~rckr orulBwl. Vol. 24.pp.651 to 662 Pergamon Press Lfd 1979.Printed m Great Britain
ENZYMIC
ACTIVITIES IN THE AQUEOUS OF HUMAN DENTAL PLAQUE A.
TATEVOSSIAN*
PHASE
and E. NEWBRIJN
Dept. Oral Medicine and Hospital Dentistry, University of California, San Francisco, CA 94143, U.S.A.
Summary-The activity of some enzymes in human dental plaque fluid was measured. Plaque of 24 h growth was collected from 9 adults, and plaque of indeterminate age obtained from children attending the Pedodontic Clinic at UCSF. Resting parotid saliva samples from the adult subjects were also assayed. Amylase activity was lower and lysozyme activity higher in plaque fluid than in saliva. The optimum pH for both enzymes in both fluids was between 6.5 and 7.5. Salivary and plaque fluid amylase was not inactivated by short incubations with two bacterial proteases. Trace levels of acid phosphatase and low alkaline phosphatase activity were detectable in plaque fluid, the latter being labile during incubation and storage. No glucosyltransferase activity synthesizing insoluble polysaccharide from sucrose was detectable in plaque fluid. Protease activity against bovine serum albumin and human serum IgA, IgG and IgM was detectable in plaque fluid.
INTRODUCTION The protein
components
of dental
plaque
constitute
three pools: bacterial cell proteins and enzymes closely associated with the bacterial cell walls; denatured and structural matrix proteins; and soluble proteins in the aqueous extracellular phase. The aqueous phase has hitherto been studied by extraction (Silverman and Kleinberg, 1967; Fox and Dawes, 1970a) and yields several enzyme activities. Among these are: amylase (Slider, 1969; Holt, 1975), decarboxylases (Hayes and Hyatt, 1974), L-fucosidase (Shizukuishi et nl., 1977), hyaluronidases (Slider, 1969; Nord and Siider, 1972), lysozyme (Sader et nl., 1971; Holt, 1975) neuraminidase (Tuyau and Sims, 1975), alkaline phosphatase (Paunio, 1969, 1970), acid and alkaline pyrophosphatase (Vreven and Frank, 1974), “sucrolytic” enzymes (Birkhed, 1975; Aksnes, 1976) including dextransucrase, levan sucrase and invertase activities (Birkhed and Dahlqvist, 1975) and proteases (SGder, 1969, 1972). The form and reactivity, in the native state, of the components obtained from plaque by extraction remains a matter for conjecture as the pH, ionic strength and composition of the plaque aqueous phase is changed during the extraction procedure; these variables affect the extraction of protein from plaque (Fox and Dawes, 1979b). Effective concentrations of the extractants from the intact plaque cannot be obtained. The development of a method to separate plaque fluid by centrifugation (Tatevossian and Gou-ld, 1976a) allows quantitation of the average activity of some enzymes in the soluble phase of dental plaque. MATERIALS AND METHODS Human dental plaquefluid
(a) Plaque was collected with perspex spatulae at least 45 min after breakfast from 9 adult volunteers * Present address: Physiology Department, University College, Card8 CF1 1XL, Wales, U.K. 0.1,.2419%B
who had not brushed their teeth in the previous 24 h. All available surfaces were sampled, except the lingual lower anterior region and areas of gingival inflammation. Contamination by saliva was minimized by instructing the subject to swallow. The procedure for pooling the plaque and separating the aqueous phase was as previously described (Tatevossian and Gould, 1976a), or modified, to avoid freeze-thawing the plaques, by maintaining the collected samples at 5°C throughout the procedure. (b) Plaque of itdeterminate age was collected from children aged 3-14 yr attending the Pedodontic Clinic for treatment, and was processed similarly. Resting parotid saliva
Resting parotid saliva was collected into glass capillary tubes directly from the parotid duct opening of the adult subjects. The samples were maintained at 5°C for short periods before use. Enzyme assays 4-glucanohydrolase, Amylase (g-1,4 glucan E.C.3.2.1.1.). The activity was measured by a modifi-
cation of the method described by Jamieson, Pruitt and Caldwell (1969). Samples (1 ~1 of saliva or plaque fluid) were incubated with 50 ~1 of substrate (1 per cent starch in 0.02 M phosphate buffer, pH 6.9, 6 mM NaCl) in a total volume of 100~1. Enzyme reaction which proceeded at room temperature was terminated (after 5 min for saliva, 10 min for plaque fluid) by the addition of 100 ~1 dinitrosalicylic acid (DNS) reagent (Hosteller et al., 1951). After boiling (5 min) and dilution, the reducing activity was read at 470nm and compared to maltose standards. The results were expressed as the rate of release of reducing activity per ~1 of plaque fluid or saliva. Lysozyme (mucopeptide N-acetylmuramylhydrolase, E.C.3.2.1.17). The method of Jollb et al. (1965) was used. Micrococcus luteus (ATCC 4698) was grown as a
batch culture in Enriched Nutrient Broth (American Type Culture Collection, 1972). Growth was increased
A. Tatevossian and E. Newbrun
658
at 37°C which was used in preference to the recommended temperature of 20°C. The cells were sedimented at SOOOg, 5°C and washed three times; an acetone powder was separated by filtration, dried and stored at 5°C. The acetone powder was suspended in buffer (0.067 M sodium phosphate pH 6.2:1 per cent sodium chloride, in the ratio 9:l) to an absorbancy of about 0.7 at 540 nm, then 1 ml was incubated with either 2 ~1 saliva, 1 ~1 plaque fluid, or standards (hen egg lysozyme) for 0.5 h at 37°C. The samples were vortexed at 1Omin intervals and their absorbance, which decreased with lysis of the suspension, was reread. Enzyme activity was expressed as the concentration of hen egg lysozyme standard with equivalent activity after identical incubation conditions. Synthesis of insoluble carbohydrate polymers (u-1,32-glucosyltransferase aCtiUity)
(1976) was used. Substrate (100 pg protein/ml in 0.15 M sodium chloride) was adsorbed onto polystyrene Petri dishes during 30min contact at room temperature. Unbound substrate was rinsed off with 0.15 M sodium chloride. Plaque fluid (l-5 ~1) was placed on the plate and incubated at 37°C for 15 min in a humid chamber. The zones of solubilized substrate were visualized in the dried and washed Petri dishes by the condensation of steam from a water bath, the droplet size difference clearly demarcating the zone of digested substrate. Materials Egg white lysozyme, Protease VI (from Streptomyces griseus) and Protease VIII (from Bacillus subtilis) were obtained from Sigma Chemical Co., St.
Louis. Human serum immunoglobulin IgA, IgG and IgM were purchased from Cappel Laboratories, A method modified from that of Robrish, Reid and Cochransville, Pa. Enriched Nutrient Broth was prepared according to the American Type Culture ColKrichevsky (1972) was used to measure the production of insoluble glucan. Plaque fluid samples (5 ~1) lection (1972). were incubated for up to 3 h at 37°C in 100~1 1OmM sucrose in 0.025 M potassium phosphate buffer, pH RESULTS 6.8; 20 mM sodium fluoride; and 20 &i/ml U-i4CAmylase sucrose. At appropriate time intervals, 10 ~1 fractions The activity of amylase in plaque fluid was lower were filtered through 0.45 pm Millipore filters and than in an equivalent volume of resting saliva from washed twice with 10 ml of distilled water. The radioactivity incorporated into insoluble polymer was the parotid duct (Table 1). The pH activity profile measured by liquid scintillation spectrometry. The from both fluids was similar when they were incusupernatant fluid from a 24 h culture of Streptococcus bated in 0.1 M imidazole/succinate buffer in the range of pH 4.5 to 8.5; peak activity was in the range of pH mutans serotype e (S27 A-l, by courtesy of C. Hoover) 6.5 to 7.0. Amylase in plaque fluid and saliva was not in Brain Heart Infusion (Difco) was used as a positive inactivated by two bacterial proteases (Protease Type control for glucosyltransferase activity, the procedures and volumes being the same as those for plaque fluid VI and Type VIII) under the test conditions (Table 2). These proteases hydrolysed bovine serum albumin, assays. IgA and IgM and partially inactivated invertase (see Alkaline and acid phosphatase (orthophosphoric below).
ghCan:D-fruCtoSf?
monoester phosphohydrolase, E.C.3.1.3.1 and 3.1.3.2)
The fluorometric method of Vaughan, Guilbault and Hackney (1971) was used. Naphthol AS-B1 phosphate in ethylene glycol (0.3 x 10m4M) was incubated for up to 24 h at 37°C with 1 ~1 samples and 3.0 ml buffer (0.1 M tris, pH 9.0 for alkaline phosphatase; 0.1 M citrate, pH 5.5, for acid phosphatase). Fluorescence was measured with a Turner fluorimeter (lex 405 nm, lem 515 nm). The results were expressed as the rate of substrate converted per ~1 of plaque fluid. “Sucrase” (Inuertase) activity (B-D-fructofuranoside fructohydrolase, E.C.3.2.1.26)
Samples (2 ~1) were incubated at 37°C in 1 ml 0.125 M sucrose buffered with 0.025 M phosphate, pH 6.8. Fractions of lOO$ were removed from the mixture before, and at appropriate intervals during, incubation. Each fraction was added to lOO$ DNS reagent and the reducing activity compared to maltose standards. The glucose content was also estimated by the Glucostat (Sigma) assay according to the recommended procedures. The results were expressed as the rate of release of reducing activity per ~1 of plaque fluid. Proteolytic E.C.3.4.4)
activity
(peptidyl
peptide
hydrolases,
The method of Elwing, Nilsson and Ouchterlony
Lysozyme
The activity of lysozyme in plaque fluid was greater than in an equivalent volume of resting parotid saliva (Table 1). The optimum pH of egg white, salivary and plaque fluid lysozyme was similar, in the range of pH 6.5 to 7.5 (Fig. l), as measured by a decreased absorbance at 540 nm with increased lysis. Synthesis of insoluble carbohydrate polymer No glucosyltransferase activity was detectable in plaque fluid, although significant amounts could be detected in the control samples of supernatant of broth culture from Strep. mutans S27 A-l, tested concurrently. The plaque fluid samples tested were unfrozen and kept at 5°C for less than 1 h before assay. Acid and alkaline phosphatase
Trace acid phosphatase activity was demonstrable in plaque fluid, at the detection limit of the method (Table 1). Formation of product did not significantly increase during the 30min to 24 h incubation time. Alkaline phosphatase activity was demonstrable in plaque fluid (Table 1). The activity diminished with increasing incubation time and was less than 20 per cent after 24 h incubation (Table 3), with excess substrate remaining. Purified calf alkaline phosphatase was stable during incubations up to 60 min, at a time when plaque fluid enzyme activity had fallen an aver-
Plaque
fluid enzymes
659
Table 1. Enzymic activity in plaque fluid and resting parotid saliva. Mean f SD (n)
24 h growth
Plaque fluid Indeterminate age
18.2 k 10.6* (17)
15.5 + 4.1* (9)
221.8 k 73.1* (20)
385 f lOl* (6)
Enzyme Amylase (pg maltose/$/min) Lysozyme (pg/ml hen egg lysozyme equivalent) l,~oluble carbohydrate polymer synthesis Acid phosphatase Alkaline phosphatase (n mole substrate converted/&min)
Trace (13) 0.248 i 0.024 (7)
17.3 t_ 8.9* (26) 89.9 + 5.3 (22) 259.5 k 105.1* (26) 65.3 If: 32.6(15)
-
ND (6)
Resting parotid saliva
Combined
ND (6)
Trace (5) 0.267 k 0.081 (12)
Trace (18) 0.260 _t 0.070 (19)
= not detectable. * p < 0.001 when compared with saliva by the Student t test.
ND
age of 15 per cent (range 8.2 to 26.9 per cent) below activity at 30 min (Table 3). By freeze-thawing plaque fluid samples 4 to 6 times and storing them at -20°C f-Jr about 2 weeks, the activity found after 30min incubation was lower than in unstored samples (Table 3, c vs b). These samples showed an average reduction in activity of 38 per cent when incubation continued for 120 min (Table 3, c at 30 min vs 120 min). “Sucrase” activity “Sucrase” activity, detected as the release of reducing sugar, was present in plaque fluid (Table 4). The activity was diminished by the addition of glucosyltransferase purified from Strep. sunguis culture fluid, and by bacterial proteases. Attempts to differentiate invertase activity from that of other enzymes capable of releasing reducing groups were unsuccessful because of the presence of substantial invertase activity in the commercially available glucose oxidase used in assay kits for glucose. This problem has not been reported by previous authors and may not be apparent if sucrose is not included in the blanks and standards incubated with glucose oxidase. Protease activity
DISCUSSION
Amylase and sucrase
Jacobsen, Melvaer and Hensten-Petersen (1972) and Birkhed (1974) found that the activity of amylase in dental plaque extracts was lower than in saliva. Similarly, the amylase activity in plaque fluid was about 25 per cent of that found in the resting parotid saliva from our subjects. The level found in plaque fluid indicated that parotid salivary contamination of the plaque used as a source of plaque fluid was limited and relatively reproducible. Submandibular and sublingual saliva also have lower amylase activity than the parotid secretion (Schneyer, 1956). The activity in plaque fluid may have originated from saliva during the formation of the plaque since amylase activity is demonstrable in undisturbed dental plaque in situ (Neff, 1967). The optimum pH and resistance to inactivation of the amylase suggested that this activity could have been derived from saliva. However, certain microorganisms in plaque have also been demonstrated to hydrolyse starch (Ruby and Gerencser, 1974). Later work on the electrophoretic mobility of the activity
The presence of proteolytic activity was demonstrable in plaque fluid against bovine serum albumin and human serum IgA, IgG and IgM substrates. Protease VI and VIII (1 mg/ml) gave zones of lysis against bovine serum albumin, IgA and IgM, but not IgG. However, reliable methodology was not obtained for quantitative assays.
Table 2. Effect of two proteases on plaque fluid and salivary amylase activity (pg maltose/pl/min). Mean + SD (n) Saliva Amylase Amylase + Protease VI Amylase + Protease VIII
4
5
51.3 + 9.1 (5) 19.4 f 1.6(5) 55.1 k 3.0(5) 18.8 + 0.6 (5) 50.1 f 3.5 (4) 19.1 + 0.5 (4)
6
7
8
9
PH
Plaque fluid Fig.
1. Activity
profile,
measured as AAS4&/pg proand plaque fluid lysozyme incubated with Micrococcus luteus acetone powder in 0.1 M imidazole-succinate buffer of different pH values at 37°C. 0 = hen egg lysozyme. o = plaque fluid. x = parotid .. sauva.
tein, for hen egg, salivary
660
A. Tatevossian and E. Newbrun Table 3. Lability of alkaline phosphatase activity in plaque fluid. Mean k SD (n) Incubation
(a)
time
Plaque of indeterminate age (n mole substrate converted/pl/min) (b) 24 h plaque (n mole substrate converted/pl/min) (c) 24 h plaque, freezethawed 46 x , stored _ 2 wk (n mole substrate converted/pl/min)
30 min 0.304 f 0.120(5)
120 min
60 min 0.263 k 0.117(5)
-
0.244 * 0.022 (14)
-
-
0.120 f 0.020 (5)
-
24h -
0.034 k 0.006t (14)
0.075 f 0.02* (5)
-
* p < 0.01 when compared to 30min sample by Student’s t test. t p < 0.001 when compared to 30 min sample by Student’s t test.
(to be published) indicated that the mobility of amylase in plaque fluid was similar to that in saliva and B. subtilis. No evidence was found for the bacterial amylase of low molecular weight reported by Seder (1969) in plaque extracts. The lack of susceptibility to exogenous protease attack found in the present experiments did not preclude the possibility that other proteases present in dental plaque were active against amylase, or that longer incubations of the enzymes with the proteases may eventually have led to degradation not observed here. More reducing activity was released by 1~1 plaque fluid per unit time from starch (Table 1) than from sucrose (Table 4). Neff (1967) reported that the pH drop produced in plaque by starch is smaller than with sucrose. The major sucrase activity in plaque was therefore in the bacterial phase (Birkhed, 1975; Aksnes, 1976) while that of amylase was in the aqueous phase. However, /3-D-fructofuranoside fructohydrolase (E.C.3.2.1.26; invertase) activity of plaque microorganisms may have been on the cell surface and not readily released into the plaque fluid. Clearly plaque organisms such as Strep. mutuns are capable of scission of sucrose (Panzer, Chassy and Krichevsky, 1972). The assay used does not exclude the possibility that soluble and filterable polymers were formed. Lysozyme
The lysozyme concentration in resting parotid saliva was similar to those reported by Hoerman, Englander and Shklair (1956) and Helderman (1976), higher than that reported by Brandtzaeg and Mann
(1964) in whole saliva, and lower than that found by Raeste and Tuompo (1976) in whole saliva. Higher activity might be expected in mixed whole saliva than in duct saliva because it contains additional lysozyme, bound to the salivary sediment fraction and to dislodged plaque. Since the quantitation relied on a comparison with the activity displayed by crystallized enzyme preparations against a particular preparation of cultured Micrococcus luteus, some of the variations recorded (which range from 8.2 to 200 pg/ml hen egg lysozyme equivalent) were probably based on the specific activity of the enzyme and the relative susceptibility of different batch cultures to lysis. Lysozyme activity reported in dental plaque extracts by Holt (1975), equivalent to about Spg/ml hen egg lysozyme, is much lower than that found in plaque fluid. This may have been due to dilution of plaque activity by the extractant. Nord, Seder and Lindqvist (1969) reported that about 70 per cent of the lysozyme activity in dental plaque occurs in the extractable fraction. The sources of oral lysozyme are the salivary glands and oral leucocytes which migrate through the gingival crevice (Wright, 1964; Raeste, 1972). The concentration of lysozyme in crevicular fluid is higher than in the salivary secretions (but not necessarily than in whole saliva) or blood (Brandtzaeg and Mann, 1964; Helderman, 1976). The concentration of lysozyme in plaque fluid is higher than in saliva (Cole et al., 1978). In our present studies the concentration of lysozyme in plaque fluid, as well as that in resting parotid saliva, was very close to those reported by Helderman (1976) for crevicular fluid and
Table 4. “Sucrase” activity in plaque fluid. Mean + SD (n) pg reducing activity released/d/min 24 h 24 h 24 h 24 h GT
Plaque Plaque Plaque Plaque
fluid fluid + Protease VI fluid + Protease VIII fluid + GT
1.256 k 0.364 (6) 0.799 + 0.326* (5) 0.609 f 0.313t (5) 0.600 (mean of 3) 0.085 (mean of 3)
* p < 0.05 when compared to 24 h plaque fluid by Student’s t test. t p < 0.01 when compared to 24 h plaque fluid by Student’s t test.
Plaque fluid enzymes parotid saliva respectively, but could not be directly compared with the plaque fluid lysozyme determined by the Osserman technique (Cole et a/., 1978). Helderman (1976) suggested that the high crevicular fluid level arose partly from the effect of the paper point on diapedesis of leucocytes across the gingival epithelium and their attachment to the sampling point. Similarly, t le stimulating effect of oral microorganisms in the gingival crevice may account for the high lysozyme activity in plaque fluid. The high plaque fluid lysozyme activity was interesting in the context of the ionic composition of plaque fluid, which is relatively high in potassium (‘Tatevossian and Gould, 1976b). Whether oral microorganisms are susceptible to the lytic action of lysozyme is not clear. Gibbons, de Stoppelaar and Harden (1966) concluded that oral microorganisms are not, while Coleman, Van de Rijn and Bleiweis (1971), Iwamoto et al. (1971) and Bladen et a[. (1973) found evidence that they are. The lysis of cells could account for the high potassium concentration in plaque fluid, but to what extent this arises from the lysis of leucocyte and host cells, by lysozyme action on plaque microorganisms or by the preparative technique for obtaining plaque fluid, has not been resolved. It is possible to demonstrate an increase in plaque fluid potassium caused by freezethaw damage to the plaque, when compared with samples kept at 5°C during and after collection (Tate\.ossian and Gould, 1976a). It is also evident from the metabolic activity demonstrable in plaque that the organisms remaining intact in the plaque are not susc,eptible to the enzyme. The optimum pH of the lysozyme of different origin in the series was similar to (Fig. l), but higher than the pH 5 reported by Nord, SGder and Lindqvist (1969), and closer to that reported by Hartsell (1948) for egg lysozyme and by Polyzois et al. (1976) for human salivary and for egg lysozyme. The plaque tluid between meals is apparently at a pH near the optimum for lysozyme activity in the plaque (Tatevos:.ian, 1977). The observation that the activity was higher in plaque of indeterminate age, presumably older than 24 h, is consistent with the hypothesis that increased leucocyte diapedesis and gingival fluid flow occurs in areas of more prolonged plaque accumulation and incipient gingival inflammation, although I he limited number of samples tested in this group does not allow this conclusion to be drawn. F’hosphatases and other enzymes Several enzymes possessing an cc-naphthyl phosphate-hydrolysing activity are demonstrable in plaque extracts (Paunio, 1969), the optimum activity being above pH 8.0 in the crude extract, but with optima around pH 6.0 when separated Sephadex G-100 fractions were tested. Enzymes hydrolysing p-nitrophenyl phosphate in alkaline buffer, pH 8.8, were reported by Paunio (1970), with a low activity (about 0.5-1.5 x 10e8 mol substrate hydrolysed/min) which necessitated a 22 h incubation period for assay. However, the plaque:extractant ratio was not stated. The plaque fluid activity found could effect millimolar concentration changes in phosphate (e.g. from Table 1: 0.26nmol P released/pl/min is equivalent to a concentration change of 0.26 mM/min).
661
The presence of a pool of organic phosphates in dental plaque and the alkaline phosphatase activity seem to explain the maintenance of inorganic phosphate in plaque fluid at higher concentrations than in saliva (Tatevossian and Gould, 1976b). The factors which affect the balance between substrate availability and alkaline phosphatase activity in dental plaque are not known. However, the lability of the enzyme in vitro may be of interest if it also occurs in vivo. The inability to detect glucosyltransferase (GT) activity synthesising insoluble polysaccharide was not likely to be related to the presence of inhibitory levels of soluble polysaccharide in plaque fluid (Tatevossian and Gould, 1976b). Possibly GT activity in plaque binds to insoluble matrix polysaccharide and was therefore sedimented during centrifugation to separate the plaque fluid. The finding that the “sucrase” activity in plaque fluid was reduced by the addition of glucosyltransferase (Table 4) indicated that some of the substrate was utilized by the GT. Unlike the amylase, the “sucrase” activity was susceptible to bacterial protease activity, and may be due to invertase, glucosyltransferase and fructosyltransferase (Gibbons, 1972; Tanzer, Brown and McInerney, 1973; Kuramitsu, 1973; Sharma, Dhillon and Newbrun, 1974; Chassy et al., 1976). However, the concentration in plaque fluid is small and the major activity is in the bacterial residue (Birkhed, 1975; Aksnes, 1976). The presence of proteolytic activity in plaque (SBder and Frostell, 1966; Sijder, 1972) could also be demonstrated in plaque fluid. However, quantitation by the absorbed layer method was inadequate. This area warrants further work and is potentially useful in understanding both the pathogenicity and the ecology of dental plaque. Acknowledgement-This work was supported by an AADR Senior Foreign Dental Fellowship. Dr. M. Morris kindly cooperated in extending use of the Pedodontic Clinic and access to the patients. The editorial geline Leash is much appreciated.
assistance
of Ms. Evan-
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Schneyer L. H. 1956. Amylase content of separate salivary gland secretions of man. J. appl. Physiol. 9, 453-455. Sharma M., Dhillon A. S. and Newbrun E. 1974. Cellbound glucosyltransferase activity of Streptococcus sanguis strain 804, Archs oral Biol. 19, 1063-1072. Shizukuishi S., Nakamura R., Tsunemitsu A. and Uesugi Y. 1977. a-L-Fucosidase activity in human saliva and dental plaque. Archs oral Biol. 22, 721-722. Silverman G. and Kleinberg 1. 1967. Fractionation of human dental plaque and the characterisation of its cellular and acellular components. Archs oral Bio[. 12, 1387-1405. SGder P-O. 1969. The molecular size of enzymes from dental plaque material. J. periodont. Res. 4, i15-219. Sijder P-O. 1972. Proteolvtic activitv in the oral cavitv: proteolytic enzymes from huma; saliva and den&l plaque material. J. dent. Res. 51, 389-393. SGder P-O. and Frostell G. 1966. Proteolytic activity of dental plaque material. I. Action of dental plaque material on azocoll, casein and gelatin. Acta odont. Stand. 24. 501-516.
SGder P-O., Nord C-E. and Linder L. 1971. Separation of enzymes from 2-day-old dental plaque material. Stand. J. dent. Res. 79, 5 15-5 17. Tanzer J. M., Chassy B. M. and Krichevsky M. I. 1972. Sucrose metabolism by Streprococcus mutans, SL- 1. Biochim. biophys. Acta 261, 379-387.
Tanzer J. M., Brown A. T. and McInerney M. F. 1973. Identification, preliminary characterisation and evidence for regulation of invertase in Streptococcus mutans. J. Bact. 116, 192-202.
Tatevossian A. and Gould C. T. 1976a. Methods of sampling and analyzing the aqueous phase of human dental plaque. Archs oral Biol. 21, 3 13-317. Tatevossian A. and Gould C. T. 1976b. The composition of the aqueous phase in human dental plaque. Archs oral Biol. 21, 319-323. Tatevossian A. 1977. Buffering capacity in human dental plaque fluid. Caries Res. 11, 216-222. Tuyau J. E. and Sims W. 1975. Occurrence of Haemophili in dental plaque and their association with neuraminidase activity. J. dent. Res. 54, 737-739. Vaughan A., Guilbault G. G. and Hackney D. 1971. Fluorometric methods for analysis of acid and alkaline phosphatase. Analvt. Chem. 43, 721-724. V&en J. and crank R. M. 1974. Activitks pyrophosphatiques acide et alcaline de la plaque dentaire humaine. Archs oral Biol. 19, 203-208. Wright D. E. 1964. The source and rate of entry of leucocytes in the human mouth. Archs oral Biol. 9, 321-329.
ENZYMIC
ACTIVITIES IN THE AQUEOUS OF HUMAN DENTAL PLAQUE A.
TATEVOSSIAN*
PHASE
and E. NEWBRIJN
Dept. Oral Medicine and Hospital Dentistry, University of California, San Francisco, CA 94143, U.S.A.
Summary-The activity of some enzymes in human dental plaque fluid was measured. Plaque of 24 h growth was collected from 9 adults, and plaque of indeterminate age obtained from children attending the Pedodontic Clinic at UCSF. Resting parotid saliva samples from the adult subjects were also assayed. Amylase activity was lower and lysozyme activity higher in plaque fluid than in saliva. The optimum pH for both enzymes in both fluids was between 6.5 and 7.5. Salivary and plaque fluid amylase was not inactivated by short incubations with two bacterial proteases. Trace levels of acid phosphatase and low alkaline phosphatase activity were detectable in plaque fluid, the latter being labile during incubation and storage. No glucosyltransferase activity synthesizing insoluble polysaccharide from sucrose was detectable in plaque fluid. Protease activity against bovine serum albumin and human serum IgA, IgG and IgM was detectable in plaque fluid.
INTRODUCTION The protein
components
of dental
plaque
constitute
three pools: bacterial cell proteins and enzymes closely associated with the bacterial cell walls; denatured and structural matrix proteins; and soluble proteins in the aqueous extracellular phase. The aqueous phase has hitherto been studied by extraction (Silverman and Kleinberg, 1967; Fox and Dawes, 1970a) and yields several enzyme activities. Among these are: amylase (Slider, 1969; Holt, 1975), decarboxylases (Hayes and Hyatt, 1974), L-fucosidase (Shizukuishi et nl., 1977), hyaluronidases (Slider, 1969; Nord and Siider, 1972), lysozyme (Sader et nl., 1971; Holt, 1975) neuraminidase (Tuyau and Sims, 1975), alkaline phosphatase (Paunio, 1969, 1970), acid and alkaline pyrophosphatase (Vreven and Frank, 1974), “sucrolytic” enzymes (Birkhed, 1975; Aksnes, 1976) including dextransucrase, levan sucrase and invertase activities (Birkhed and Dahlqvist, 1975) and proteases (SGder, 1969, 1972). The form and reactivity, in the native state, of the components obtained from plaque by extraction remains a matter for conjecture as the pH, ionic strength and composition of the plaque aqueous phase is changed during the extraction procedure; these variables affect the extraction of protein from plaque (Fox and Dawes, 1979b). Effective concentrations of the extractants from the intact plaque cannot be obtained. The development of a method to separate plaque fluid by centrifugation (Tatevossian and Gou-ld, 1976a) allows quantitation of the average activity of some enzymes in the soluble phase of dental plaque. MATERIALS AND METHODS Human dental plaquefluid
(a) Plaque was collected with perspex spatulae at least 45 min after breakfast from 9 adult volunteers * Present address: Physiology Department, University College, Card8 CF1 1XL, Wales, U.K. 0.1,.2419%B
who had not brushed their teeth in the previous 24 h. All available surfaces were sampled, except the lingual lower anterior region and areas of gingival inflammation. Contamination by saliva was minimized by instructing the subject to swallow. The procedure for pooling the plaque and separating the aqueous phase was as previously described (Tatevossian and Gould, 1976a), or modified, to avoid freeze-thawing the plaques, by maintaining the collected samples at 5°C throughout the procedure. (b) Plaque of itdeterminate age was collected from children aged 3-14 yr attending the Pedodontic Clinic for treatment, and was processed similarly. Resting parotid saliva
Resting parotid saliva was collected into glass capillary tubes directly from the parotid duct opening of the adult subjects. The samples were maintained at 5°C for short periods before use. Enzyme assays 4-glucanohydrolase, Amylase (g-1,4 glucan E.C.3.2.1.1.). The activity was measured by a modifi-
cation of the method described by Jamieson, Pruitt and Caldwell (1969). Samples (1 ~1 of saliva or plaque fluid) were incubated with 50 ~1 of substrate (1 per cent starch in 0.02 M phosphate buffer, pH 6.9, 6 mM NaCl) in a total volume of 100~1. Enzyme reaction which proceeded at room temperature was terminated (after 5 min for saliva, 10 min for plaque fluid) by the addition of 100 ~1 dinitrosalicylic acid (DNS) reagent (Hosteller et al., 1951). After boiling (5 min) and dilution, the reducing activity was read at 470nm and compared to maltose standards. The results were expressed as the rate of release of reducing activity per ~1 of plaque fluid or saliva. Lysozyme (mucopeptide N-acetylmuramylhydrolase, E.C.3.2.1.17). The method of Jollb et al. (1965) was used. Micrococcus luteus (ATCC 4698) was grown as a
batch culture in Enriched Nutrient Broth (American Type Culture Collection, 1972). Growth was increased
A. Tatevossian and E. Newbrun
658
at 37°C which was used in preference to the recommended temperature of 20°C. The cells were sedimented at SOOOg, 5°C and washed three times; an acetone powder was separated by filtration, dried and stored at 5°C. The acetone powder was suspended in buffer (0.067 M sodium phosphate pH 6.2:1 per cent sodium chloride, in the ratio 9:l) to an absorbancy of about 0.7 at 540 nm, then 1 ml was incubated with either 2 ~1 saliva, 1 ~1 plaque fluid, or standards (hen egg lysozyme) for 0.5 h at 37°C. The samples were vortexed at 1Omin intervals and their absorbance, which decreased with lysis of the suspension, was reread. Enzyme activity was expressed as the concentration of hen egg lysozyme standard with equivalent activity after identical incubation conditions. Synthesis of insoluble carbohydrate polymers (u-1,32-glucosyltransferase aCtiUity)
(1976) was used. Substrate (100 pg protein/ml in 0.15 M sodium chloride) was adsorbed onto polystyrene Petri dishes during 30min contact at room temperature. Unbound substrate was rinsed off with 0.15 M sodium chloride. Plaque fluid (l-5 ~1) was placed on the plate and incubated at 37°C for 15 min in a humid chamber. The zones of solubilized substrate were visualized in the dried and washed Petri dishes by the condensation of steam from a water bath, the droplet size difference clearly demarcating the zone of digested substrate. Materials Egg white lysozyme, Protease VI (from Streptomyces griseus) and Protease VIII (from Bacillus subtilis) were obtained from Sigma Chemical Co., St.
Louis. Human serum immunoglobulin IgA, IgG and IgM were purchased from Cappel Laboratories, A method modified from that of Robrish, Reid and Cochransville, Pa. Enriched Nutrient Broth was prepared according to the American Type Culture ColKrichevsky (1972) was used to measure the production of insoluble glucan. Plaque fluid samples (5 ~1) lection (1972). were incubated for up to 3 h at 37°C in 100~1 1OmM sucrose in 0.025 M potassium phosphate buffer, pH RESULTS 6.8; 20 mM sodium fluoride; and 20 &i/ml U-i4CAmylase sucrose. At appropriate time intervals, 10 ~1 fractions The activity of amylase in plaque fluid was lower were filtered through 0.45 pm Millipore filters and than in an equivalent volume of resting saliva from washed twice with 10 ml of distilled water. The radioactivity incorporated into insoluble polymer was the parotid duct (Table 1). The pH activity profile measured by liquid scintillation spectrometry. The from both fluids was similar when they were incusupernatant fluid from a 24 h culture of Streptococcus bated in 0.1 M imidazole/succinate buffer in the range of pH 4.5 to 8.5; peak activity was in the range of pH mutans serotype e (S27 A-l, by courtesy of C. Hoover) 6.5 to 7.0. Amylase in plaque fluid and saliva was not in Brain Heart Infusion (Difco) was used as a positive inactivated by two bacterial proteases (Protease Type control for glucosyltransferase activity, the procedures and volumes being the same as those for plaque fluid VI and Type VIII) under the test conditions (Table 2). These proteases hydrolysed bovine serum albumin, assays. IgA and IgM and partially inactivated invertase (see Alkaline and acid phosphatase (orthophosphoric below).
ghCan:D-fruCtoSf?
monoester phosphohydrolase, E.C.3.1.3.1 and 3.1.3.2)
The fluorometric method of Vaughan, Guilbault and Hackney (1971) was used. Naphthol AS-B1 phosphate in ethylene glycol (0.3 x 10m4M) was incubated for up to 24 h at 37°C with 1 ~1 samples and 3.0 ml buffer (0.1 M tris, pH 9.0 for alkaline phosphatase; 0.1 M citrate, pH 5.5, for acid phosphatase). Fluorescence was measured with a Turner fluorimeter (lex 405 nm, lem 515 nm). The results were expressed as the rate of substrate converted per ~1 of plaque fluid. “Sucrase” (Inuertase) activity (B-D-fructofuranoside fructohydrolase, E.C.3.2.1.26)
Samples (2 ~1) were incubated at 37°C in 1 ml 0.125 M sucrose buffered with 0.025 M phosphate, pH 6.8. Fractions of lOO$ were removed from the mixture before, and at appropriate intervals during, incubation. Each fraction was added to lOO$ DNS reagent and the reducing activity compared to maltose standards. The glucose content was also estimated by the Glucostat (Sigma) assay according to the recommended procedures. The results were expressed as the rate of release of reducing activity per ~1 of plaque fluid. Proteolytic E.C.3.4.4)
activity
(peptidyl
peptide
hydrolases,
The method of Elwing, Nilsson and Ouchterlony
Lysozyme
The activity of lysozyme in plaque fluid was greater than in an equivalent volume of resting parotid saliva (Table 1). The optimum pH of egg white, salivary and plaque fluid lysozyme was similar, in the range of pH 6.5 to 7.5 (Fig. l), as measured by a decreased absorbance at 540 nm with increased lysis. Synthesis of insoluble carbohydrate polymer No glucosyltransferase activity was detectable in plaque fluid, although significant amounts could be detected in the control samples of supernatant of broth culture from Strep. mutans S27 A-l, tested concurrently. The plaque fluid samples tested were unfrozen and kept at 5°C for less than 1 h before assay. Acid and alkaline phosphatase
Trace acid phosphatase activity was demonstrable in plaque fluid, at the detection limit of the method (Table 1). Formation of product did not significantly increase during the 30min to 24 h incubation time. Alkaline phosphatase activity was demonstrable in plaque fluid (Table 1). The activity diminished with increasing incubation time and was less than 20 per cent after 24 h incubation (Table 3), with excess substrate remaining. Purified calf alkaline phosphatase was stable during incubations up to 60 min, at a time when plaque fluid enzyme activity had fallen an aver-
Plaque
fluid enzymes
659
Table 1. Enzymic activity in plaque fluid and resting parotid saliva. Mean f SD (n)
24 h growth
Plaque fluid Indeterminate age
18.2 k 10.6* (17)
15.5 + 4.1* (9)
221.8 k 73.1* (20)
385 f lOl* (6)
Enzyme Amylase (pg maltose/$/min) Lysozyme (pg/ml hen egg lysozyme equivalent) l,~oluble carbohydrate polymer synthesis Acid phosphatase Alkaline phosphatase (n mole substrate converted/&min)
Trace (13) 0.248 i 0.024 (7)
17.3 t_ 8.9* (26) 89.9 + 5.3 (22) 259.5 k 105.1* (26) 65.3 If: 32.6(15)
-
ND (6)
Resting parotid saliva
Combined
ND (6)
Trace (5) 0.267 k 0.081 (12)
Trace (18) 0.260 _t 0.070 (19)
= not detectable. * p < 0.001 when compared with saliva by the Student t test.
ND
age of 15 per cent (range 8.2 to 26.9 per cent) below activity at 30 min (Table 3). By freeze-thawing plaque fluid samples 4 to 6 times and storing them at -20°C f-Jr about 2 weeks, the activity found after 30min incubation was lower than in unstored samples (Table 3, c vs b). These samples showed an average reduction in activity of 38 per cent when incubation continued for 120 min (Table 3, c at 30 min vs 120 min). “Sucrase” activity “Sucrase” activity, detected as the release of reducing sugar, was present in plaque fluid (Table 4). The activity was diminished by the addition of glucosyltransferase purified from Strep. sunguis culture fluid, and by bacterial proteases. Attempts to differentiate invertase activity from that of other enzymes capable of releasing reducing groups were unsuccessful because of the presence of substantial invertase activity in the commercially available glucose oxidase used in assay kits for glucose. This problem has not been reported by previous authors and may not be apparent if sucrose is not included in the blanks and standards incubated with glucose oxidase. Protease activity
DISCUSSION
Amylase and sucrase
Jacobsen, Melvaer and Hensten-Petersen (1972) and Birkhed (1974) found that the activity of amylase in dental plaque extracts was lower than in saliva. Similarly, the amylase activity in plaque fluid was about 25 per cent of that found in the resting parotid saliva from our subjects. The level found in plaque fluid indicated that parotid salivary contamination of the plaque used as a source of plaque fluid was limited and relatively reproducible. Submandibular and sublingual saliva also have lower amylase activity than the parotid secretion (Schneyer, 1956). The activity in plaque fluid may have originated from saliva during the formation of the plaque since amylase activity is demonstrable in undisturbed dental plaque in situ (Neff, 1967). The optimum pH and resistance to inactivation of the amylase suggested that this activity could have been derived from saliva. However, certain microorganisms in plaque have also been demonstrated to hydrolyse starch (Ruby and Gerencser, 1974). Later work on the electrophoretic mobility of the activity
The presence of proteolytic activity was demonstrable in plaque fluid against bovine serum albumin and human serum IgA, IgG and IgM substrates. Protease VI and VIII (1 mg/ml) gave zones of lysis against bovine serum albumin, IgA and IgM, but not IgG. However, reliable methodology was not obtained for quantitative assays.
Table 2. Effect of two proteases on plaque fluid and salivary amylase activity (pg maltose/pl/min). Mean + SD (n) Saliva Amylase Amylase + Protease VI Amylase + Protease VIII
4
5
51.3 + 9.1 (5) 19.4 f 1.6(5) 55.1 k 3.0(5) 18.8 + 0.6 (5) 50.1 f 3.5 (4) 19.1 + 0.5 (4)
6
7
8
9
PH
Plaque fluid Fig.
1. Activity
profile,
measured as AAS4&/pg proand plaque fluid lysozyme incubated with Micrococcus luteus acetone powder in 0.1 M imidazole-succinate buffer of different pH values at 37°C. 0 = hen egg lysozyme. o = plaque fluid. x = parotid .. sauva.
tein, for hen egg, salivary
660
A. Tatevossian and E. Newbrun Table 3. Lability of alkaline phosphatase activity in plaque fluid. Mean k SD (n) Incubation
(a)
time
Plaque of indeterminate age (n mole substrate converted/pl/min) (b) 24 h plaque (n mole substrate converted/pl/min) (c) 24 h plaque, freezethawed 46 x , stored _ 2 wk (n mole substrate converted/pl/min)
30 min 0.304 f 0.120(5)
120 min
60 min 0.263 k 0.117(5)
-
0.244 * 0.022 (14)
-
-
0.120 f 0.020 (5)
-
24h -
0.034 k 0.006t (14)
0.075 f 0.02* (5)
-
* p < 0.01 when compared to 30min sample by Student’s t test. t p < 0.001 when compared to 30 min sample by Student’s t test.
(to be published) indicated that the mobility of amylase in plaque fluid was similar to that in saliva and B. subtilis. No evidence was found for the bacterial amylase of low molecular weight reported by Seder (1969) in plaque extracts. The lack of susceptibility to exogenous protease attack found in the present experiments did not preclude the possibility that other proteases present in dental plaque were active against amylase, or that longer incubations of the enzymes with the proteases may eventually have led to degradation not observed here. More reducing activity was released by 1~1 plaque fluid per unit time from starch (Table 1) than from sucrose (Table 4). Neff (1967) reported that the pH drop produced in plaque by starch is smaller than with sucrose. The major sucrase activity in plaque was therefore in the bacterial phase (Birkhed, 1975; Aksnes, 1976) while that of amylase was in the aqueous phase. However, /3-D-fructofuranoside fructohydrolase (E.C.3.2.1.26; invertase) activity of plaque microorganisms may have been on the cell surface and not readily released into the plaque fluid. Clearly plaque organisms such as Strep. mutuns are capable of scission of sucrose (Panzer, Chassy and Krichevsky, 1972). The assay used does not exclude the possibility that soluble and filterable polymers were formed. Lysozyme
The lysozyme concentration in resting parotid saliva was similar to those reported by Hoerman, Englander and Shklair (1956) and Helderman (1976), higher than that reported by Brandtzaeg and Mann
(1964) in whole saliva, and lower than that found by Raeste and Tuompo (1976) in whole saliva. Higher activity might be expected in mixed whole saliva than in duct saliva because it contains additional lysozyme, bound to the salivary sediment fraction and to dislodged plaque. Since the quantitation relied on a comparison with the activity displayed by crystallized enzyme preparations against a particular preparation of cultured Micrococcus luteus, some of the variations recorded (which range from 8.2 to 200 pg/ml hen egg lysozyme equivalent) were probably based on the specific activity of the enzyme and the relative susceptibility of different batch cultures to lysis. Lysozyme activity reported in dental plaque extracts by Holt (1975), equivalent to about Spg/ml hen egg lysozyme, is much lower than that found in plaque fluid. This may have been due to dilution of plaque activity by the extractant. Nord, Seder and Lindqvist (1969) reported that about 70 per cent of the lysozyme activity in dental plaque occurs in the extractable fraction. The sources of oral lysozyme are the salivary glands and oral leucocytes which migrate through the gingival crevice (Wright, 1964; Raeste, 1972). The concentration of lysozyme in crevicular fluid is higher than in the salivary secretions (but not necessarily than in whole saliva) or blood (Brandtzaeg and Mann, 1964; Helderman, 1976). The concentration of lysozyme in plaque fluid is higher than in saliva (Cole et al., 1978). In our present studies the concentration of lysozyme in plaque fluid, as well as that in resting parotid saliva, was very close to those reported by Helderman (1976) for crevicular fluid and
Table 4. “Sucrase” activity in plaque fluid. Mean + SD (n) pg reducing activity released/d/min 24 h 24 h 24 h 24 h GT
Plaque Plaque Plaque Plaque
fluid fluid + Protease VI fluid + Protease VIII fluid + GT
1.256 k 0.364 (6) 0.799 + 0.326* (5) 0.609 f 0.313t (5) 0.600 (mean of 3) 0.085 (mean of 3)
* p < 0.05 when compared to 24 h plaque fluid by Student’s t test. t p < 0.01 when compared to 24 h plaque fluid by Student’s t test.
Plaque fluid enzymes parotid saliva respectively, but could not be directly compared with the plaque fluid lysozyme determined by the Osserman technique (Cole et a/., 1978). Helderman (1976) suggested that the high crevicular fluid level arose partly from the effect of the paper point on diapedesis of leucocytes across the gingival epithelium and their attachment to the sampling point. Similarly, t le stimulating effect of oral microorganisms in the gingival crevice may account for the high lysozyme activity in plaque fluid. The high plaque fluid lysozyme activity was interesting in the context of the ionic composition of plaque fluid, which is relatively high in potassium (‘Tatevossian and Gould, 1976b). Whether oral microorganisms are susceptible to the lytic action of lysozyme is not clear. Gibbons, de Stoppelaar and Harden (1966) concluded that oral microorganisms are not, while Coleman, Van de Rijn and Bleiweis (1971), Iwamoto et al. (1971) and Bladen et a[. (1973) found evidence that they are. The lysis of cells could account for the high potassium concentration in plaque fluid, but to what extent this arises from the lysis of leucocyte and host cells, by lysozyme action on plaque microorganisms or by the preparative technique for obtaining plaque fluid, has not been resolved. It is possible to demonstrate an increase in plaque fluid potassium caused by freezethaw damage to the plaque, when compared with samples kept at 5°C during and after collection (Tate\.ossian and Gould, 1976a). It is also evident from the metabolic activity demonstrable in plaque that the organisms remaining intact in the plaque are not susc,eptible to the enzyme. The optimum pH of the lysozyme of different origin in the series was similar to (Fig. l), but higher than the pH 5 reported by Nord, SGder and Lindqvist (1969), and closer to that reported by Hartsell (1948) for egg lysozyme and by Polyzois et al. (1976) for human salivary and for egg lysozyme. The plaque tluid between meals is apparently at a pH near the optimum for lysozyme activity in the plaque (Tatevos:.ian, 1977). The observation that the activity was higher in plaque of indeterminate age, presumably older than 24 h, is consistent with the hypothesis that increased leucocyte diapedesis and gingival fluid flow occurs in areas of more prolonged plaque accumulation and incipient gingival inflammation, although I he limited number of samples tested in this group does not allow this conclusion to be drawn. F’hosphatases and other enzymes Several enzymes possessing an cc-naphthyl phosphate-hydrolysing activity are demonstrable in plaque extracts (Paunio, 1969), the optimum activity being above pH 8.0 in the crude extract, but with optima around pH 6.0 when separated Sephadex G-100 fractions were tested. Enzymes hydrolysing p-nitrophenyl phosphate in alkaline buffer, pH 8.8, were reported by Paunio (1970), with a low activity (about 0.5-1.5 x 10e8 mol substrate hydrolysed/min) which necessitated a 22 h incubation period for assay. However, the plaque:extractant ratio was not stated. The plaque fluid activity found could effect millimolar concentration changes in phosphate (e.g. from Table 1: 0.26nmol P released/pl/min is equivalent to a concentration change of 0.26 mM/min).
661
The presence of a pool of organic phosphates in dental plaque and the alkaline phosphatase activity seem to explain the maintenance of inorganic phosphate in plaque fluid at higher concentrations than in saliva (Tatevossian and Gould, 1976b). The factors which affect the balance between substrate availability and alkaline phosphatase activity in dental plaque are not known. However, the lability of the enzyme in vitro may be of interest if it also occurs in vivo. The inability to detect glucosyltransferase (GT) activity synthesising insoluble polysaccharide was not likely to be related to the presence of inhibitory levels of soluble polysaccharide in plaque fluid (Tatevossian and Gould, 1976b). Possibly GT activity in plaque binds to insoluble matrix polysaccharide and was therefore sedimented during centrifugation to separate the plaque fluid. The finding that the “sucrase” activity in plaque fluid was reduced by the addition of glucosyltransferase (Table 4) indicated that some of the substrate was utilized by the GT. Unlike the amylase, the “sucrase” activity was susceptible to bacterial protease activity, and may be due to invertase, glucosyltransferase and fructosyltransferase (Gibbons, 1972; Tanzer, Brown and McInerney, 1973; Kuramitsu, 1973; Sharma, Dhillon and Newbrun, 1974; Chassy et al., 1976). However, the concentration in plaque fluid is small and the major activity is in the bacterial residue (Birkhed, 1975; Aksnes, 1976). The presence of proteolytic activity in plaque (SBder and Frostell, 1966; Sijder, 1972) could also be demonstrated in plaque fluid. However, quantitation by the absorbed layer method was inadequate. This area warrants further work and is potentially useful in understanding both the pathogenicity and the ecology of dental plaque. Acknowledgement-This work was supported by an AADR Senior Foreign Dental Fellowship. Dr. M. Morris kindly cooperated in extending use of the Pedodontic Clinic and access to the patients. The editorial geline Leash is much appreciated.
assistance
of Ms. Evan-
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