from top to bottom.
ELECTROPHORESIS
14,000) apparently specific to sunflower honey was noticeably less prominent in centrifuged samples (Fig. 1). DISCUSSION
Silver staining and 2-D electrophoresis of the proteins of honey has not previously been reported. SDS-PAGE and Coomassie brilliant blue staining has detected up to 11 protein bands in honey (5) but our method offers the following advantages: (i) t-eproducible detection of at least 19 protein bands in unconcentrated native honey (Fig. 1) without concentration of sugar-free samples (5) and (ii) multiple sample analysis (200 samples/l 5 identical precast gels) with optimal resolution over a wide M, range (lO,OOO500,000) using polyacrylamide gradient gels (Fig. 1) rather than linear 10% polyacrylamide rod gels (5). The 2-D electrophoresis patterns were of poor resolution (Fig. 2) relative to those we routinely obtain with human body fluids (results not shown). Our methodology currently omits IEF gel SDS equilibration ( 12,13) but recourse to equilibration (1 I, 15), with or without inclusion of 2-mercaptoethanol ( 16) only marginally improved resolution. The problem may arise from the heterogeneous nature of the proteins, their predominance in specific p1 and A4, ranges, or the complexing/interfering effect of other honey constituents. However, the use of 2-D electrophoresis pertains to the physicochemical characterization of the proteins (and their identification) and not routine analysis-SDS-PAGE is technically simpler and less time-consuming. In accordance with previous observations the consistency and reproducibility of the
303
OF HONEY
patterns that we have obtained with different honeys suggests the protein constituents are predominantly of bee origin. Bee a-amylases have been implicated (4) and it is of interest to note that, with our methodology, human a-amylase gives multiple strings of spots in the same pl and M, range as the major honey component G (12,13). However, the sunflower-specific component (Fig. 1) which is reduced in amount following sample centrifugation may be of pollen origin. REFERENCES I. White, J. W. (1978) Adv. Food Rex 24,287-374. White, J. W. (1978) J. Apic. Rex 17, 234-238. Lee, C. Y., Smith, N. L., Kime, R. W., and Morse, R. A. (1985) J. Apic. Res. 24, 190-194. 4. Stadelmeier, M., and Bergner, K. G. (1986) Z. Lebensm. Unters. Forsch. 182, 196-199. 5. Croft, L. R., Mistry, R. P., and Washington, R. J. (1986) in Electrophoresis ‘86 (Dunn, M. J., Ed.), pp. 338-339. VCH Publishers, Deerlield Beach, FL. 6. Kerenyi, L., and Gallyas, F. (1972) C/in. Chim. Acta 2. 3.
38,465-467.
7. Switzer, R. C., Merril, R. C., and Shifiin, S. (1979) Anal. Biochem. 98,231-237. 8. Laemmli, U. K. (1970) Nature (London) 227, 680-685.
Marshall. T. (1984) Clin. Chem. 30,475-479. 10. Marshall, T., and Williams, K. M. (1985) BioTechniques 3, 350-352. 11. O’Farrell, P. H. (1975) .I. Biol. Chem. 250, 9.
4007-4021.
12. Marshall, T. (1984) Electrophoresis 5, 245-250. 13. Marshall, T., and Williams, K. M. (1986) in Electrophoresis ‘86 (Dunn, M. J., Ed.), pp. 523-537, VCH Publishers, Deerfield Beach, FL. 14. Marshall, T. (1984) Anal. Biochem. 136,340-346. 15. Latner. A. L., Marshall, T., and Gambie, M. (1980) Clin. Chim. Acta 103, 51-59. 16. Marshall, T., and Latner, A. L. ( I98 1) Electrophoresis 2,228-235.
BIOCHEMISTRY
167,30
I-303
( 1987)
Electrophoresis of Honey: Characterization of Trace Proteins from a Complex Biological Matrix by Silver Staining Biochemistry
Research
THOMAS MARSHALL AND KATHERINE M. WILLIAMS Laboratory, Biology Department, University of Ulster, ColeraineBTj2
IsA,
Northern
Ireland
Received June 29, 1987 The proteins of unconcentrated honey have been detected with the methylamine-incorporating silver stain (T. Marshall, 1984, Anal. Biochem. 136, 340-346) following sodium dodecyl sulfate-polyacrylamide gel electrophoresis or high-resolution two-dimensional electrophoresis. The former consistently reveals at least 19 protein bands in a variety of Australian honeys. Two-dimensional electrophoresis gave patterns of poor resolution but proved useful for further characterization of the major protein constituents. The protein patterns were unaffected by centrifugation of the Samples prior to preparation fOt’ electrophoresis. 0 1987 Academic POW. IN. KEY WORDS: honey; proteins; SDS-PAGE: two-dimensional electrophoresis: silver staining.
The production and composition of honey have been extensively reviewed (1). It contains approximately 0.2% protein (2), of bee and plant (pollen/nectar) origin (3-5), including bee cY-amylase (4) and constituents capable of clarifying apple juice (3). The protein content and distribution could prove useful for electrophoretic monitoring of the adulteration and misrepresentation of honey (5); studies are currently complicated by its high sugar and low protein content which necessitate extensive sample manipulation, e.g., ultrafiltration, column chromatography, concentration, and lyophilization (3-5). We demonstrate avoidance of these problems by ultrasensitive silver staining (67) which, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)’ of unconcentrated native honey, reveals an unprecedented number of protein constituents. We also demonstrate high-resolution two-dimensional (2-D) electrophoresis of honey for further characterization of these proteins, an application not previously reported. ’ Abbreviations used: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 2-D, two-dimensional; IEF, isoelectric focusing. 301
MATERIALS
AND METHODS
Sample preparation. A variety of commercially available (“Epicure”) Australian honeys were purchased locally. In each case, 1 g of sample was diluted with 1 ml of ultrapure water (MilhQ, Millipore, UK), thoroughly mixed, centrifuged (Microfuge), and mixed with an equal volume of sampledenaturing solution (0.0625 M Tris-HCl, pH 6.8, containing 2% (w/v) SDS, 5% (v/v) 2mercaptoethanol, and 20% (w/v) glycerol) prior to heating at 95°C for 5 min. Duplicate samples were prepared without centrifugation. SDS-PAGE (8). The samples (l-2.5 ~1 SDS mixture) were loaded in agarose wells precast on the upper surface of 4-20% (w/v) polyacrylamide gradient gels (75 X 75 X 3 mm) and electrophoresed (without a stacking gel) at 50 mA/gel for 1 h in 0.025 M Tris containing 0.2 M glycine and 0.1% (w/v) SDS (9, IO). 2-D electrophoresis (11). Isoelectric focusing (IEF, first dimension) in 4% (w/v) polyacrylamide gel cylinders (65 X 3 mm) containing 9 M urea, 0.5 or 2% (w/v) Nonidet P-40 (NP-40), and 2% (w/v) Ampholine (pH range 2.5-4, 3.5-10; 2:9; v/v) was followed, 0003-2697187
$3.00
Copyright 0 1987 by Academic F’req Inc. All rights of reproduction in any form reserved.
302
MARSHALL
AND WILLIAMS
FIG. I. Silver-stained SDS-PAGE protein patterns of 300 pg (lanes 1-8) or 750 ~g (lanes 9-12) of strawberry clover (lanes 1, 2, 9) sunflower (lanes 3,4. 10). coolibah (lanes 5, 6, 1I), and orange blossom (lanes 7, 8. 12) Australian honey. Samples centrifuged prior to SDS denaturation correspond to lanes 2,4,6, and 8; the respective noncentrifuged samples correspond to lanes I, 3, 5, and 7 (and 9-12 at higher protein loads). The major constituents are lettered A-S and an additional sunflower component is marked with an asterisk. M, indicates relative molecular weight X 1O-3
distinctive bands (denoted A-S) were reproducibly detected following SDS-PAGE (Fig. 1). These proteins were further characterized by 2-D electrophoresis-the respective constituents being tentatively identified by their prominence and M, position (Fig. 2). The major constituents, in order of prominence, comprised G (Mr 56,000; pZ 4.8-7.7), F (M, 68,000; pZ 5.2-6), S (Mr 10,500; pZ 5.1), N (A& 23,000; pZ 4.8-6.1) H (Mr 45,000), E (Ad, 79,000; pZ 7.2), C (A4r 12 1,000; pZ 5.2-5.8), and D (M, 102,000; pZ 5.2-5.8). Thus while S and E were quite homogeneous and C and D were diffuse over a narrow pZ range, the other major constituents were highly heterogeneous and apparently comprised of poorly resolved multiple strings of spots. Component G was particularly complex consisting of at least two parallel strings of prominent spots and a minor string of lower M,. Very high-molecular-weight components A (M, 500,000) and B (Mr 350,000) were also detected in all samples (Fig. 1). A low-molecular-weight component (M,
with or without IEF gel equilibration, by SDS-PAGE as described above ( 12,13). Silver staining (6,7). The electrophoresis gels were soaked for 48 h in 50% methanol, 10% acetic acid, and the proteins were detected using the overstain/destain strategy of the methylamine-incorporating silver stain ( 14). RESULTS
Representative silver-stained SDS-PAGE and 2-D electrophoresis patterns of honey are shown in Figs. 1 and 2, respectively. Both methods gave highly reproducible patterns which were remarkably similar for each of the honey types analyzed. Centrifugation of the samples prior to SDS denaturation did not significantly affect the protein patterns (Fig. 1). In all samples analyzed 19 clearly
FIG. 2. Silver-stained 2-D electrophoresis pattern of 3 mg of orange blossom Australian honey. The major constituents have been tentatively identified according to prominence and M, with respect to Fig. 1. The anode of the IEF gel is to the left and electrophoresis performed is from top to bottom.
ELECTROPHORESIS
14,000) apparently specific to sunflower honey was noticeably less prominent in centrifuged samples (Fig. 1). DISCUSSION
Silver staining and 2-D electrophoresis of the proteins of honey has not previously been reported. SDS-PAGE and Coomassie brilliant blue staining has detected up to 11 protein bands in honey (5) but our method offers the following advantages: (i) t-eproducible detection of at least 19 protein bands in unconcentrated native honey (Fig. 1) without concentration of sugar-free samples (5) and (ii) multiple sample analysis (200 samples/l 5 identical precast gels) with optimal resolution over a wide M, range (lO,OOO500,000) using polyacrylamide gradient gels (Fig. 1) rather than linear 10% polyacrylamide rod gels (5). The 2-D electrophoresis patterns were of poor resolution (Fig. 2) relative to those we routinely obtain with human body fluids (results not shown). Our methodology currently omits IEF gel SDS equilibration ( 12,13) but recourse to equilibration (1 I, 15), with or without inclusion of 2-mercaptoethanol ( 16) only marginally improved resolution. The problem may arise from the heterogeneous nature of the proteins, their predominance in specific p1 and A4, ranges, or the complexing/interfering effect of other honey constituents. However, the use of 2-D electrophoresis pertains to the physicochemical characterization of the proteins (and their identification) and not routine analysis-SDS-PAGE is technically simpler and less time-consuming. In accordance with previous observations the consistency and reproducibility of the
303
OF HONEY
patterns that we have obtained with different honeys suggests the protein constituents are predominantly of bee origin. Bee a-amylases have been implicated (4) and it is of interest to note that, with our methodology, human a-amylase gives multiple strings of spots in the same pl and M, range as the major honey component G (12,13). However, the sunflower-specific component (Fig. 1) which is reduced in amount following sample centrifugation may be of pollen origin. REFERENCES I. White, J. W. (1978) Adv. Food Rex 24,287-374. White, J. W. (1978) J. Apic. Rex 17, 234-238. Lee, C. Y., Smith, N. L., Kime, R. W., and Morse, R. A. (1985) J. Apic. Res. 24, 190-194. 4. Stadelmeier, M., and Bergner, K. G. (1986) Z. Lebensm. Unters. Forsch. 182, 196-199. 5. Croft, L. R., Mistry, R. P., and Washington, R. J. (1986) in Electrophoresis ‘86 (Dunn, M. J., Ed.), pp. 338-339. VCH Publishers, Deerlield Beach, FL. 6. Kerenyi, L., and Gallyas, F. (1972) C/in. Chim. Acta 2. 3.
38,465-467.
7. Switzer, R. C., Merril, R. C., and Shifiin, S. (1979) Anal. Biochem. 98,231-237. 8. Laemmli, U. K. (1970) Nature (London) 227, 680-685.
Marshall. T. (1984) Clin. Chem. 30,475-479. 10. Marshall, T., and Williams, K. M. (1985) BioTechniques 3, 350-352. 11. O’Farrell, P. H. (1975) .I. Biol. Chem. 250, 9.
4007-4021.
12. Marshall, T. (1984) Electrophoresis 5, 245-250. 13. Marshall, T., and Williams, K. M. (1986) in Electrophoresis ‘86 (Dunn, M. J., Ed.), pp. 523-537, VCH Publishers, Deerfield Beach, FL. 14. Marshall, T. (1984) Anal. Biochem. 136,340-346. 15. Latner. A. L., Marshall, T., and Gambie, M. (1980) Clin. Chim. Acta 103, 51-59. 16. Marshall, T., and Latner, A. L. ( I98 1) Electrophoresis 2,228-235.