ANALYTICAL
72, 315-319 (1976)
BIOCHEMISTRY
A Rapid Technique for the Detection of Amylase lsoenzymes Using an Enzyme Sensitive “Test-Paper” P. E. BURDETT,
ANN E. KIPPS, AND P. H. WHITEHEAD
Home Ojjjce Aldermaston,
Central Reading,
Received November
Research Be&s,
Establishment United Kingdom
3, 1975: accepted January 8, 1976
A new technique for the detection of amylase isoenzymes is described. An amylase sensitive test paper is applied to the surface of a flat bed electrophoresis gel and the isoenzyme bands rapidly appear as well defined white lines on a pink background. The electrophoresis gel is undamaged by this treatment and can be further stained with other reagents.
It has been reported by a number of workers that salivary amylase isoenzyme patterns are subject to genetic control (l-3). Such a polymorphism is of interest to forensic scientists as a potential means of typing saliva traces left at a scene of crime, since amylase is stable for some months in dried saliva stains (4). Amylase isoenzymes were studied using flat bed isoelectric focusing because of the very high resolving power of this technique, but initial work showed that the high resolution of the separation method was frequently lost during the staining procedure. A number of methods have been described for localizing amylase isoenzymes after electrophoresis. The simplest involve soaking the electrophoretic medium in a starch solution and staining with iodine so that the amylase bands appear yellow on a stained blue background (5). This method has been modified by a number of workers and the starch applied as a thin film (6), a starch-agar film (7) and a starch-polyacrylamide layer (8). A saccharogenic staining method to detect the products of amylase action has also been described (9), as well as an immunofixation technique (10). Recently, insoluble dyed starch substrates have been developed for amylase assay and these have been incorporated into agar gel for isoenzyme detection (11,12). These substrates have the advantage of being more specific for amylase than the starch iodide techniques (13,14) and simpler than the saccharogenic and immunofixation methods. The method described in the present paper uses a soluble dyed starch substrate which is precipitated on to Whatman 3MM chromatography 315 CopyrIght All
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1976
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BURDETT,
KIPPS AND WHITEHEAD
paper to give a pink amylase%tilsitive test paper. The method has all the advantages of the specificit: of dyed starch substrates but is simpler, quicker, and offers better resolution than dyed starch in agar methods. EXPERIMENTAL Materials
and Methods
Various protein solutions were obtained for analysis of their amylase isoenzyme patterns. Whole unstimulated saliva was used fresh without further treatment. Hyland Multienzyme reference serum C (1140 IU amylase)’ was obtained as a freeze dried preparation from Travenol Laboratories Ltd. (Thetford, Norfolk), and was made up at 5 times the recommended concentration. Bacterial cr-amylase was obtained from BDH (Poole, Dorset) as a powder with an activity as defined by the manufacturer of 10 EU/mg,l and a-amylase from pig pancreas, also from BDH, was obtained freeze dried with activity of 250 EU/mg.l Isolectric
Focusing
Isoelectric focusing was accomplished using the LKB 2117 Multiphor equipment. The gels were prepared with a final concentration of 5.25% acrylamide of which 5% was the monomer and 0.25% N,N’methylenebisacrylamide (BDH, Poole, Dorset) and with 1.2% (w/v) of Ampholine buffers. (LKB Instruments Ltd, 232 Addington Road, Croydon, Surrey). Equal amounts of Ampholines of pH ranges 5 to 7 and 7 to 9 were used which gave a nominal range of pH 5 to 8.5. Following the addition of 0.5 ml of riboflavin solution (10 mg%, w/v) per 60 ml of gel solution, photopolymerization was allowed to proceed for 2 hr. The cathode strip was soaked in 1% diaminoethane, whilst the anode strip was soaked in 1% acetic acid. Samples were applied directly to the gel by a Partigen dispenser (Hoechst Pharmaceuticals, Hounslow, Middlesex) in amounts ranging from 1 to 10 ~1. They were applied 3 cm from the anode. Saliva was used undiluted, whilst solutions of bacterial and pig pancreas amylases were applied in solutions of 20 and 1 mg/ml, respectively. Power was applied to the gel from the LKB power supply, following the manufacturer’s instructions. After 2lh hr, when the run was terminated, a current of 14 mA at 1150 V was being passed through the gel. 1 Amylase levels are those quoted by the manufacturer. An International Unit (IU) is that amount of enzyme which will catalyze the transformation of one micromole of substrate per minute per liter. One enzyme unit (EU) is that amount of enzyme which liberates one micromol of reducing sugar as maltose per minute at 25°C in acetate buffer pH 4.8 with B.D.H. Lintner soluble starch as substrate.
DETECTION
OF AMYLASE
ISOENZYMES
317
The preparation of the amylase sensitive paper, which was originally developed for detecting saliva stains in forensic science investigations, has been described previously (4). A solution of lyosine red (0.4% w/v) in phosphate buffer, pH 4.9 (15) was precipitated on to Whatman 3MM chromatography paper with ethylenegiycolmonomethylether and zinc sulphate solutions. At the end of the isoelectric focusing run a dry piece of amylasesensitive test paper was placed carefully on top of the gel. The band development was watched by viewing the paper through the gel and when the white bands on the pink background could be seen clearly (lo- 15 min), the paper was wetted with distilled water and carefully peeled off the gel. The paper was then washed in an aqueous solution of sodium hexametaphosphate (20% w/v) to inhibit further amylase action and air dried to provide a permanent record of the amylase isoenzyme pattern. The gel was subsequently stained by an iodometric method (5).
FIG. 1. Amyiase zymogram, following visualisation using the test paper. The grainy ap pearance is due to the texture of the filter paper. Samples A and H: Bacterial amylase 5 /.&I; I: Bacterial amylase, 1 ~1; C: Pig pancreas amylase 1 ~1; E, Hyland serum, 10 ~1; F and G: Human salivary amylase, 1 and 5 ~1, respectively. B: A mixture of bacterial amylase 1 ~1 with pig pancreas amylase. 1 ~4; D: A mixture of pig pancreas amyiase 5 PLI, with a trace of bacterial amyiase. Note that Horizontal distortion of the pH gradient, gives the false impression that the band with the most acidic pI point of samples B and C are different.
318
BURDETT.
KIPPS AND WHITEHEAD
RESULTS AND DISCUSSION
The patterns of the amylaseisoenzymes, as visualized by the amylase test paper are shown in Fig. 1. The zymograms produced by amylase from bacteria, pig pancreas,human saliva, and Hyland serum show a diverse rangeof isoelectric points (about 2.5 pH units). The fine resolving power of the combination of the isoelectric focusing technique and the test paper detection system is demonstratedby the definition of the threeminor bandsseenin the sampleof Hyland serum(sampleE, Fig. 1). Salivary amylase was found to produce at least 10 isoenzyme bands (samplesF and G). The amyloclastic nature of all the bandsseenin Fig. 1 has beenconfirmed by using the iodometric staining method on the same gel. More isoenzyme bands are shown here than have been previously reported (12). The insoluble Phadebas-substratehas been used dispersedin agar gel asan overlay for amylaseisoenzymesseparatedin polyacrylamidegel(12). It is reported that this technique requires a 4 hr incubation period in which to develop the isoenzyme bands from 10 ~1 neat human saliva. With the test paper that is describedhere the isoenzymebandsfrom 5 ~1 of saliva are well resolved in IO- 15 min. This short incubation period has the advantagethat diffusion of the enzyme does not impair resolution The test paper would be equally suitable for detecting amylase isoenzymepatternsproducedby other techniquessuchasagargeland conventional polyacrylamide gel electrophoresis. The test paperis sensitiveto the low levels of amylasefound in normal urine and serum and could thus be used to detect amylase isoenzymes in clinical samples.Modification of the sampleloadingsandthe zymogram incubation time would make the technique suitable for use with samples containing concentrations of amylase either lower or higher than those used here. In conclusion, the test paper can be recommendedas a rapid, simple methodfor the detection of amylaseisoenzymes.It is specific and hasthe additional advantages, unlike Phadebas or the starch methods, of simplicity and of producing a permanentrecord. REFERENCES 1. Boettcher, B., and de la Lande, F. A. (1969)Ausr. J. Exp. Biol. Med. Sci. 47,97-103. 2. Wolf, R, O., Taylor, L. L., Niswander, J. D., and Schwartz, J. T., (1971) Archs. Oral Biol. 16, 1357- 1359. 3. Skude, G. (1971) Humangenetik 12, 255-256. 4. Whitehead, P. H. and Kipps, A. E. (1975)J. Forens. Sci. SW. 15, 36-42. 5. Beottcher, B., and de la Lande, F. A. (1%9)Anai. Biochem. 28, 510-514. 6. Robinson, L. A., Churchill, C. L., and White, T. T. (1970)Biochim. Biophys.Acfa 222, 390-395. 7. Wolf, R. O., and Taylor, L. L. (1968) Amer. J. Clin. Pathol. 38, 871-876.
DETECTION 8. 9. 10. 11. 12. 13. 14. 15.
OF AMYLASE
ISOENZYMES
Doane, W. W. (1967) Sabco. J. 3, 63-68. Joseph, R. R., Olivero, E., and Ressler, N. (1966) Gastroenterology 51, 377-382. Skude, G. (1970) Hereditas 65, 277-284. Rosalki, S. B. (197O)J. C/in. Path. 23, 373-374. Wadstrom, T., and Smyth, C. J. (1973) Sci. Tools 20, 17-21. Wilding, P. (1965) C/in. Chim. Acta 12, 97-104. Longprt, and Hamel, M (1973) C/in. Biochem. 6, 71-81. Sax, S. M., Bridgewater, A. B., and Moore, J. J. (1971) Clin. Chem. 17, 311-315.
319
72, 315-319 (1976)
BIOCHEMISTRY
A Rapid Technique for the Detection of Amylase lsoenzymes Using an Enzyme Sensitive “Test-Paper” P. E. BURDETT,
ANN E. KIPPS, AND P. H. WHITEHEAD
Home Ojjjce Aldermaston,
Central Reading,
Received November
Research Be&s,
Establishment United Kingdom
3, 1975: accepted January 8, 1976
A new technique for the detection of amylase isoenzymes is described. An amylase sensitive test paper is applied to the surface of a flat bed electrophoresis gel and the isoenzyme bands rapidly appear as well defined white lines on a pink background. The electrophoresis gel is undamaged by this treatment and can be further stained with other reagents.
It has been reported by a number of workers that salivary amylase isoenzyme patterns are subject to genetic control (l-3). Such a polymorphism is of interest to forensic scientists as a potential means of typing saliva traces left at a scene of crime, since amylase is stable for some months in dried saliva stains (4). Amylase isoenzymes were studied using flat bed isoelectric focusing because of the very high resolving power of this technique, but initial work showed that the high resolution of the separation method was frequently lost during the staining procedure. A number of methods have been described for localizing amylase isoenzymes after electrophoresis. The simplest involve soaking the electrophoretic medium in a starch solution and staining with iodine so that the amylase bands appear yellow on a stained blue background (5). This method has been modified by a number of workers and the starch applied as a thin film (6), a starch-agar film (7) and a starch-polyacrylamide layer (8). A saccharogenic staining method to detect the products of amylase action has also been described (9), as well as an immunofixation technique (10). Recently, insoluble dyed starch substrates have been developed for amylase assay and these have been incorporated into agar gel for isoenzyme detection (11,12). These substrates have the advantage of being more specific for amylase than the starch iodide techniques (13,14) and simpler than the saccharogenic and immunofixation methods. The method described in the present paper uses a soluble dyed starch substrate which is precipitated on to Whatman 3MM chromatography 315 CopyrIght All
rights
0 of
1976
by
reproduction
Academic in
any
Press.
Inc.
form
raewed.
316
BURDETT,
KIPPS AND WHITEHEAD
paper to give a pink amylase%tilsitive test paper. The method has all the advantages of the specificit: of dyed starch substrates but is simpler, quicker, and offers better resolution than dyed starch in agar methods. EXPERIMENTAL Materials
and Methods
Various protein solutions were obtained for analysis of their amylase isoenzyme patterns. Whole unstimulated saliva was used fresh without further treatment. Hyland Multienzyme reference serum C (1140 IU amylase)’ was obtained as a freeze dried preparation from Travenol Laboratories Ltd. (Thetford, Norfolk), and was made up at 5 times the recommended concentration. Bacterial cr-amylase was obtained from BDH (Poole, Dorset) as a powder with an activity as defined by the manufacturer of 10 EU/mg,l and a-amylase from pig pancreas, also from BDH, was obtained freeze dried with activity of 250 EU/mg.l Isolectric
Focusing
Isoelectric focusing was accomplished using the LKB 2117 Multiphor equipment. The gels were prepared with a final concentration of 5.25% acrylamide of which 5% was the monomer and 0.25% N,N’methylenebisacrylamide (BDH, Poole, Dorset) and with 1.2% (w/v) of Ampholine buffers. (LKB Instruments Ltd, 232 Addington Road, Croydon, Surrey). Equal amounts of Ampholines of pH ranges 5 to 7 and 7 to 9 were used which gave a nominal range of pH 5 to 8.5. Following the addition of 0.5 ml of riboflavin solution (10 mg%, w/v) per 60 ml of gel solution, photopolymerization was allowed to proceed for 2 hr. The cathode strip was soaked in 1% diaminoethane, whilst the anode strip was soaked in 1% acetic acid. Samples were applied directly to the gel by a Partigen dispenser (Hoechst Pharmaceuticals, Hounslow, Middlesex) in amounts ranging from 1 to 10 ~1. They were applied 3 cm from the anode. Saliva was used undiluted, whilst solutions of bacterial and pig pancreas amylases were applied in solutions of 20 and 1 mg/ml, respectively. Power was applied to the gel from the LKB power supply, following the manufacturer’s instructions. After 2lh hr, when the run was terminated, a current of 14 mA at 1150 V was being passed through the gel. 1 Amylase levels are those quoted by the manufacturer. An International Unit (IU) is that amount of enzyme which will catalyze the transformation of one micromole of substrate per minute per liter. One enzyme unit (EU) is that amount of enzyme which liberates one micromol of reducing sugar as maltose per minute at 25°C in acetate buffer pH 4.8 with B.D.H. Lintner soluble starch as substrate.
DETECTION
OF AMYLASE
ISOENZYMES
317
The preparation of the amylase sensitive paper, which was originally developed for detecting saliva stains in forensic science investigations, has been described previously (4). A solution of lyosine red (0.4% w/v) in phosphate buffer, pH 4.9 (15) was precipitated on to Whatman 3MM chromatography paper with ethylenegiycolmonomethylether and zinc sulphate solutions. At the end of the isoelectric focusing run a dry piece of amylasesensitive test paper was placed carefully on top of the gel. The band development was watched by viewing the paper through the gel and when the white bands on the pink background could be seen clearly (lo- 15 min), the paper was wetted with distilled water and carefully peeled off the gel. The paper was then washed in an aqueous solution of sodium hexametaphosphate (20% w/v) to inhibit further amylase action and air dried to provide a permanent record of the amylase isoenzyme pattern. The gel was subsequently stained by an iodometric method (5).
FIG. 1. Amyiase zymogram, following visualisation using the test paper. The grainy ap pearance is due to the texture of the filter paper. Samples A and H: Bacterial amylase 5 /.&I; I: Bacterial amylase, 1 ~1; C: Pig pancreas amylase 1 ~1; E, Hyland serum, 10 ~1; F and G: Human salivary amylase, 1 and 5 ~1, respectively. B: A mixture of bacterial amylase 1 ~1 with pig pancreas amylase. 1 ~4; D: A mixture of pig pancreas amyiase 5 PLI, with a trace of bacterial amyiase. Note that Horizontal distortion of the pH gradient, gives the false impression that the band with the most acidic pI point of samples B and C are different.
318
BURDETT.
KIPPS AND WHITEHEAD
RESULTS AND DISCUSSION
The patterns of the amylaseisoenzymes, as visualized by the amylase test paper are shown in Fig. 1. The zymograms produced by amylase from bacteria, pig pancreas,human saliva, and Hyland serum show a diverse rangeof isoelectric points (about 2.5 pH units). The fine resolving power of the combination of the isoelectric focusing technique and the test paper detection system is demonstratedby the definition of the threeminor bandsseenin the sampleof Hyland serum(sampleE, Fig. 1). Salivary amylase was found to produce at least 10 isoenzyme bands (samplesF and G). The amyloclastic nature of all the bandsseenin Fig. 1 has beenconfirmed by using the iodometric staining method on the same gel. More isoenzyme bands are shown here than have been previously reported (12). The insoluble Phadebas-substratehas been used dispersedin agar gel asan overlay for amylaseisoenzymesseparatedin polyacrylamidegel(12). It is reported that this technique requires a 4 hr incubation period in which to develop the isoenzyme bands from 10 ~1 neat human saliva. With the test paper that is describedhere the isoenzymebandsfrom 5 ~1 of saliva are well resolved in IO- 15 min. This short incubation period has the advantagethat diffusion of the enzyme does not impair resolution The test paper would be equally suitable for detecting amylase isoenzymepatternsproducedby other techniquessuchasagargeland conventional polyacrylamide gel electrophoresis. The test paperis sensitiveto the low levels of amylasefound in normal urine and serum and could thus be used to detect amylase isoenzymes in clinical samples.Modification of the sampleloadingsandthe zymogram incubation time would make the technique suitable for use with samples containing concentrations of amylase either lower or higher than those used here. In conclusion, the test paper can be recommendedas a rapid, simple methodfor the detection of amylaseisoenzymes.It is specific and hasthe additional advantages, unlike Phadebas or the starch methods, of simplicity and of producing a permanentrecord. REFERENCES 1. Boettcher, B., and de la Lande, F. A. (1969)Ausr. J. Exp. Biol. Med. Sci. 47,97-103. 2. Wolf, R, O., Taylor, L. L., Niswander, J. D., and Schwartz, J. T., (1971) Archs. Oral Biol. 16, 1357- 1359. 3. Skude, G. (1971) Humangenetik 12, 255-256. 4. Whitehead, P. H. and Kipps, A. E. (1975)J. Forens. Sci. SW. 15, 36-42. 5. Beottcher, B., and de la Lande, F. A. (1%9)Anai. Biochem. 28, 510-514. 6. Robinson, L. A., Churchill, C. L., and White, T. T. (1970)Biochim. Biophys.Acfa 222, 390-395. 7. Wolf, R. O., and Taylor, L. L. (1968) Amer. J. Clin. Pathol. 38, 871-876.
DETECTION 8. 9. 10. 11. 12. 13. 14. 15.
OF AMYLASE
ISOENZYMES
Doane, W. W. (1967) Sabco. J. 3, 63-68. Joseph, R. R., Olivero, E., and Ressler, N. (1966) Gastroenterology 51, 377-382. Skude, G. (1970) Hereditas 65, 277-284. Rosalki, S. B. (197O)J. C/in. Path. 23, 373-374. Wadstrom, T., and Smyth, C. J. (1973) Sci. Tools 20, 17-21. Wilding, P. (1965) C/in. Chim. Acta 12, 97-104. Longprt, and Hamel, M (1973) C/in. Biochem. 6, 71-81. Sax, S. M., Bridgewater, A. B., and Moore, J. J. (1971) Clin. Chem. 17, 311-315.
319
