Antibacterial Effect of Salivary Peroxidases on a Cariogenic Strain of Streptococcus mutans JORMA TENOVUO and MATTI L. E. KNUUTTILA
Institute of Dentistry, University of Turku, Turku, Finland
The antibacterial effect of purified human salivary lactoperoxidase on a cariogenic strain of Streptococcus mutans was demonstrated while another oral peroxidase, probably of leukocytic origin, did not affect the growth. Lactoperoxidase was rapidly adsorbed by bacterial cells indicating the necessity of the contact between the enzyme and the cells before inhibition.
media containing glucose or xylitol or no added carbohydrate. Further experiments were carried out in order to study the necessity of the contact between the enzymes and bacterial cells during inhibition. Materials and Methods
PARTIAL PURIFICATION OF SALIVARY PEROXIDASES. - The partial purification of peroxidases from human whole saliva was performed An antibacterial system in saliva and milk, in principle according to a method described which consists of the enzyme lactoperoxidase, earlier. '4 In the present study, the original macofactor thiocyanate, and hydrogen peroxide, terial comprised 940-ml paraffin-stimulated has been described.' 6 The role of hydrogen whole saliva from 30 persons. The material was peroxide was established byjago and Morrison2 concentrated with an Amicon Ultrafiltration and of thiocyanate ions by Reiter et al.3 Anti- Apparatus TCF-10 (Membrane UM20E) to bacterial activity has been shown against Lacto- 40 ml, desalted with a Sephadex* G-25 column bacillus casei ATCC 4646,' L acidophilus (32 x 5.5 cm) and applied to a DEAE ATCC 4357,8 and L plantarum ATCC 8014.9 celluloset column (Fig 1). The active fractions Some strains of streptococci, e.g. Streptococcus of the DEAE cellulose chromatography were cremoris strain 972,2 S pyogenes,3 S faecalis,'O pooled and focused as described earlier.t4 The and S mutans C-57-1" were also sensitive to the pools (P1 and P2) made after focusing were used lactoperoxidase system. as enzyme preparations for testing the antibacAlthough Morrison et al'2 have shown that terial activity of salivary peroxidases. all lactoperoxidases from different sources are The specific peroxidase activities of the pools immunologically identical, Morrison and were 672 U/mg for P1 and 5.3 U/mg for P2. Steele'3 observed that bovine lactoperoxidase ASSAY PROCEDURES. - Peroxidase activity had only a slight effect on some cariogenic was determined with the guaiacol+ method as strains of S mutans (cariogenic in hamster and suggested by Chance and Maehly.1'The specifrat) while pig lactoperoxidase showed inhibi- ic activity and the guaiacol units were calcution of the same strains of S mutans. lated as described earlier.16 All enzyme assays The objective of the present study was to were performed with a Hitachi Perkin Elmer show whether peroxidases purified from human UV-VIS Spectrophotometer. Total protein was saliva have an antibacterial effect on a cario- estimated by the Lowry method, 17 using bovine genic strain of S mutans grown in various serum albumin§ as a standard. Thiocyanate ions were determined coloriReceived for publication September 28, 1976. metrically according to Powell."8 The producAccepted for publication February 1, 1977. tion of hydrogen peroxide by test bacteria was This investigation was financially supported by a grant from the estimated by measuring the increase in extincFinnish Dental Society. tion at 470 nm due to the peroxidase-catalyzed *Pharmacia Fine Chemicals, Uppsala, Sweden. t230-270 mesh, Schleicher & Schull, Dassel/Kr., Einbeck, Ger- oxidation of guaiacol by hydrogen peroxide. many. CULTIVATION OF S MUTANS. - S mutans (strain tAnalytical grade, BDH Chemicals Ltd., Poole, England. §Sigma Chemical Company, St. Louis, Mo., U.S.A. Ingbritt) was originally isolated from the hu-
J Dent Res 56(12): 1608-1613, December 1977.
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Vol. 56 No. 12 ANTIBACTERIAL EFFECT OF SALIVARY PEROXIDASES ON S MUTANS
1609
10
120
z L-
I-
u
0
n
a-
40
>
L
0.5
0.2
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N
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0.3 Z C]
0.1 70
012 ,,
80
0.1
100 0
50
100 FRACTION NUMBER
0
I
o
O
150
FIG 1. - DEAE-cellulose chromatography of the peroxidases of the supernatant fluid of human whole mouth saliva. Sample: 40 ml of concentrated saliva solution; column: 38 x 1.8 cm; elution buffer: 0.01 M f3-dimethylglutarate buffer, pH 7.2; NaCl gradient: linear from 0 to 0.5 M; mixing volume: 200 + 200 ml; hydrostatic pressure in packing and elution: 120 cm; temperature: 4 C.
oral cavity and donated to this laboratory by Prof. Bo Krasse (University of Goethenburg, Sweden). The biochemical characterization, maintenance and the cultivation of the cells has been described earlier.'9 Some of the experiments were carried out with cells of S mutans which had been maintained for 28 months as stab cultures containing xylitol* (0.25 gm/100 ml) instead of glucose.t Cultures which were first maintained for 22 months in a xylitol medium and thereafter for 6 months in a medium without any added carbohydrates were also used. All cultivations were carried out aerobically (without agitation) at 37 C and the turbidity was measured. The pH was measured potentioman
*Analytical grade, Finnish Sugar Co., Helslinki, Finland. tAnalytical grade, BDH Chemicals Ltd., Poole, England. 1KIetl-Summersson colorimeter (filter no. 62). §Sigma Chemical Company, St. Louis, U.S.A.
metrically with calomel and glass electrodes at 25 C. DETERMINATION OF ANTIBACTERIAL EFFECT OF PEROXIDASES. - The streptococci were first precultured in the above-described media for 15 hours and then transferred to the final medium, containing the components of the lactoperoxidase system. The activities of both commercial lactoperoxidase§ and the main salivary peroxidase (PI) were adjusted to the level normally found in human saliva (3 U/ml).20-21 However, the activity of salivary P2 in the medium was only 0.18 U/ml. The concentrations of SCN- and H202 in the media were 90 mg/l and 2.8 mM respectively. These studies were carried out with organisms grown in all three types of media. Additionally, commercial lactoperoxidase was used at a concentration corresponding to the activity of saliva of persons on a xylitol diet.'6 The dif-
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TENO VUO & KNUUTTILA
1610
ference between the inhibitory action of the lactoperoxidase system and H202 alone was tested using the combinations presented in Figure 3. The cells from 0.5 ml culture were harvested by centrifugation* at 4 C for 10 min at 12,000 x g at the end of the exponential growth phase. The pellet was washed with 0.9% NaCl, with water and then suspended in 30 ml water; 1.0 ml of lactoperoxidase was added to the suspension (activity in the suspension 3 U/ml). The suspension was divided into seven 1 ml aliquots and 1 ml of different concentrations of phosphate buffer, pH 7.2 (0.025, 0.05, 0.1, 0.5, 1.0, and 2.0 M) or of distilled water were added to these seven test tubes. Immediately after centrifugation at 4 C (10 minutes, 12,000 x g) the enzyme activity of the supernatant fluids of the suspensions was determined. The necessity of the contact between the enzyme system and the cells was tested as follows. A cultivation of 50 ml containing lactoperoxidase (1 ml at the concentration of 3 U/ml in the medium) enclosed in a dialyze sac, SCN- (90 mg/l) and H202 (2.8 mmoles), was done. The turbidity and pH were compared to the growth of the cells without the presence of the enzyme and its cofactors. Results
PARTIAL PURIFICATION OF SALIVARY PEROXIDASES. - Ion exchange chromatography on DEAE cellulose yielded two peroxidase peaks (Fig 1). Peak I (PI) was not adsorbed to DEAE cellulose under the conditions used. The perox*Sorvall Superspeed RC-2B centrifuge,
rotor SS-34.
j Dent Res December 1977 idase of the second enzyme peak was eluted from the column at approximately 0.04 M NaCl. Isoelectric focusing showed that the isoelectric point (IP) of salivary lactoperoxidase (PI, cf. 14) is about 8.1. The specific activity of PI was much higher than that of P2. The specific activities at different purification steps are presented in Table 1. COMPARISON OF ANTIMICROBIAL EFFECTS OF SALIVARY PEROXIDASES AND COMMERCIAL LAC TOPEROXIDASE. - The growth curves in the presence of glucose, xylitol, or no added carbohydrate (Fig 2) indicated: (a) The presence of either salivary lactoperoxidase, P,, or the commercial peroxidase resulted in no change in turbidity for the first 2 hours. Thereafter the rate of increase in turbidity rose until the level of the control culture was reached after 7 to 9 hours. (b) The other salivary peroxidase, P,, did not affect the growth, and the growth curve was identical with that found in the control medium. The changes in pH in the media during the growth corresponded quite closely to the alterations of turbidity including the delay phase between 2 and 4 hours growth. (c) The results were similar in spite of the variations in the content of the test media. The only difference was the much lower growth rate of cells in the control culture of xylitol-containing medium or of the medium without any added carbohydrates. The above results, concerning the similarity of the inhibitory effect of human salivary lactoperoxidase and commercial lactoperoxidase, made it possible to study the effect of the enzyme concentration by using only the latter enzyme preparation. In all three media (B, C, and D) (Fig 2), the turbidity remained at nearly
100 80
FIG 2. -The growth of the cells of S mutans in the presence of various peroxidase preparations (A) and in the medium containing either glucose (A, B) or xylitol (C) or without any added carbohydrate (D). Symbols: salivary peroxidase, Pi, *-*; salivary peroxidase, P2, * --; commercial milk lactoperoxidase, V-V (3 U/ml) or A1-z (6 U/ml); control, 0-0. The components of the peroxidase system were simultaneously added to the culture after 2 (A) or 0 (B, C, D) hours of growth.
ct 60
c
Dn '
40 20
1L 30 I0 g
20 10
0
0
2
4
6
2 4 8 10 0 INCUBATION TIME (h)
6
8
10
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Vol. 56 No. 12 ANTIBACTERIAL EFFECT OF SALIVARY PEROXIDASES ON S MUTANS
nents that the ability of these cells to produce H202 themselves was found to be nil. ADSORPTION OF ENZYME TO CELLS. - When the components of the lactoperoxidase system were added to the growth medium and both the activity of the enzyme and the concentration of SCN were followed in the supernatant fluid during the growth, it appeared that the concentration of thiocyanate was not changed but the enzyme activity disappeared almost immediately after the addition of the enzyme. Figure 4 presents the extracellular enzyme activity after adding water (A) or phosphate buffer (pH 7.2) with different concentrations of phosphate to the suspension consisting of the cells (suspended in water) and the enzyme. The commercial lactoperoxidase activity was almost totally absent in buffers up to 0.025 M or 0.050 M phosphate. Thereafter, the activity in the extracellular fluid increased significantly with increasing concentration of phosphate. However, the highest concentration (1.0 M) was slightly less effective than the 0.5 M concentration. The salivary lactoperoxidase (P1) was not adsorbed as effectively as the commercial lactoperoxidase. The phosphate ions released the activity by 28% of the initial activity in the case of commercial lactoperoxidase and by 14% when sali(P1) was used. The activity of vary peroxidase the P2 also disappeared, but it could not be released with the buffers used. These buffers did not have any detectable effect on the activity of the enzymes. The commercial lactoperoxidase enclosed in the dialyze sac did not affect the growth of the cells.
the sarnne level for 5 hours when the lower concentrat ion (3 U/ml) of lactoperoxidase was used. Akfter this time the growth rate began to increas;e. This point of increase occurred later when tthe higher concentration (6 U/ml) was used, a,nd this was again obtained in all media. H20 2 alone also inhibited the growth of the cells bh it the effect deviated from that caused by the lac toperoxidase system as shown in Figure 3. Th e combination of lactoperoxidase and SCN- (caused so mild an inhibition during the 90 mirnutes after the addition of the compo-
100 cr -0 C)
H-
3x:
0
80 60 40 20 0
10 6 8 2 4 INCUBATION TIME (h ) . iOE virdrl-JlacEoperoxicaase fe OI _ - Tp i L.. ne eIIect rlG 3. system
0
(commercial LPO
+ KSCN +
H202, v-Y) and
H202 alone (V-V) on the growth of the cells of S mutans. The control the components.
culture, 0-0, contained
none
1611
of
1L 1~~~~~~~~~~~~L
50 HFIG 4. -The release of the peroxidases, bound to the bacteria cells, after adding water (A) and the various concentrations of phosphate to the cell suspension of S mutans. Salivary peroxidase, Pi, L; commercial milk LPO, 1.
LU
2 100 N
z LLl
130
A
1.0 0.025 Q050 0125 025 0.5 PHOSPHATE CONCENTRATION (M)
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1612
TENO VUO & KNUUTTILA
j Dent Res December 1977 TABLE 1
Discussion
Marked variations in the inhibitory action of lactoperoxidase on different strains of S mutans have been found. 13.20 The results of the present study showed inhibition of a cariogenic strain of S mutans by lactoperoxidase isolated from human saliva. The human salivary lactoperoxidase acted similarly to commercial lactoperoxidase purified from milk. Conversely, the second enzyme peak isolated from human saliva did not have any noticeable inhibitory effect on the same bacteria. This enzyme peak (P2) most likely contained leukocyte peroxidases and/or some heterogenic forms of salivary lactoperoxidase. 14.16 Iwamoto et a122 observed that heterogenic forms of human parotid peroxidases, adsorbed on DEAE cellulose, had a slight inhibitory effect on the growth of L casei and that the minor peroxidase fraction of centrifuged whole saliva, adsorbed on DEAE cellulose, had an antibacterial effect on L plantarum. Purified peroxidase (evidently salivary lactoperoxidase) from human parotid saliva inhibited the growth of L acidophilus.23 All these experiments, including the results of the present study, have shown that human salivary lactoperoxidase inhibits the growth of lactobacilli and a cariogenic strain of S mutans. Other peroxidases isolated from human saliva have a much smaller antibacterial effect on the bacteria tested. Hoogendoorn20 has assumed that inadequate inhibition of streptococci in the mouth is the result of too low a concentration of H202 produced by oral flora. He found that some (but not all) oral strains of S mutans produce H202. The cariogenic strain used in the present study did not have any noticeable H202 production. The lack of sufficient H202 production may be a limiting factor in the growth inhibition of some cariogenic strains of S mutans by the lactoperoxidase system in vivo. Morrison and Steele'3 have presented evidence that it is necessary to have a contact between the enzyme and the cell for the inhibition to occur. However, Hoogendoorn20 achieved inhibition of acid production by streptococci though the direct contact between the enzyme and the cells was impossible. He assumed that an inhibitor with low molecular weight formed by the enzymatic oxidation of thiocyanate was responsible for inhibition. Hogg andJago24 suggested the inhibitor to be HO2SCN or HO3SCN which can be formed also by the nonenzymatic oxidation of thiocyanate by hydrogen peroxide. In the present study, no inhibition was found if
SPECIFIC ACTIVITIES OF DIFFERENT PURIFICATION STEPS OF HUMAN SALIVARY PEROXIDASES.
(1) Centrifuged whole saliva (2) Concentrated sample (3) Pool I (P,) from DEAE chromatography (4) Isoelectric focusing of (3) (5) Pool 11 (P2) from DEAE chromatography (6) Isoelectric focusing of (5)
19.9* 32.6 15.9 672 4.5 5.3
*The values are given as enzvme units/mg protein (U/mg).
contact between the enzyme and the cells was prevented. It was also observed that the enzyme was adsorbed very rapidly to the cells suspended in distilled water and that the activity could be partly returned using increasing concentrations of phosphates. These results indicated that a direct contact between the enzyme and the cells was needed for the inhibition of the growth of the cells. Conclusions
Lactoperoxidase purified from human saliva was shown to be antibacterial against a cariogenic strain of S mutans. In case of S mutans, the inhibition was achieved only when lactoperoxidase and the cells were in contact. Peroxidases adsorbed rapidly to the bacterial cells and partly desorbed when sodium phosphate was added to the reaction mixture. In spite of different growth rates, no differences were found between the cells grown in the presence of glucose, xylitol or no added carbohydrate in the susceptibility to be inhibited by lactoperoxidase system. However, the lactoperoxidase activity of xylitol-consuming persons was more effective than average salivary activity to inhibit the growth of S mutans. The authors wish to exstcid their thaikis to Prof. KaLiko Makinien conlstructive suggestiotis. Thie aLuthors are also ittdebted to Miss Rautti Suomitteii atid NMrs. Aila l ahteenrnakl for their reliable techilical assistatlce. tor
References 1. WRIGHT, R. C., and TRAMER, J.: Factors Influencing the Activity of Cheese Starters. The Role of Milk Peroxidase, J Dairy Res 25:104-118, 1958. 2. JAGO,
G. R.,
and
MORRISON,
M.:
Anti-Streptococcal Activity of Lactoperoxidase III, Proc Soc Exp Biol Med 1 11:585 -588, 1962. 3. REITER, B.; PICKERING, A.; and ORAM, J. D.: in Microbial Inhibitors in Food, by MOLIN, N.,
(ed), Stockholm, Almqvist and Wiksell, 1964, p 297.
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Vol. 56 No. 12 ANTIBACTERIAL EFFECT OF SALIVARY PEROXIDASES ON S MUTANS 4. KLEBANOFF, S. J., and LUEBKE, R. G.: The
Antilactobacillus System of Saliva. Role of Salivary Peroxidase, Proc Soc Exfp Biol Med 118:483-486, 1965. 5. KLEBANOFF, S. J.; CLEM, W. H.; and LUEBKE, R. G.: The Peroxidase-Thiocyanate-Hydrogen Peroxide Antimicrobial System, Biochim BiophysActa 117:63-72, 1966. 6. ORAM, J. D., and REITER, B.: The Inhibition of Streptococci by Lactoperoxidase, Thiocyanate and Hydrogen Peroxide. The Effect of the Inhibitory System on Susceptible and Resistant Strains of Group N Streptococci, J Biochem 100:373-381, 1966. 7. DOGON, I. L.; KERR, A. C.; and AMDUR, B. H.: Characterization of an Antibacterial Factor in Human Parotid Secretions, Active Against Lactobacillus case4, Arch Oral Biol 7:81 -90, 1962. 8. ZELDOW, B. J.: Studies on the Antibacterial Action of Human Saliva. III. Cofactor Requirements of a Lactobacillus BactericidinJ Immunol 90:12-16, 1963. 9. IWAMOTO, Y., and MATSUMURA, T.: Purification
and Characterization of the Salivary Antibacterial Factor (S.A. Factor), Arch Oral Bzol 11:667-676, 1966. 10. KLEBANOFF, S. J.; LUEBKE, R. G.; and CLEM, W. H.: Studies on an Antibacterial System of Saliva, IADR Abstracts No 199, p 86, 1965. 11. HOOGENDOORN, H., and MOORER, W. R.: Lactoperoxidase in the Prevention of Plaque Ac-
cumulation, Gingivitis and Dental Caries (I), Odontol Rety 24:355-366, 1973. 12, MORRISON, M.; ALLEN, P. Z.; BRIGHT, J.; and JAYASINGHE, W.: Lactoperoxidase. V. Identification and Isolation of Lactoperoxidase from Salivary Gland, Arch Biocheem Biophys 111:126-133, 1965. 13. MORRISON, M., and STEELE, W. F.:
Lactoperoxidase, the Peroxidase in the Salivary Gland, in Biology of the Mouth, PERSON, P., (ed), Washington, 1968, pp 89- 110. 14. MAKINEN,
K. K.,
and
TENOVUO,
J.:
Chromatographic Separation of Human Salivary
1613
Peroxidases, Acta Odont Scand 34:141-150, 1976. 15. CHANCE, B., and MAEHLY, A. C.: in Methods in Enzymology, Vol II. Academic Press, New York, 1964, p 764. 16. MXKINEN, K. K.; TENOVUO, J.; and SCHEININ, A.: Xylitol-Induced Elevation of Lactoperoxidase Activity,J Dent Res 55:652 -660, 1976. 17. LOWRY, 0. H.; ROSEBROUGH, N. J.; FARR, A. L.; and RANDALL, R. J.: Protein Measurement with the Folin Phenol Reagent,J Biol Chem 193:265-275, 1951. 18. POWELL, W. N.: Photoelectric Determination of Blood Thiocyanates without Precipitation of Proteins,J Lab Cliin Med 30:1071-1075, 1945. 19. KNUUTTILA, M. L. E., and MXKINEN, K. K.: Purification and Characterization of a Phospha-
Specifically Hydrolyzing p-Nitrophenyl Phosphate from an Oral Strain of Streptococcus mutans, Arch Biochem Biophys 152:685-701, 1972. of The Effect H.: 20. HOOGENDOORN, Lactoperoxidase-Thiocyanate-Hydrogen Peroxide on the Metabolism of Cariogenic Microorganisms In Vitro and In the Oral Cavity, Academic dissertation, Haag, Holland, 1974. tase
21. TENOVUO,
J.,
and
VALTAKOSKI,
J.:
The
Correlation Between Salivary Peroxidase Activity, Salivary Flow Rate, and the Oxidation-Reduction Potentials of Human Saliva and Dental Plaque Suspensions, A cta Odont Scand 34:169-176, 1976. 22. IWAMOTO, Y.; NAKAMURA, R.; TSUNEMITSU, A.;
and MATSUMURA, T.: The Heterogeneity of Human Salivary Peroxidase, Arch Oral Biol 13:1015-1018, 1968. 23. SLOWEY, R. R.; EIDELMAN, S.; and KLEBANOFF, S. J.: Antibacterial Activity of the Purified Peroxidase from Human Parotid Saliva, J Bacteriol 96:575-579, 1968. 24. HOGG, D.McC., and JAGO, G. R.: The Antibacterial Action of Lactoperoxidase. The Nature of the Bacterial Inhibitor, Biochem J 117:779-790, 1970.
Downloaded from jdr.sagepub.com at FLORIDA INTERNATIONAL UNIV on June 19, 2015 For personal use only. No other uses without permission.
Institute of Dentistry, University of Turku, Turku, Finland
The antibacterial effect of purified human salivary lactoperoxidase on a cariogenic strain of Streptococcus mutans was demonstrated while another oral peroxidase, probably of leukocytic origin, did not affect the growth. Lactoperoxidase was rapidly adsorbed by bacterial cells indicating the necessity of the contact between the enzyme and the cells before inhibition.
media containing glucose or xylitol or no added carbohydrate. Further experiments were carried out in order to study the necessity of the contact between the enzymes and bacterial cells during inhibition. Materials and Methods
PARTIAL PURIFICATION OF SALIVARY PEROXIDASES. - The partial purification of peroxidases from human whole saliva was performed An antibacterial system in saliva and milk, in principle according to a method described which consists of the enzyme lactoperoxidase, earlier. '4 In the present study, the original macofactor thiocyanate, and hydrogen peroxide, terial comprised 940-ml paraffin-stimulated has been described.' 6 The role of hydrogen whole saliva from 30 persons. The material was peroxide was established byjago and Morrison2 concentrated with an Amicon Ultrafiltration and of thiocyanate ions by Reiter et al.3 Anti- Apparatus TCF-10 (Membrane UM20E) to bacterial activity has been shown against Lacto- 40 ml, desalted with a Sephadex* G-25 column bacillus casei ATCC 4646,' L acidophilus (32 x 5.5 cm) and applied to a DEAE ATCC 4357,8 and L plantarum ATCC 8014.9 celluloset column (Fig 1). The active fractions Some strains of streptococci, e.g. Streptococcus of the DEAE cellulose chromatography were cremoris strain 972,2 S pyogenes,3 S faecalis,'O pooled and focused as described earlier.t4 The and S mutans C-57-1" were also sensitive to the pools (P1 and P2) made after focusing were used lactoperoxidase system. as enzyme preparations for testing the antibacAlthough Morrison et al'2 have shown that terial activity of salivary peroxidases. all lactoperoxidases from different sources are The specific peroxidase activities of the pools immunologically identical, Morrison and were 672 U/mg for P1 and 5.3 U/mg for P2. Steele'3 observed that bovine lactoperoxidase ASSAY PROCEDURES. - Peroxidase activity had only a slight effect on some cariogenic was determined with the guaiacol+ method as strains of S mutans (cariogenic in hamster and suggested by Chance and Maehly.1'The specifrat) while pig lactoperoxidase showed inhibi- ic activity and the guaiacol units were calcution of the same strains of S mutans. lated as described earlier.16 All enzyme assays The objective of the present study was to were performed with a Hitachi Perkin Elmer show whether peroxidases purified from human UV-VIS Spectrophotometer. Total protein was saliva have an antibacterial effect on a cario- estimated by the Lowry method, 17 using bovine genic strain of S mutans grown in various serum albumin§ as a standard. Thiocyanate ions were determined coloriReceived for publication September 28, 1976. metrically according to Powell."8 The producAccepted for publication February 1, 1977. tion of hydrogen peroxide by test bacteria was This investigation was financially supported by a grant from the estimated by measuring the increase in extincFinnish Dental Society. tion at 470 nm due to the peroxidase-catalyzed *Pharmacia Fine Chemicals, Uppsala, Sweden. t230-270 mesh, Schleicher & Schull, Dassel/Kr., Einbeck, Ger- oxidation of guaiacol by hydrogen peroxide. many. CULTIVATION OF S MUTANS. - S mutans (strain tAnalytical grade, BDH Chemicals Ltd., Poole, England. §Sigma Chemical Company, St. Louis, Mo., U.S.A. Ingbritt) was originally isolated from the hu-
J Dent Res 56(12): 1608-1613, December 1977.
1608 Downloaded from jdr.sagepub.com at FLORIDA INTERNATIONAL UNIV on June 19, 2015 For personal use only. No other uses without permission.
Vol. 56 No. 12 ANTIBACTERIAL EFFECT OF SALIVARY PEROXIDASES ON S MUTANS
1609
10
120
z L-
I-
u
0
n
a-
40
>
L
0.5
0.2
II
50
0.4 > I-
N
z
LU60
0.3 Z C]
0.1 70
012 ,,
80
0.1
100 0
50
100 FRACTION NUMBER
0
I
o
O
150
FIG 1. - DEAE-cellulose chromatography of the peroxidases of the supernatant fluid of human whole mouth saliva. Sample: 40 ml of concentrated saliva solution; column: 38 x 1.8 cm; elution buffer: 0.01 M f3-dimethylglutarate buffer, pH 7.2; NaCl gradient: linear from 0 to 0.5 M; mixing volume: 200 + 200 ml; hydrostatic pressure in packing and elution: 120 cm; temperature: 4 C.
oral cavity and donated to this laboratory by Prof. Bo Krasse (University of Goethenburg, Sweden). The biochemical characterization, maintenance and the cultivation of the cells has been described earlier.'9 Some of the experiments were carried out with cells of S mutans which had been maintained for 28 months as stab cultures containing xylitol* (0.25 gm/100 ml) instead of glucose.t Cultures which were first maintained for 22 months in a xylitol medium and thereafter for 6 months in a medium without any added carbohydrates were also used. All cultivations were carried out aerobically (without agitation) at 37 C and the turbidity was measured. The pH was measured potentioman
*Analytical grade, Finnish Sugar Co., Helslinki, Finland. tAnalytical grade, BDH Chemicals Ltd., Poole, England. 1KIetl-Summersson colorimeter (filter no. 62). §Sigma Chemical Company, St. Louis, U.S.A.
metrically with calomel and glass electrodes at 25 C. DETERMINATION OF ANTIBACTERIAL EFFECT OF PEROXIDASES. - The streptococci were first precultured in the above-described media for 15 hours and then transferred to the final medium, containing the components of the lactoperoxidase system. The activities of both commercial lactoperoxidase§ and the main salivary peroxidase (PI) were adjusted to the level normally found in human saliva (3 U/ml).20-21 However, the activity of salivary P2 in the medium was only 0.18 U/ml. The concentrations of SCN- and H202 in the media were 90 mg/l and 2.8 mM respectively. These studies were carried out with organisms grown in all three types of media. Additionally, commercial lactoperoxidase was used at a concentration corresponding to the activity of saliva of persons on a xylitol diet.'6 The dif-
Downloaded from jdr.sagepub.com at FLORIDA INTERNATIONAL UNIV on June 19, 2015 For personal use only. No other uses without permission.
TENO VUO & KNUUTTILA
1610
ference between the inhibitory action of the lactoperoxidase system and H202 alone was tested using the combinations presented in Figure 3. The cells from 0.5 ml culture were harvested by centrifugation* at 4 C for 10 min at 12,000 x g at the end of the exponential growth phase. The pellet was washed with 0.9% NaCl, with water and then suspended in 30 ml water; 1.0 ml of lactoperoxidase was added to the suspension (activity in the suspension 3 U/ml). The suspension was divided into seven 1 ml aliquots and 1 ml of different concentrations of phosphate buffer, pH 7.2 (0.025, 0.05, 0.1, 0.5, 1.0, and 2.0 M) or of distilled water were added to these seven test tubes. Immediately after centrifugation at 4 C (10 minutes, 12,000 x g) the enzyme activity of the supernatant fluids of the suspensions was determined. The necessity of the contact between the enzyme system and the cells was tested as follows. A cultivation of 50 ml containing lactoperoxidase (1 ml at the concentration of 3 U/ml in the medium) enclosed in a dialyze sac, SCN- (90 mg/l) and H202 (2.8 mmoles), was done. The turbidity and pH were compared to the growth of the cells without the presence of the enzyme and its cofactors. Results
PARTIAL PURIFICATION OF SALIVARY PEROXIDASES. - Ion exchange chromatography on DEAE cellulose yielded two peroxidase peaks (Fig 1). Peak I (PI) was not adsorbed to DEAE cellulose under the conditions used. The perox*Sorvall Superspeed RC-2B centrifuge,
rotor SS-34.
j Dent Res December 1977 idase of the second enzyme peak was eluted from the column at approximately 0.04 M NaCl. Isoelectric focusing showed that the isoelectric point (IP) of salivary lactoperoxidase (PI, cf. 14) is about 8.1. The specific activity of PI was much higher than that of P2. The specific activities at different purification steps are presented in Table 1. COMPARISON OF ANTIMICROBIAL EFFECTS OF SALIVARY PEROXIDASES AND COMMERCIAL LAC TOPEROXIDASE. - The growth curves in the presence of glucose, xylitol, or no added carbohydrate (Fig 2) indicated: (a) The presence of either salivary lactoperoxidase, P,, or the commercial peroxidase resulted in no change in turbidity for the first 2 hours. Thereafter the rate of increase in turbidity rose until the level of the control culture was reached after 7 to 9 hours. (b) The other salivary peroxidase, P,, did not affect the growth, and the growth curve was identical with that found in the control medium. The changes in pH in the media during the growth corresponded quite closely to the alterations of turbidity including the delay phase between 2 and 4 hours growth. (c) The results were similar in spite of the variations in the content of the test media. The only difference was the much lower growth rate of cells in the control culture of xylitol-containing medium or of the medium without any added carbohydrates. The above results, concerning the similarity of the inhibitory effect of human salivary lactoperoxidase and commercial lactoperoxidase, made it possible to study the effect of the enzyme concentration by using only the latter enzyme preparation. In all three media (B, C, and D) (Fig 2), the turbidity remained at nearly
100 80
FIG 2. -The growth of the cells of S mutans in the presence of various peroxidase preparations (A) and in the medium containing either glucose (A, B) or xylitol (C) or without any added carbohydrate (D). Symbols: salivary peroxidase, Pi, *-*; salivary peroxidase, P2, * --; commercial milk lactoperoxidase, V-V (3 U/ml) or A1-z (6 U/ml); control, 0-0. The components of the peroxidase system were simultaneously added to the culture after 2 (A) or 0 (B, C, D) hours of growth.
ct 60
c
Dn '
40 20
1L 30 I0 g
20 10
0
0
2
4
6
2 4 8 10 0 INCUBATION TIME (h)
6
8
10
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Vol. 56 No. 12 ANTIBACTERIAL EFFECT OF SALIVARY PEROXIDASES ON S MUTANS
nents that the ability of these cells to produce H202 themselves was found to be nil. ADSORPTION OF ENZYME TO CELLS. - When the components of the lactoperoxidase system were added to the growth medium and both the activity of the enzyme and the concentration of SCN were followed in the supernatant fluid during the growth, it appeared that the concentration of thiocyanate was not changed but the enzyme activity disappeared almost immediately after the addition of the enzyme. Figure 4 presents the extracellular enzyme activity after adding water (A) or phosphate buffer (pH 7.2) with different concentrations of phosphate to the suspension consisting of the cells (suspended in water) and the enzyme. The commercial lactoperoxidase activity was almost totally absent in buffers up to 0.025 M or 0.050 M phosphate. Thereafter, the activity in the extracellular fluid increased significantly with increasing concentration of phosphate. However, the highest concentration (1.0 M) was slightly less effective than the 0.5 M concentration. The salivary lactoperoxidase (P1) was not adsorbed as effectively as the commercial lactoperoxidase. The phosphate ions released the activity by 28% of the initial activity in the case of commercial lactoperoxidase and by 14% when sali(P1) was used. The activity of vary peroxidase the P2 also disappeared, but it could not be released with the buffers used. These buffers did not have any detectable effect on the activity of the enzymes. The commercial lactoperoxidase enclosed in the dialyze sac did not affect the growth of the cells.
the sarnne level for 5 hours when the lower concentrat ion (3 U/ml) of lactoperoxidase was used. Akfter this time the growth rate began to increas;e. This point of increase occurred later when tthe higher concentration (6 U/ml) was used, a,nd this was again obtained in all media. H20 2 alone also inhibited the growth of the cells bh it the effect deviated from that caused by the lac toperoxidase system as shown in Figure 3. Th e combination of lactoperoxidase and SCN- (caused so mild an inhibition during the 90 mirnutes after the addition of the compo-
100 cr -0 C)
H-
3x:
0
80 60 40 20 0
10 6 8 2 4 INCUBATION TIME (h ) . iOE virdrl-JlacEoperoxicaase fe OI _ - Tp i L.. ne eIIect rlG 3. system
0
(commercial LPO
+ KSCN +
H202, v-Y) and
H202 alone (V-V) on the growth of the cells of S mutans. The control the components.
culture, 0-0, contained
none
1611
of
1L 1~~~~~~~~~~~~L
50 HFIG 4. -The release of the peroxidases, bound to the bacteria cells, after adding water (A) and the various concentrations of phosphate to the cell suspension of S mutans. Salivary peroxidase, Pi, L; commercial milk LPO, 1.
LU
2 100 N
z LLl
130
A
1.0 0.025 Q050 0125 025 0.5 PHOSPHATE CONCENTRATION (M)
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1612
TENO VUO & KNUUTTILA
j Dent Res December 1977 TABLE 1
Discussion
Marked variations in the inhibitory action of lactoperoxidase on different strains of S mutans have been found. 13.20 The results of the present study showed inhibition of a cariogenic strain of S mutans by lactoperoxidase isolated from human saliva. The human salivary lactoperoxidase acted similarly to commercial lactoperoxidase purified from milk. Conversely, the second enzyme peak isolated from human saliva did not have any noticeable inhibitory effect on the same bacteria. This enzyme peak (P2) most likely contained leukocyte peroxidases and/or some heterogenic forms of salivary lactoperoxidase. 14.16 Iwamoto et a122 observed that heterogenic forms of human parotid peroxidases, adsorbed on DEAE cellulose, had a slight inhibitory effect on the growth of L casei and that the minor peroxidase fraction of centrifuged whole saliva, adsorbed on DEAE cellulose, had an antibacterial effect on L plantarum. Purified peroxidase (evidently salivary lactoperoxidase) from human parotid saliva inhibited the growth of L acidophilus.23 All these experiments, including the results of the present study, have shown that human salivary lactoperoxidase inhibits the growth of lactobacilli and a cariogenic strain of S mutans. Other peroxidases isolated from human saliva have a much smaller antibacterial effect on the bacteria tested. Hoogendoorn20 has assumed that inadequate inhibition of streptococci in the mouth is the result of too low a concentration of H202 produced by oral flora. He found that some (but not all) oral strains of S mutans produce H202. The cariogenic strain used in the present study did not have any noticeable H202 production. The lack of sufficient H202 production may be a limiting factor in the growth inhibition of some cariogenic strains of S mutans by the lactoperoxidase system in vivo. Morrison and Steele'3 have presented evidence that it is necessary to have a contact between the enzyme and the cell for the inhibition to occur. However, Hoogendoorn20 achieved inhibition of acid production by streptococci though the direct contact between the enzyme and the cells was impossible. He assumed that an inhibitor with low molecular weight formed by the enzymatic oxidation of thiocyanate was responsible for inhibition. Hogg andJago24 suggested the inhibitor to be HO2SCN or HO3SCN which can be formed also by the nonenzymatic oxidation of thiocyanate by hydrogen peroxide. In the present study, no inhibition was found if
SPECIFIC ACTIVITIES OF DIFFERENT PURIFICATION STEPS OF HUMAN SALIVARY PEROXIDASES.
(1) Centrifuged whole saliva (2) Concentrated sample (3) Pool I (P,) from DEAE chromatography (4) Isoelectric focusing of (3) (5) Pool 11 (P2) from DEAE chromatography (6) Isoelectric focusing of (5)
19.9* 32.6 15.9 672 4.5 5.3
*The values are given as enzvme units/mg protein (U/mg).
contact between the enzyme and the cells was prevented. It was also observed that the enzyme was adsorbed very rapidly to the cells suspended in distilled water and that the activity could be partly returned using increasing concentrations of phosphates. These results indicated that a direct contact between the enzyme and the cells was needed for the inhibition of the growth of the cells. Conclusions
Lactoperoxidase purified from human saliva was shown to be antibacterial against a cariogenic strain of S mutans. In case of S mutans, the inhibition was achieved only when lactoperoxidase and the cells were in contact. Peroxidases adsorbed rapidly to the bacterial cells and partly desorbed when sodium phosphate was added to the reaction mixture. In spite of different growth rates, no differences were found between the cells grown in the presence of glucose, xylitol or no added carbohydrate in the susceptibility to be inhibited by lactoperoxidase system. However, the lactoperoxidase activity of xylitol-consuming persons was more effective than average salivary activity to inhibit the growth of S mutans. The authors wish to exstcid their thaikis to Prof. KaLiko Makinien conlstructive suggestiotis. Thie aLuthors are also ittdebted to Miss Rautti Suomitteii atid NMrs. Aila l ahteenrnakl for their reliable techilical assistatlce. tor
References 1. WRIGHT, R. C., and TRAMER, J.: Factors Influencing the Activity of Cheese Starters. The Role of Milk Peroxidase, J Dairy Res 25:104-118, 1958. 2. JAGO,
G. R.,
and
MORRISON,
M.:
Anti-Streptococcal Activity of Lactoperoxidase III, Proc Soc Exp Biol Med 1 11:585 -588, 1962. 3. REITER, B.; PICKERING, A.; and ORAM, J. D.: in Microbial Inhibitors in Food, by MOLIN, N.,
(ed), Stockholm, Almqvist and Wiksell, 1964, p 297.
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Vol. 56 No. 12 ANTIBACTERIAL EFFECT OF SALIVARY PEROXIDASES ON S MUTANS 4. KLEBANOFF, S. J., and LUEBKE, R. G.: The
Antilactobacillus System of Saliva. Role of Salivary Peroxidase, Proc Soc Exfp Biol Med 118:483-486, 1965. 5. KLEBANOFF, S. J.; CLEM, W. H.; and LUEBKE, R. G.: The Peroxidase-Thiocyanate-Hydrogen Peroxide Antimicrobial System, Biochim BiophysActa 117:63-72, 1966. 6. ORAM, J. D., and REITER, B.: The Inhibition of Streptococci by Lactoperoxidase, Thiocyanate and Hydrogen Peroxide. The Effect of the Inhibitory System on Susceptible and Resistant Strains of Group N Streptococci, J Biochem 100:373-381, 1966. 7. DOGON, I. L.; KERR, A. C.; and AMDUR, B. H.: Characterization of an Antibacterial Factor in Human Parotid Secretions, Active Against Lactobacillus case4, Arch Oral Biol 7:81 -90, 1962. 8. ZELDOW, B. J.: Studies on the Antibacterial Action of Human Saliva. III. Cofactor Requirements of a Lactobacillus BactericidinJ Immunol 90:12-16, 1963. 9. IWAMOTO, Y., and MATSUMURA, T.: Purification
and Characterization of the Salivary Antibacterial Factor (S.A. Factor), Arch Oral Bzol 11:667-676, 1966. 10. KLEBANOFF, S. J.; LUEBKE, R. G.; and CLEM, W. H.: Studies on an Antibacterial System of Saliva, IADR Abstracts No 199, p 86, 1965. 11. HOOGENDOORN, H., and MOORER, W. R.: Lactoperoxidase in the Prevention of Plaque Ac-
cumulation, Gingivitis and Dental Caries (I), Odontol Rety 24:355-366, 1973. 12, MORRISON, M.; ALLEN, P. Z.; BRIGHT, J.; and JAYASINGHE, W.: Lactoperoxidase. V. Identification and Isolation of Lactoperoxidase from Salivary Gland, Arch Biocheem Biophys 111:126-133, 1965. 13. MORRISON, M., and STEELE, W. F.:
Lactoperoxidase, the Peroxidase in the Salivary Gland, in Biology of the Mouth, PERSON, P., (ed), Washington, 1968, pp 89- 110. 14. MAKINEN,
K. K.,
and
TENOVUO,
J.:
Chromatographic Separation of Human Salivary
1613
Peroxidases, Acta Odont Scand 34:141-150, 1976. 15. CHANCE, B., and MAEHLY, A. C.: in Methods in Enzymology, Vol II. Academic Press, New York, 1964, p 764. 16. MXKINEN, K. K.; TENOVUO, J.; and SCHEININ, A.: Xylitol-Induced Elevation of Lactoperoxidase Activity,J Dent Res 55:652 -660, 1976. 17. LOWRY, 0. H.; ROSEBROUGH, N. J.; FARR, A. L.; and RANDALL, R. J.: Protein Measurement with the Folin Phenol Reagent,J Biol Chem 193:265-275, 1951. 18. POWELL, W. N.: Photoelectric Determination of Blood Thiocyanates without Precipitation of Proteins,J Lab Cliin Med 30:1071-1075, 1945. 19. KNUUTTILA, M. L. E., and MXKINEN, K. K.: Purification and Characterization of a Phospha-
Specifically Hydrolyzing p-Nitrophenyl Phosphate from an Oral Strain of Streptococcus mutans, Arch Biochem Biophys 152:685-701, 1972. of The Effect H.: 20. HOOGENDOORN, Lactoperoxidase-Thiocyanate-Hydrogen Peroxide on the Metabolism of Cariogenic Microorganisms In Vitro and In the Oral Cavity, Academic dissertation, Haag, Holland, 1974. tase
21. TENOVUO,
J.,
and
VALTAKOSKI,
J.:
The
Correlation Between Salivary Peroxidase Activity, Salivary Flow Rate, and the Oxidation-Reduction Potentials of Human Saliva and Dental Plaque Suspensions, A cta Odont Scand 34:169-176, 1976. 22. IWAMOTO, Y.; NAKAMURA, R.; TSUNEMITSU, A.;
and MATSUMURA, T.: The Heterogeneity of Human Salivary Peroxidase, Arch Oral Biol 13:1015-1018, 1968. 23. SLOWEY, R. R.; EIDELMAN, S.; and KLEBANOFF, S. J.: Antibacterial Activity of the Purified Peroxidase from Human Parotid Saliva, J Bacteriol 96:575-579, 1968. 24. HOGG, D.McC., and JAGO, G. R.: The Antibacterial Action of Lactoperoxidase. The Nature of the Bacterial Inhibitor, Biochem J 117:779-790, 1970.
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