Proteolytic Activity in the Oral Cavity:
Proteolytic Enzymes from Human Saliva and Dental
Plaque Material
PER-OSTEN SODER
Odontological Faculty, Karolinska Institutet, Stockholm, Sweden
The presence of proteolytic activity within mined in whole saliva before centrifugation
the oral cavity is well known.'-3 Protease and in the pellet and supernatant after cenactivity in rat and mouse submandibular trifugation. The supernatants were dialyzed
glands has been demonstrated,45 and pro- against 1% glycine at 4 C for 24 hours beteolytic activity in salivary glands of rodents fore separation.
DENTAL PLAQUE MATERIAL.-Two-dayhas been studied.5'6
Although protease activity in human old dental plaque material was collected
saliva also has been demonstrated," 7 the from ten persons (five men and five
origin and chemical characteristics of pro- women), according to a method described
teolytic enzymes in human saliva have not by Sdder and Frostell." The material (985.3
been studied sufficiently. The presence of mg wet weight) was pooled and suspended
proteolytic enzymes in dental plaque ma- in 1% glycine and homogenized in a Virterial has been reported by many investiga- chow glass mortar. The suspension was centors.8,9 Leukocytes, regularly found in hu- trifuged at 20,000 x g for 15 minutes at
man saliva, generally increase in number 4 C. The supernatant was dialyzed against
during development of gingivitis. Proteolytic 1% glycine at 4 C for 24 hours and then
enzymes in human leukocytes'0 have been was used for separation and enzyme determinations.
demonstrated.
ENZYME ASSAY.-Proteolytic activity was
The aim of this investigation was to
demonstrate and separate proteolytic en- tested on the natural substrates gelatin* and
zymes of different origin in the oral cavity urea-denatured bovine hemoglobin, and on
of humans and to describe some character- the synthetic substrate poly-L-lysine hydroistics of enzymes from human saliva and bromide (PLL).t
dental plaque material.
Gelatin splitting activity was assayed by
the change in viscosity of the gelatin soluMaterials and Methods
according to the method of Hultin'2
SALIVA.-Parotid saliva was collected with tion,
used
by Lundblad'3 and described previLashley cups from ten healthy persons, ously.14 One milliliter of the enzyme fracpooled (42 ml), and stored frozen. Secre- tion was mixed with 3.0 ml of a 4% gelatin
tion was stimulated by placing citric acid solution in 0.1 M Tris-HCl buffer, pH 8.1,
on the posterior part of the tongue. Sub- that contained 0.01 % thymerosal§ as a bacmaxillary saliva was collected from the same tericidal agent. The outflow times of the subpersons with a suction pump and with cotton strate-enzyme mixtures were measured in
rolls in the vestibulum of the upper and Ostwald viscosimeters at 37 C for different
lower jaw. The saliva was pooled (44 ml) periods of time of as much as 20 hours, and
and stored frozen. Stimulated whole saliva the proteolytic activity was calculated ac(50 ml from each person) was collected
from eight persons, stored frozen, and ana- cording to Hultin's formula.'2-14 Values oblyzed individually.
* USP, Fischer Scientific Co., NJ.
Parotid, submaxillary, and whole saliva
t Difco, Detroit, Mich.
was centrifuged at 20,000 x g for 15 minT Pilot Chemical Inc., Watertown, Mass.
§ Merthiolate.
utes at 4 C. Proteolytic activity was deter389
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390
tained were multiplied by 109 and called
Hultin units (HU).
Proteolysis of hemoglobin was measured
by the method of Anson,'5 with the use of
urea-denatured bovine hemoglobin. The reaction mixture consisted of 1.0 ml enzyme
fraction, 2.0 ml 0.2 M Tris-HCl buffer, pH
7.5, and 1.0 ml of a 4% hemoglobin solution. The reaction mixture was incubated in
a water bath at 37 C for 20 hours.
The enzymatic reaction was stopped by
adding 5.0 ml 0.3 M trichloroacetic acid
(TCA). The amount of amino acids released
was determined spectrophotometrically at
280 nm with the use of tyrosine as a standard.
PLL was made into a 2% stock solution
in 0.20 M NaCl. The reaction mixture consisted of 1.50 ml stock substrate solution,
1.50 ml 0.2 M glycine-NaOH buffer, pH
9.1, and either 1.0 ml of the test solution
or 1.0 ml corresponding buffer. The viscosimetric method was used for enzyme determination, the outflow times of the substrateenzyme mixtures were measured in Ostwald
viscosimeters at 37 C, and the activity was
calculated according to Hultin's formula'2
and expressed in Hultin units (HU/PLL).16
ISOELECTRIC FOCUSING.-Isoelectric focusing was carried out in an Ampholine
column 8100.* The pH of the fractions
was determined with the use of a pH meter. t
GEL FILTRATION.-Sephadex G-100 superfine columns were used in the gel filtration experiments, and the gels were treated
according to manufacturer's instructions.
The elution medium consisted of 0.005 M
Tris-HCl buffer, pH 8.1, in 0.5 M NaCl and
2% butanol as a bactericidal agent. The elutions were made at room temperature, and
the changes in extinctions were followed
continuously by use of an ultraviolet absorption meter.t The optical density of the fractions was measured at 260 and 280 nm in a
Zeiss spectrophotometer PMQ II.§
To estimate the molecular weight of the
enzymes found, the column was standardized against the following substances: blue
dextran (mol wt 800,000), y-globulin (mol
wt 158,000), ovalbumin (mol wt 45,000),
ac-chymotrypsin (mol wt 25,000), cytochrome C (mol wt 12,400), vitamin B12
LB, Stockholm, Sw.
Type PHM 28, Radiometer, Copenhagen, Den.
: LKB, Stockholm, Sw.
§ Spectrophotometer PMQ II.
*
t
J Dent Res Supplement to No. 2
S6DER
(mol wt 1,355), and sucrose (mol wt 342).
The Kav of these substances was determined
and compared with the Kay of the enzymes:
= (Ve -VO)/ (Vt- Vo),
Ve
is the elution volume, V0 is the
where
void volume, and Vt is the total bed volume.
Laurent and Killander'7 found a correlation
between the Kav and the logarithm of the
molecular weights. The method and results
have been reported.'8
Ka
Results
SALIVA.-Proteolytic activity against gelatin was present in whole human saliva. The
proteolytic activity in whole saliva from
eight persons varied between 15 and 25
HU/ml, and about 45% of the activity was
recovered in the supernatant after centrifugation. The proteolytic activity that remained in the pellet after centrifugation
could not have been washed out.
The separation of the proteases from
whole saliva from one person after electrofocusing the supernatant is shown in Figure
1; similar results were obtained with the
saliva of the other seven people. The enzymes were separated into at least six or
seven different proteases. Four isoelectric
points (pi) determined were at pH 4.0,
about pH 5.0, pH 6.0, and pH 6.4.
The distribution of proteolytic enzymes
found after electrofocusing the supernatant
pH A
H
12.0 48
4
100 2400
40
8.0 -.2
U-m!
?2
4.0 .61
~~A80
2.0
10
20
30
40
Fraction No (3.0 ml)
FIG 1.-Isoelectric focusing of supernatant
from whole human saliva (50 ml) on 110 ml
column; range of carrier ampholytes, pH 4 to
6 (72 hours, 400 v, -0.5 mA). Solid circles,
pH of effluent; solid line, A280; clear circles,
proteolytic activity in HU/ml.
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391
PROTEOLYTIC ACTIVITY IN THE ORAL CAVITY
Vol 51 1972
of pooled submaxillary saliva is shown in
Figure 2. The proteases were distributed in
four peaks, with p1 around pH 4.0, pH 5.0,
pH 6.0, and pH 6.4.
Electrofocusing the supernatant of pooled
parotid saliva showed low, but detectable
proteolytic activity, with a pl around pH
3.8 to 4.2.
DENTAL PLAQUE MATERIAL.-Separation
of proteolytic enzymes found after electrofocusing the supernatant of pooled dental
plaque material is shown in Figure 3. At
least five proteolytic enzymes were visible
when gelatin was used as substrate. The p1
of these enzymes were at pH 4.0, 4.3, 4.6,
5.9, and 6.8.
The first (I) and second (II) enzyme in
fractions 6 to 10, the third enzyme (III) in
fractions 13, 14, and the fifth enzyme (V)
in fractions 37 to 40 were tested on their
proteolysis and also on hemoglobin, poly-Llysin, and the effect on addition of Ca2 +.
The fourth enzyme (IV) was present in fractions 32 and 33. Enzymes I and II were
pooled before testing. The pooled enzyme I
and II could hydrolyze gelatin, hemoglobin,
and poly-L-lysine (Fig 4). The enzymes
were strongly activated by the addition of
Ca2+, and the activity increased fivefold
if Ca2+ was added to the enzyme substrate
in 0.1 M final concentration. Enzyme III
could hydrolyze all the substrate used, and
when Ca2+ was added, the activity increased from 3 to 42 HU/ml. Enzyme V
30
FIG 3.-Isoelectric focusing of supernatant
from two-day-old dental plaque material (985.3
mg wet weight) on 110 ml column; range of
carrier ampholytes, pH 4 to 6 (42 hours, 500
v, 1 mA). Solid circles, pH of the effluent;
solid line, A2.0; clear circles, proteolytic activity
in HU/ml.
could hydrolyze gelatin and poly-L-lysine
but could not hydrolyze hemoglobin and
was not activated by the addition of Ca2+.
The pooled enzymes I and II, enzyme
III, and enzyme V were separated on a
Sephadex G-100 superfine column (Table).
After separation, the pooled enzyme fractions I and II were separated into two
proteolytic enzymes, one with molecular
weight of more than 150,000 and one of
about 10,000. Enzyme III had a molecular
weight of about 20,000, and enzyme V had
a molecular weight of 24,000.
-H.U ml ygmt'h'H.ULI
40 -20
20
30
Fraction No (3.0 ml)
FIG 2.-Isoelectric focusing of pooled submaxillary saliva (44 ml) on 110 ml column;
range of carrier ampholytes, pH 4 to 7 (65
hours, -500 v, 1.0 mA). Solid circles, pH of
the effluent; solid line, A280; clear circles, proteolytic activity in HU/ml.
40
Fraction No (30 ml )
_uq-mt'Kl
(PLLL)-
_2.0
30 -15
1.5
20-10
1.0
10 5
0.5
I+
Je
\Gelatin
I
I
dSTY|1
l /Vemoglobin PolyL-Lysin
FIG 4.-Proteolytic activity in different fractions with different substrates. I + II, fractions
6 to 10; 111, fractions 13 to 14; V, fractions 37
to 40. Shaded bar, gelainase activity without
activation; clear bar, activation with Ca2+, 0.1
M final concentration with gelatin as substrate.
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392
J Dent Res Supplement to No. 2
SODER
TABLE
PI, Ka , AND CALCULATED MOLECULAR
WEIGHTS OF ENZYMES
Calculated
Fraction
pI
Enzyme
Kav
I
II
III
V
4.0
4.3
4.6
6.8
Protease
Protease
Protease
Protease
0.0
0.77
~0.45
0.38
Molecular
Weight
> 150,000
10,000
~20,000
~24,000
Discussion and Conclusions
Because about 55% of the proteolytic
activity in human saliva remains in the
pellet after centrifugation, other proteolytic
enzymes that are insoluble or of high molecular weight could be present in the pellet
or enzymes could be bound to components
in saliva.
The proteolytic enzymes, after isoelectric
focusing of the supernatants of whole human
saliva (Fig 1) from eight persons, showed
similar patterns. The pI ranged between pH
4 and 6, and most of the activity was found
at a low pH, about 4. Similar enzyme fractions were present in submaxillary saliva
(Fig 2). In the submaxillary saliva, submandibular and sublingual saliva was present.
The fractions with p1 around pH 4 also
were present in parotid saliva with low proteolytic activity.
The great amount of proteins in the first
fractions after isoelectric focusing (Figs 1,
2, fractions 1 to 10; Fig 3, fractions 1 to 5)
could depend on the presence of proteins
with low pI in saliva or changes of the protein pattern during the elution of the column.
The first five fractions (Figs 1-3) were
not tested for proteolytic activity, because
the fractions were too diluted by buffers to
allow enzyme determination when the pH
increases from 2.0 to 8.1.
The proteolytic activity in dental plaque
material (Fig 3, Table) was in agreement
with earlier findings.18 The proteolytic enzyme (I) with a p1 4.0 had a molecular
weight over 150,000. This enzyme had the
same pI as one of the enzymes found in
whole and submaxillary saliva (Figs 1, 2).
The proteolytic enzyme (IV) in dental
plaque material (Fig 3, fractions 31 to 34)
had a pI about pH 5.9 and could be compared with the enzyme found in whole and
submaxillary saliva with the p1 about pH
6 (Figs 1, 2). The proteolytic enzyme (III)
in dental plaque material with a pI of 4.6
and molecular weight around 20,000 had
been found earlier and called protease II,.18
The proteolytic enzyme (V) with the pI
about 6.8 and molecular weight about 24,000
also had been found earlier and called protease IIb.Th
Some of the enzymes found in dental
plaque material may be of bacterial origin,
because the proteolytic activity increases as
the amount of dental plaque increases.19
References
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2. S6DER, P.-O.; LUNDBLAD, G.; and LIDQUIST, L.: Proteolytic Activity of Dental
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3. FULLMER, H.M., and GIBSON, W.A.: Collagenolytic Activity in the Gingiva of Man,
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13. LUNDBLAD, G.: Proteolytic Activity and
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PROTEOLYTIC ACTIVITY IN THE ORAL CAVITY
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393
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17. LAURENT, T.C., and KILLANDER, J.: A
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in press.
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