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Facts and artifacts in acid phosphatase and esterase localization in disc electrophoresis of human saliva
Archs oral BioL
Vol. 14, pp. 1-6, 1969. Pergamon Press. Printed in Gt. Britain.
FACTS A N D ARTIFACTS IN ACID PHOSPHATASE A N D ESTERASE LOCALIZATION IN DISC ELECTROPHORESIS OF H U M A N SALIVA E. WEINSTEINand I. D. MANDEL Columbia University, School of Dental and Oral Surgery, NewYork, NewYork 10032, U.S.A. Summary--The present study demonstrates that caution must be used in interpreting the results of histochemical staining of polyacrylamide gels for acid phosphatase activity. Nonspecific staining and artifacts present serious hazards and suitable controls must be utilized. The presence of acid phosphatase activity appears to be localized to the Rm.3 area. Esterase staining proved more reliable and several areas of activity were found in both submaxillary and parotid saliva in areas Rm.2-.4, .55, .65 and .85. Several proteins can co-exist in areas which appear on polyacrylamide gels to contain only a single band.
DURING the past several years, important strides have been made in the separation and characterization of the salivary proteins. The technique of disc electrophoresis on polyacrylamide gels is perhaps one of the most important means of studying the properties of these proteins. Through the application of this method, numerous laboratories have reported excellent separation of salivary proteins into as many as 20 bands (MANDEL, 1966; CALDWELLand PIGMAN, 1965). Use of Coomassie blue B as a protein stain has extended the application of the method to examination of as little as 0-025 ml of unconcentrated saliva. The salivary protein profile developed by disc electrophoresis would be more meaningful to investigators if staining procedures could be employed which not only localized, but identified components. There have been several reports on identification of glyco- and lipo-proteins in saliva (GIFFORD and YUKIN, 1965). Saliva is known to contain numerous enzymes, among which are acid phosphatases and esterases. Because of the possible implication of these enzymes in dental health and disease, we have investigated the localization of these components on polyacrylamide gels by standard histochemical staining. METHODS
AND RESULTS
Fresh parotid and submandibular saliva was collected by means of a modified Curby cup and a customized submaxillary collection device (BLOCKand BROTMAN, 1962). The saliva was analyzed by disc electrophoresis according to the technique of ORNSTEIN and DAVIS (1962), using a 7.5 per cent polyacrylamide gel and tris-HC1 buffer o f p H 8.3. A 5 per cent gel was also used on occasion, but proved more difficult ^.o.~. 14/1~A
1
2
E. WEINSTEIN AND I. D . MANDEL
to handle than the 7.5 per cent gel. Current was adjusted to supply 4 mA per gel column for a period of approximately 2 hr (until the marking dye reached the end of the column). Electrophoresis was conducted at 4°C to minimize protein denaturation and to limit diffusion and loss of enzyme activity. For most of the experiments 0.2 ml of saliva was applied to the gel. When a heavier concentration of sample was desired, either 1 mg of lyophilized saliva was reconstituted in 0.2 ml of distilled water or the samples were concentrated by ultrafiltration. At the completion of electrophoresis, the gels were removed under ice, washed briefly in distilled water, and treated by various histochemical means to locate zones of enzyme activity. Because 0.2 ml samples were used for most of the examinations, control columns were stained for proteins with Amidoschwarz 10B rather than with Coomassie blue B.
Acid phosphatase--technique In the initial effort to localize acid phosphatase activity, the Barka technique was employed (BARKA, 1961). This procedure calls for the use of sodium-alpha-naphthyl acid phosphate in a pH 5.0 medium as the substrate and 4 per cent hexazonium pararosaniline for colour development. Since electrophoresis was conducted at pH 8.3, the gels were soaked in a buffer at pH 5- 0 for ½ hr prior to substrate incubation in order to bring the pH of the gels closer to the optimum for enzyme reaction. They were then incubated in the buffer-substrate solution until colour development was noted. RESULTS When saliva samples were treated with a 4 per cent pararosaniline solution as recommended, very dense banding and a heavy background stain were observed. The concentration of pararosaniline was then reduced to 0.4 per cent which resulted in a much lighter background stain. This seemed to be a more desirable technique because it permitted easier identification of the enzyme bands. The numerous parotid and submaxillary saliva samples that were treated in this manner all gave enzyme activity staining in the Rm.3 area (Fig. 1). The term (Rm), relative mobility refers to the position of banding relative to the total length of the gel column. It is computed by dividing the distance the leading edge of the band has travelled from the origin by the total length of the gel column. It is a useful term because it allows comparisons of the bands on one gel column with those of another. N a F is known to inhibit acid phosphatase activity (PEARSE, 1960). The addition of NaF, 10-a M to the substrate solution gave only a suggestion of inhibition of enzyme activity. Because a more complete inhibition was anticipated, these findings prompted a scrutiny of the technique of acid phosphatase localization. In order to test the reliability of the Barka method as a means of localizing acid phosphatase activity, several gels were incubated in a control media which contained all of the ingredients except the substrate. Surprisingly, these control gels also showed banding (See Fig. 1). At this point it was recognized that the Barka technique as outlined for serum enzyme analysis was not suitable for acid phosphase detection on
FACTS AND ARTIFACTS IN ACID PHOSPHATASE
3
gels of salivary secretions. Similar non-specific staining and artifacts have recently been recognized by other investigators and present serious problems in enzyme localization studies (G~n'L~ON, 1966).
Technique-H Several other techniques that have been employed for acid phosphatase activity localization were then tested. The Gomori technique using lead nitrate and sodium beta-glycerophosphate was tried (Go~ORI, 1955). This method resulted in the production of several bands of activity at several locations, including the Rm.2-Rm.3 area. Realizing the non-specificity of the previous technique, control experiments were run which omitted the substrate from the incubation medium. Here, again, the control gels showed similar staining patterns. This indicated very dearly the non-specificity of this reaction. It would appear that careful control procedures would be necessary to make this a meaningful reaction. Technique-Ill The use of diazo-garnet (GBC salt) and alphanaphthyl phosphate as an incubating media was also tried (ALLEN, 1965). This method produced some light banding in the Rm.3 area in both the parotid and submaxillary saliva gels. When the substrate was omitted from the medium, the control gel showed some light banding which became visible several hours after the saliva-containing gels displayed banding. This method seemed to be somewhat more specific than techniques-I and -II, provided suitable time controls were used. Technique-IV Studies have indicated that acid phosphatase is indeed present in saliva (C~trNc~e et aL 1954). In our laboratory no difficulty is encountered in assaying for acid phosphatase using para-nitro-phenyl phosphate in salivary secretions. The sorrowful results experienced in attempting to localize phosphatase activity in acrylamide gel columns by some of the standard techniques merely indicated that a more rigorous test for enzyme detection in gels is needed. Because of the uncertainty of enzyme localization with the several histochemical techniques for acid phosphatase activity, a biochemical localization test was conducted using para-nitrophenyl phosphate. After electrophoresis of parotid and submaxillary saliva, the gels were removed from the apparatus and cut crossways in ¼in. sections. These sections were eluted in distilled water and the eluates incubated with para-nitro-phenyl phosphate. The standard Sigma Colorimetric technique was used for quantitation (Sigma Technical Bull., 1963). The results of these tests indicated that the acid phosphatase activity seemed to be localized to the Rm.2Rm.3 area. Esterase In order to localize esterase activity, the gels were incubated in solutions containing either alpha-naphthyl acetate or butyrate and fast Blue RR salt buffered at pH 7.4 (HUNTERand MAYNARD, 1962; MAYNARDand HUNTER, 1959). The gels were
4
E. WEINSTEINANDI. D. MANDEL
presoaked for ½ hr in a buffer solution at pH 7.0 prior to incubation to adjust the pH to a level where the action proceeds more readily. After an overnight incubation, the gels were examined. Strong banding was evident in the regions of Rm.2-Rm.4 with some lesser activity at Rm.55, .65 and .85 (Fig. 2). Alpha-naphthyl-butyrate tended to give stronger banding than did alphanaphthyl-acetate. Recognizing the need for suitable controls, the substrate was eliminated from the incubating media. No banding was evident in these control gels, thus indicating specificity of the reaction. An attempt was made to determine which, if any, of these esterase bands was cholinesterase. Eserine at 10 -4 M can selectively inhibit cholinesterase activity (MAg~ERT and HUNTER, 1959). When the gels were soaked in eserine, some slight fading was noted around the Rm.2 area, but it was so slight that no definite conclusions could be drawn. A thiocholine iodide technique as a direct colouring method for determining cholinesterase activity was also tried (K_~rtNOVSKYand ROOTS, 1964). The results of this test were also equivocal. DISCUSSION Enzyme localization using histochemical techniques on polyacrylamide gel can give rise to spurious results unless suitable control procedures are used. This was demonstrated in our study of acid phosphatase, where, with some of the procedures, the patterns obtained with and without substrate were very similar. Both the gel electrophoresis techniques for separating the enzymes and the histochemical staining techniques for their localization present difficulties that must be considered when attempts are made to combine the two. The pH in the gel column during electrophoresis may vary from 8.3 to 9.3. Such alterations may affect enzyme activity. The temperature of the gel during electrophoresis may rise to levels which might effect the activity of the enzyme. Conducting the electrophoresis at 4°C may not have been sufficient to prevent this temperature rise within the gel. The chemical composition of the polyacrylamide gel itself may alter the enzymes in saliva. Sodium persulphate which is one of the ingredients of the gel, is known to inhibit some enzyme activity. While we were cautious in adjusting the persulphate concentration to its very minimum, it is possible that this precaution was not sufficient. Riboflavin has been substituted for the persulphate in some gel systems and has been recommended as a means of avoiding this problem (BREWER, 1967). We have not, as yet, explored the use of such systems in this application. The fact that we were able to demonstrate acid phosphatase activity by biochemical means in eluates from the gel would suggest that difficulties inherent in the electrophoresis procedure p e r se do not completely inactivate the enzyme. The amount of activity noted in the eluate, however, was much less than exhibited by parotid and submaxillary saliva examined directly with the para-nitrophenyl phosphate procedure.
FACTS AND ARTIFAC'TS I N ACID PHOSPHATASE
5
Histochemical staining problems could arise from a variety of sources. Impurities in the pararosanaline are not uncommon and might account for the heavy background stain and some of the non-specific reactions (BARICAand A~qDERSON, 1963, p. 243). The spurious results with the Gomori technique may be due to the non-specific binding of heavy metals. This problem has been discussed in numerous histochemical texts (PEARSE, 1960, p. 434; BARI(Aand ANDERSON, 1963, p. 239--241). Although the technique utilizing the G.B.C. salt might be used, with suitable controls, to localize salivary acid phosphatase on acrylamide gels, a truly reliable localization would require techniques other than the standard histochemical methods. The results obtained in localizing esterase activity were somewhat more successful than with acid phosphatase. Control reactions (no substrate added) were almost always negative. Although no biochemical elution tests have been attempted as yet, it would seem that there are multiple areas of esterase activity in both parotid and submaxillary saliva. These observations are supported by the recent studies of PAUNIO, et al., (1966) who found several peaks of esterase activity in human parotid saliva fractionated on DEAE---cellulose and CM--cellulose columns. In all of our experiments we were impressed with the overlapping of esterase and acid-phosphatase activity in the Rm.2-Rm.3 areas. Other experiments have demonstrated that amylase activity and P.A.S. staining are also present in these areas (REILLY, DAVIS and DELLER, 1968; CALDWELLand PIGMAN, 1965; WEINSTEIN and MANDEL, 1968). It would seem that this area is a region rich in enzyme activity and glycoprotein and that numerous proteins can co-exist in what appears to be an individual band. Some of these overlapping bands can be separated by extending the time of electrophoresis. We have not, as yet, applied this technique to enzyme activity localization.
Acknowledgements--This investigation was supported in part by Public Health Service Grant DE-01554 and the Bristol-Myers Co. R6sumg---La pr6sente 6tude montre qu'il faut ~.tre prudent en interpr6tant les r6sultats histochimiques, obtenus h l'aide de gels de polyacrylamide, pour d6terminer l'activit6 en phosphatase acide. Des colorations non-sp6cifiques et des art6facts montrent qu'il faut ~tre tr6s prudent, en utilisant des contr61es appropri6s. L'activit6 en phosphatase acide paralt limit6e b. la r6gion Rm.3. La coloration de l'est6rase parait plus sore et plusieurs zones d'activit6 ont 6t6 trouv6es dans la salive parotidienne et sous-maxillaire, dans les r6gions Rm.2-.4,.55,.65
et .85. Plusieurs prot6ines peuvent co6xister dans des r6gions qui semblent constitu6es une simple bande sur des gels de polyacrylamide. Zusammenfassung--Diese Untersuchung zeigt, wie vorsichtig die Ergebnisse nach histochemischer Anf~irbung yon Polyacrylamid-Gel zum Nachweis der Aktivit~it saurer Phosphatasen interpretiert werden m0ssen. Unspezifische F~irbung und Artefakte ffihren zu schwerwiegenden T~iuschungen; entsprechende Kontrollen sind ni3tig. Die AktivitM saurer Phosphatase scheint im Rm-.3-Gebiet lokalisiert zu sein. Die Esterasef/irbung erwies sich als zuverRissiger. In den Bezirken R m .2-.4, .55, .65 und .85 wurden ffir Submandibular- und Parotisspeichel Esteraseaktivit~it gefunden. In solchen Bezirken, welche auf Polyacrylamid-Gel nur ein einzelnes Band zu enthalten scheinen, k/Snnen mehrere Proteine gemeinsam vorhanden sein.
6
E. WEINSTEINAND I. D. MANDEL REFERENCES
ALL~N, J. 1965. Private Communication, Enzyme Analysis Special Report. Canal Instrument Corp., Bethesda, Md., p. 7. BARKA,T. J. and ANDERSON,P. J. 1963. Histochemistry. Hoeber Medical Division, Harper & Row, New York. BARKA,T. J. 1961. Electrophoretic separation of acid phosphatase in rat liver on polyacrylamid¢ gels. J. Histochem. Cytochem. 9, 542-547. BLOCK,P. L. and BROTMAN,S. 1962. A method of submaxillary saliva collection without cannulation. N.Y. State Dent. J. 28, 116-118. BREWER,J. M. 1967. Artifact produced in disc dectrophoresis by ammonium persulfate. Science 156, 256. CALDWELL,R. C. and PIGMAN,W. 1965. Electrophoresis of human saliva in polyacrylamide gel. Archs Biochem. Biophys. 110, 91-96. CHAUNCEY,H. H., LIONETrI,F., WINER,R. and LISAN'n, V. F. 1954. Enzymes of human saliva. J. dent. Res. 33, 321-334. CURaY, W. A. 1953. Device for collection of human parotid saliva. J. Lab. clin. Meal. 41,493. GIFFORD, G. T. and YVrdN, L. 1965. Protein patterns in human parotid saliva. J. Chromatog. 20, 150-153. GIMPELSON,H. 1966. Electrophoresis application and interpretation. Penn. Dent. Y. 70, 8-10. GOMORI,G. 1955. Methods in Enzymology. (Edited by COLOWtCH,S. and KAPLAN,N.) Vol. 1, p. 138146. Academic Press, New York. HUNTER,R. L. and MAYNARD,E. A. 1962. Esterases in the trigeminal and superior ganglion of the rabbit. J. Histochem. Cytochem. 10, 667. KARNOVSKY,M. J. and ROOTS, L. 1964. A direct-coloring method for cholinesterases. J. Histochem. Cytochem. 12, 219-221. MANDEL,I. D. 1966. Electrophoretic studies of saliva. J. dent. Res. 45, 634-643. MARKERT,C. L. and HUNTER,R. L. 1959. The distribution of esterases in mouse tissues. J. Histochem. Cytochem. 7, 42-49. ORNSTEIN,L. and DAVIES,B. K. 1962. Disc Electrophoresis. Preprinted by Distillation Products Industries, Eastman Kodak Co. PAvhao, I. K., RIEgrdNmq, P. J. and LARMAS,M. A. 1966. Studies on esterolytic enzymes from human parotid saliva. Acta odont, scand. 24, 147-157. PEARSE, A. G. E. 1960. Histochemistry. Second Edition, Little, Brown, Boston. REILLY, P. L., DAVIS, P. S. and DELLER, D. J. 1968. Iron binding properties of saliva. Nature, Lond. 217, 68. Sigma Technical Bulletin, No. 104. 1963. Sigma Chemical Co., St. Louis, Mo. WEINSTErN, E. and MANDEL,]. D. 1968. Characterization of salivary proteins separated by disc electrophoresis. Internat. Ass. for Dent. Res. Preprinted abstracts, 46th General Meeting, Abstract 546.
FACTS AND ARTIFACTSIN ACID PHOSPHATASE
4
FIG. I. Acid phosphatase localization on 7" 5 per cent polyacrylamide gels using 0.4 per cent hexazonium pararosaniline. Gels number 1 and 2 are parotid saliva. Gels number 3 and 4 are submaxillary saliva. The substrate has been omitted from gels 2 and 4. The photograph has been cropped; the arrow indicates the area of sample application and the anode is designated. Note similarity in activity in RM.3 area in both the experimental and control gels.
PLATE 1 A.O.B.f.p. 6
E. WEINSFEINAND I. D. MANDEL
2
¢
4-
FIG. 2. Esterase localization on 7" 5 per cent polyacrylamide gels using alpha-naphthy[ acetate and fast blue R R salt. Gel 1 is parotid saliva and gel 2 is submaxillary saliva. The arrow indicates the area of sample application and the anode is designated. Note activity in RM.2-RM.4 area and at RM.55, .65 and .85.
PLATE 2
FACTS AND ARTIFACTS IN ACID PHOSPHATASE
7
r
2
3
+
FIG. 1. Acid phosphatase localization on 7- 5 per cent polyacrylamide gels using 0-4 per cent hexazonium pararosaniline. Gels number 1 and 2 are parotid saliva. Gels number 3 and 4 are submaxillary saliva. The substrate has been omitted from gels 2 and 4. The photograph has been cropped; the arrow indicates the area of sample application and the anode is designated, Note similarity in activity in RM.3 area in both the experimental and control gels.
PLATE l A.O.B. Lp. 6
E. WEINSFEINAND I. D. MANDEL
2
4FIG. 2. Esterase localization on 7" 5 per cent polyacrylamide gels using alpha-naphthyl acetate and fast blue R R salt. Gel 1 is parotid saliva and gel 2 is submaxillary saliva. The arrow indicates the area of sample application and the anode is designated. Note activity in RM.2-RM.4 area and at RM.55, .65 and .85.
PLATE 2
Vol. 14, pp. 1-6, 1969. Pergamon Press. Printed in Gt. Britain.
FACTS A N D ARTIFACTS IN ACID PHOSPHATASE A N D ESTERASE LOCALIZATION IN DISC ELECTROPHORESIS OF H U M A N SALIVA E. WEINSTEINand I. D. MANDEL Columbia University, School of Dental and Oral Surgery, NewYork, NewYork 10032, U.S.A. Summary--The present study demonstrates that caution must be used in interpreting the results of histochemical staining of polyacrylamide gels for acid phosphatase activity. Nonspecific staining and artifacts present serious hazards and suitable controls must be utilized. The presence of acid phosphatase activity appears to be localized to the Rm.3 area. Esterase staining proved more reliable and several areas of activity were found in both submaxillary and parotid saliva in areas Rm.2-.4, .55, .65 and .85. Several proteins can co-exist in areas which appear on polyacrylamide gels to contain only a single band.
DURING the past several years, important strides have been made in the separation and characterization of the salivary proteins. The technique of disc electrophoresis on polyacrylamide gels is perhaps one of the most important means of studying the properties of these proteins. Through the application of this method, numerous laboratories have reported excellent separation of salivary proteins into as many as 20 bands (MANDEL, 1966; CALDWELLand PIGMAN, 1965). Use of Coomassie blue B as a protein stain has extended the application of the method to examination of as little as 0-025 ml of unconcentrated saliva. The salivary protein profile developed by disc electrophoresis would be more meaningful to investigators if staining procedures could be employed which not only localized, but identified components. There have been several reports on identification of glyco- and lipo-proteins in saliva (GIFFORD and YUKIN, 1965). Saliva is known to contain numerous enzymes, among which are acid phosphatases and esterases. Because of the possible implication of these enzymes in dental health and disease, we have investigated the localization of these components on polyacrylamide gels by standard histochemical staining. METHODS
AND RESULTS
Fresh parotid and submandibular saliva was collected by means of a modified Curby cup and a customized submaxillary collection device (BLOCKand BROTMAN, 1962). The saliva was analyzed by disc electrophoresis according to the technique of ORNSTEIN and DAVIS (1962), using a 7.5 per cent polyacrylamide gel and tris-HC1 buffer o f p H 8.3. A 5 per cent gel was also used on occasion, but proved more difficult ^.o.~. 14/1~A
1
2
E. WEINSTEIN AND I. D . MANDEL
to handle than the 7.5 per cent gel. Current was adjusted to supply 4 mA per gel column for a period of approximately 2 hr (until the marking dye reached the end of the column). Electrophoresis was conducted at 4°C to minimize protein denaturation and to limit diffusion and loss of enzyme activity. For most of the experiments 0.2 ml of saliva was applied to the gel. When a heavier concentration of sample was desired, either 1 mg of lyophilized saliva was reconstituted in 0.2 ml of distilled water or the samples were concentrated by ultrafiltration. At the completion of electrophoresis, the gels were removed under ice, washed briefly in distilled water, and treated by various histochemical means to locate zones of enzyme activity. Because 0.2 ml samples were used for most of the examinations, control columns were stained for proteins with Amidoschwarz 10B rather than with Coomassie blue B.
Acid phosphatase--technique In the initial effort to localize acid phosphatase activity, the Barka technique was employed (BARKA, 1961). This procedure calls for the use of sodium-alpha-naphthyl acid phosphate in a pH 5.0 medium as the substrate and 4 per cent hexazonium pararosaniline for colour development. Since electrophoresis was conducted at pH 8.3, the gels were soaked in a buffer at pH 5- 0 for ½ hr prior to substrate incubation in order to bring the pH of the gels closer to the optimum for enzyme reaction. They were then incubated in the buffer-substrate solution until colour development was noted. RESULTS When saliva samples were treated with a 4 per cent pararosaniline solution as recommended, very dense banding and a heavy background stain were observed. The concentration of pararosaniline was then reduced to 0.4 per cent which resulted in a much lighter background stain. This seemed to be a more desirable technique because it permitted easier identification of the enzyme bands. The numerous parotid and submaxillary saliva samples that were treated in this manner all gave enzyme activity staining in the Rm.3 area (Fig. 1). The term (Rm), relative mobility refers to the position of banding relative to the total length of the gel column. It is computed by dividing the distance the leading edge of the band has travelled from the origin by the total length of the gel column. It is a useful term because it allows comparisons of the bands on one gel column with those of another. N a F is known to inhibit acid phosphatase activity (PEARSE, 1960). The addition of NaF, 10-a M to the substrate solution gave only a suggestion of inhibition of enzyme activity. Because a more complete inhibition was anticipated, these findings prompted a scrutiny of the technique of acid phosphatase localization. In order to test the reliability of the Barka method as a means of localizing acid phosphatase activity, several gels were incubated in a control media which contained all of the ingredients except the substrate. Surprisingly, these control gels also showed banding (See Fig. 1). At this point it was recognized that the Barka technique as outlined for serum enzyme analysis was not suitable for acid phosphase detection on
FACTS AND ARTIFACTS IN ACID PHOSPHATASE
3
gels of salivary secretions. Similar non-specific staining and artifacts have recently been recognized by other investigators and present serious problems in enzyme localization studies (G~n'L~ON, 1966).
Technique-H Several other techniques that have been employed for acid phosphatase activity localization were then tested. The Gomori technique using lead nitrate and sodium beta-glycerophosphate was tried (Go~ORI, 1955). This method resulted in the production of several bands of activity at several locations, including the Rm.2-Rm.3 area. Realizing the non-specificity of the previous technique, control experiments were run which omitted the substrate from the incubation medium. Here, again, the control gels showed similar staining patterns. This indicated very dearly the non-specificity of this reaction. It would appear that careful control procedures would be necessary to make this a meaningful reaction. Technique-Ill The use of diazo-garnet (GBC salt) and alphanaphthyl phosphate as an incubating media was also tried (ALLEN, 1965). This method produced some light banding in the Rm.3 area in both the parotid and submaxillary saliva gels. When the substrate was omitted from the medium, the control gel showed some light banding which became visible several hours after the saliva-containing gels displayed banding. This method seemed to be somewhat more specific than techniques-I and -II, provided suitable time controls were used. Technique-IV Studies have indicated that acid phosphatase is indeed present in saliva (C~trNc~e et aL 1954). In our laboratory no difficulty is encountered in assaying for acid phosphatase using para-nitro-phenyl phosphate in salivary secretions. The sorrowful results experienced in attempting to localize phosphatase activity in acrylamide gel columns by some of the standard techniques merely indicated that a more rigorous test for enzyme detection in gels is needed. Because of the uncertainty of enzyme localization with the several histochemical techniques for acid phosphatase activity, a biochemical localization test was conducted using para-nitrophenyl phosphate. After electrophoresis of parotid and submaxillary saliva, the gels were removed from the apparatus and cut crossways in ¼in. sections. These sections were eluted in distilled water and the eluates incubated with para-nitro-phenyl phosphate. The standard Sigma Colorimetric technique was used for quantitation (Sigma Technical Bull., 1963). The results of these tests indicated that the acid phosphatase activity seemed to be localized to the Rm.2Rm.3 area. Esterase In order to localize esterase activity, the gels were incubated in solutions containing either alpha-naphthyl acetate or butyrate and fast Blue RR salt buffered at pH 7.4 (HUNTERand MAYNARD, 1962; MAYNARDand HUNTER, 1959). The gels were
4
E. WEINSTEINANDI. D. MANDEL
presoaked for ½ hr in a buffer solution at pH 7.0 prior to incubation to adjust the pH to a level where the action proceeds more readily. After an overnight incubation, the gels were examined. Strong banding was evident in the regions of Rm.2-Rm.4 with some lesser activity at Rm.55, .65 and .85 (Fig. 2). Alpha-naphthyl-butyrate tended to give stronger banding than did alphanaphthyl-acetate. Recognizing the need for suitable controls, the substrate was eliminated from the incubating media. No banding was evident in these control gels, thus indicating specificity of the reaction. An attempt was made to determine which, if any, of these esterase bands was cholinesterase. Eserine at 10 -4 M can selectively inhibit cholinesterase activity (MAg~ERT and HUNTER, 1959). When the gels were soaked in eserine, some slight fading was noted around the Rm.2 area, but it was so slight that no definite conclusions could be drawn. A thiocholine iodide technique as a direct colouring method for determining cholinesterase activity was also tried (K_~rtNOVSKYand ROOTS, 1964). The results of this test were also equivocal. DISCUSSION Enzyme localization using histochemical techniques on polyacrylamide gel can give rise to spurious results unless suitable control procedures are used. This was demonstrated in our study of acid phosphatase, where, with some of the procedures, the patterns obtained with and without substrate were very similar. Both the gel electrophoresis techniques for separating the enzymes and the histochemical staining techniques for their localization present difficulties that must be considered when attempts are made to combine the two. The pH in the gel column during electrophoresis may vary from 8.3 to 9.3. Such alterations may affect enzyme activity. The temperature of the gel during electrophoresis may rise to levels which might effect the activity of the enzyme. Conducting the electrophoresis at 4°C may not have been sufficient to prevent this temperature rise within the gel. The chemical composition of the polyacrylamide gel itself may alter the enzymes in saliva. Sodium persulphate which is one of the ingredients of the gel, is known to inhibit some enzyme activity. While we were cautious in adjusting the persulphate concentration to its very minimum, it is possible that this precaution was not sufficient. Riboflavin has been substituted for the persulphate in some gel systems and has been recommended as a means of avoiding this problem (BREWER, 1967). We have not, as yet, explored the use of such systems in this application. The fact that we were able to demonstrate acid phosphatase activity by biochemical means in eluates from the gel would suggest that difficulties inherent in the electrophoresis procedure p e r se do not completely inactivate the enzyme. The amount of activity noted in the eluate, however, was much less than exhibited by parotid and submaxillary saliva examined directly with the para-nitrophenyl phosphate procedure.
FACTS AND ARTIFAC'TS I N ACID PHOSPHATASE
5
Histochemical staining problems could arise from a variety of sources. Impurities in the pararosanaline are not uncommon and might account for the heavy background stain and some of the non-specific reactions (BARICAand A~qDERSON, 1963, p. 243). The spurious results with the Gomori technique may be due to the non-specific binding of heavy metals. This problem has been discussed in numerous histochemical texts (PEARSE, 1960, p. 434; BARI(Aand ANDERSON, 1963, p. 239--241). Although the technique utilizing the G.B.C. salt might be used, with suitable controls, to localize salivary acid phosphatase on acrylamide gels, a truly reliable localization would require techniques other than the standard histochemical methods. The results obtained in localizing esterase activity were somewhat more successful than with acid phosphatase. Control reactions (no substrate added) were almost always negative. Although no biochemical elution tests have been attempted as yet, it would seem that there are multiple areas of esterase activity in both parotid and submaxillary saliva. These observations are supported by the recent studies of PAUNIO, et al., (1966) who found several peaks of esterase activity in human parotid saliva fractionated on DEAE---cellulose and CM--cellulose columns. In all of our experiments we were impressed with the overlapping of esterase and acid-phosphatase activity in the Rm.2-Rm.3 areas. Other experiments have demonstrated that amylase activity and P.A.S. staining are also present in these areas (REILLY, DAVIS and DELLER, 1968; CALDWELLand PIGMAN, 1965; WEINSTEIN and MANDEL, 1968). It would seem that this area is a region rich in enzyme activity and glycoprotein and that numerous proteins can co-exist in what appears to be an individual band. Some of these overlapping bands can be separated by extending the time of electrophoresis. We have not, as yet, applied this technique to enzyme activity localization.
Acknowledgements--This investigation was supported in part by Public Health Service Grant DE-01554 and the Bristol-Myers Co. R6sumg---La pr6sente 6tude montre qu'il faut ~.tre prudent en interpr6tant les r6sultats histochimiques, obtenus h l'aide de gels de polyacrylamide, pour d6terminer l'activit6 en phosphatase acide. Des colorations non-sp6cifiques et des art6facts montrent qu'il faut ~tre tr6s prudent, en utilisant des contr61es appropri6s. L'activit6 en phosphatase acide paralt limit6e b. la r6gion Rm.3. La coloration de l'est6rase parait plus sore et plusieurs zones d'activit6 ont 6t6 trouv6es dans la salive parotidienne et sous-maxillaire, dans les r6gions Rm.2-.4,.55,.65
et .85. Plusieurs prot6ines peuvent co6xister dans des r6gions qui semblent constitu6es une simple bande sur des gels de polyacrylamide. Zusammenfassung--Diese Untersuchung zeigt, wie vorsichtig die Ergebnisse nach histochemischer Anf~irbung yon Polyacrylamid-Gel zum Nachweis der Aktivit~it saurer Phosphatasen interpretiert werden m0ssen. Unspezifische F~irbung und Artefakte ffihren zu schwerwiegenden T~iuschungen; entsprechende Kontrollen sind ni3tig. Die AktivitM saurer Phosphatase scheint im Rm-.3-Gebiet lokalisiert zu sein. Die Esterasef/irbung erwies sich als zuverRissiger. In den Bezirken R m .2-.4, .55, .65 und .85 wurden ffir Submandibular- und Parotisspeichel Esteraseaktivit~it gefunden. In solchen Bezirken, welche auf Polyacrylamid-Gel nur ein einzelnes Band zu enthalten scheinen, k/Snnen mehrere Proteine gemeinsam vorhanden sein.
6
E. WEINSTEINAND I. D. MANDEL REFERENCES
ALL~N, J. 1965. Private Communication, Enzyme Analysis Special Report. Canal Instrument Corp., Bethesda, Md., p. 7. BARKA,T. J. and ANDERSON,P. J. 1963. Histochemistry. Hoeber Medical Division, Harper & Row, New York. BARKA,T. J. 1961. Electrophoretic separation of acid phosphatase in rat liver on polyacrylamid¢ gels. J. Histochem. Cytochem. 9, 542-547. BLOCK,P. L. and BROTMAN,S. 1962. A method of submaxillary saliva collection without cannulation. N.Y. State Dent. J. 28, 116-118. BREWER,J. M. 1967. Artifact produced in disc dectrophoresis by ammonium persulfate. Science 156, 256. CALDWELL,R. C. and PIGMAN,W. 1965. Electrophoresis of human saliva in polyacrylamide gel. Archs Biochem. Biophys. 110, 91-96. CHAUNCEY,H. H., LIONETrI,F., WINER,R. and LISAN'n, V. F. 1954. Enzymes of human saliva. J. dent. Res. 33, 321-334. CURaY, W. A. 1953. Device for collection of human parotid saliva. J. Lab. clin. Meal. 41,493. GIFFORD, G. T. and YVrdN, L. 1965. Protein patterns in human parotid saliva. J. Chromatog. 20, 150-153. GIMPELSON,H. 1966. Electrophoresis application and interpretation. Penn. Dent. Y. 70, 8-10. GOMORI,G. 1955. Methods in Enzymology. (Edited by COLOWtCH,S. and KAPLAN,N.) Vol. 1, p. 138146. Academic Press, New York. HUNTER,R. L. and MAYNARD,E. A. 1962. Esterases in the trigeminal and superior ganglion of the rabbit. J. Histochem. Cytochem. 10, 667. KARNOVSKY,M. J. and ROOTS, L. 1964. A direct-coloring method for cholinesterases. J. Histochem. Cytochem. 12, 219-221. MANDEL,I. D. 1966. Electrophoretic studies of saliva. J. dent. Res. 45, 634-643. MARKERT,C. L. and HUNTER,R. L. 1959. The distribution of esterases in mouse tissues. J. Histochem. Cytochem. 7, 42-49. ORNSTEIN,L. and DAVIES,B. K. 1962. Disc Electrophoresis. Preprinted by Distillation Products Industries, Eastman Kodak Co. PAvhao, I. K., RIEgrdNmq, P. J. and LARMAS,M. A. 1966. Studies on esterolytic enzymes from human parotid saliva. Acta odont, scand. 24, 147-157. PEARSE, A. G. E. 1960. Histochemistry. Second Edition, Little, Brown, Boston. REILLY, P. L., DAVIS, P. S. and DELLER, D. J. 1968. Iron binding properties of saliva. Nature, Lond. 217, 68. Sigma Technical Bulletin, No. 104. 1963. Sigma Chemical Co., St. Louis, Mo. WEINSTErN, E. and MANDEL,]. D. 1968. Characterization of salivary proteins separated by disc electrophoresis. Internat. Ass. for Dent. Res. Preprinted abstracts, 46th General Meeting, Abstract 546.
FACTS AND ARTIFACTSIN ACID PHOSPHATASE
4
FIG. I. Acid phosphatase localization on 7" 5 per cent polyacrylamide gels using 0.4 per cent hexazonium pararosaniline. Gels number 1 and 2 are parotid saliva. Gels number 3 and 4 are submaxillary saliva. The substrate has been omitted from gels 2 and 4. The photograph has been cropped; the arrow indicates the area of sample application and the anode is designated. Note similarity in activity in RM.3 area in both the experimental and control gels.
PLATE 1 A.O.B.f.p. 6
E. WEINSFEINAND I. D. MANDEL
2
¢
4-
FIG. 2. Esterase localization on 7" 5 per cent polyacrylamide gels using alpha-naphthy[ acetate and fast blue R R salt. Gel 1 is parotid saliva and gel 2 is submaxillary saliva. The arrow indicates the area of sample application and the anode is designated. Note activity in RM.2-RM.4 area and at RM.55, .65 and .85.
PLATE 2
FACTS AND ARTIFACTS IN ACID PHOSPHATASE
7
r
2
3
+
FIG. 1. Acid phosphatase localization on 7- 5 per cent polyacrylamide gels using 0-4 per cent hexazonium pararosaniline. Gels number 1 and 2 are parotid saliva. Gels number 3 and 4 are submaxillary saliva. The substrate has been omitted from gels 2 and 4. The photograph has been cropped; the arrow indicates the area of sample application and the anode is designated, Note similarity in activity in RM.3 area in both the experimental and control gels.
PLATE l A.O.B. Lp. 6
E. WEINSFEINAND I. D. MANDEL
2
4FIG. 2. Esterase localization on 7" 5 per cent polyacrylamide gels using alpha-naphthyl acetate and fast blue R R salt. Gel 1 is parotid saliva and gel 2 is submaxillary saliva. The arrow indicates the area of sample application and the anode is designated. Note activity in RM.2-RM.4 area and at RM.55, .65 and .85.
PLATE 2