J. Biomedical Science and Engineering, 2010, 3, 1069-1072 JBiSE
doi:10.4236/jbise.2010.311138 Published Online November 2010 (http://www.SciRP.org/journal/jbise/).
Published Online November 2010 in SciRes. http://www.scirp.org/journal/jbise
Basic peptide protamine exerts antimicrobial activity against
periodontopathic bacteria
——Growth inhibition of periodontopathic bacteria by protamine
Tadashi Miura1, Keishi Iohara2, Tetsuo Kato3, Kazuyuki Ishihara4, Masao Yoshinari1
1Division of Oral Implants Research, Oral Health Science Center, Tokyo Dental College, Mihama-ku, Japan;
2Central Research Institute, Maruha Nichiro Holdings, Inc., Tsukuba, Japan;
3Laboratory of Chemistry, Oral Health Science Center; Tokyo Dental College, Mihama-ku, Japan;
4Department of Microbiology, Oral Health Science Center; Tokyo Dental College, Mihama-ku, Japan.
Received 28 September 2010; revised 18 October 2010; accepted 21 October 2010.
ABSTRACT
Protamine was investigated for its antibacterial ac-
tivity against the periodontal pathogens, Porphyro-
monas gingivalis, Prevotella intermedia and Aggrega-
tibacter actinomycetemcomitans. We determined the
minimum inhibitory concentrations of protamine and
its hydrolysate and their bactericidal activity. Pro-
tamine inhibited the growth of all periodontopathic
bacteria tested on agar plates. Protamine, which MIC
was 6.3 × 10-7 g L-1, was most effective against P. gin-
givalis. The antibacterial effect of native protamine
was higher than that of its hydrolysate. An ATP bio-
luminescence assay revealed that protamine showed
bactericidal activity against P. gingivalis in a time-
dependent manner. These results indicate that pro-
tamine could be candidate peptide for prevention of P.
gingivalis infection.
Keywords: Antimicrobial Peptide; Periodontopathic Ba-
cteria; Implantitis; Protamine
1. INTRODUCTION
Bacterial infection arising from an accumulation of mi-
crobial plaque around dental implants is a major cause of
periodontal disease including peri-implantitis. Five spe-
cies of periodontal pathogen detected in cases where a
titanium implant was used [1]. This indicates the impor-
tance of maintaining biofilm-free surfaces on both the
sub-gingival and supra-gingival portions of dental im-
plants in preventing peri-implantitis. Porphyromonas
gingivalis [2] and Aggregatibacter actinomycetemcomi-
tans [3] are believed to be major etiologic bacteria in
many cases of human periodontitis. Prevotella interme-
dia [4] has also been associated with human periodontal
disease. Research at our laboratory has focused on de-
veloping a system of defense against infection on dental
implant surfaces [5]. Reducing plaque has been empha-
sized in the prevention of periodontal diseases, including
peri-implantitis [6]. It is possible that using antimicrobial
materials can help reduce oral bacteria.
The loading of antimicrobial peptides onto the surface
of a dental implant is an important candidate for achiev-
ing antimicrobial activity. Antimicrobial peptides, a
promising new type of antimicrobial agent, offer the
advantage of not easily acting as antigens against the
host [7]. One of the aims of our ongoing study is to cre-
ate a defense system against peri-implantitis. We are
currently exploring the antimicrobial potential of pep-
tides affixed to the surfaces of dental materials [8].
We investigated the anti-periodontopathic activity of
the antimicrobial peptide, protamine and its hydrolysate.
Protamine, a basic peptide [9], discovered from salmon
testicles by F. Miescher in 1869 was later found to be
involved in the folding of nucleic acids in salmon sperm
[10]. Protamine has been isolated from more than 50
kinds of fish, and is used as a natural food preservative.
Protamine has several characteristics, including high
stability under heat and a preservative effect in neutral or
alkaline food. It does not influence the texture, smell, or
taste of food to which it is added. It can also be eaten as
a raw material, and has been used as a food additive for
many years. An acute toxicity test of protamine in mouse
[11] and a sub-long-term toxicity test in rat [12] demon-
strated its safety and confirmed that it was an excellent
antibacterial agent in milt. The antibacterial activity of
protamine is strongest against Gram-positive bacteria
such as the Bacillus species [13] and lactobacilli [14],
whereas its activity against fungi and yeast [15] is weak.
Our previous study revealed that hydrolyzed protamine,

T. Miura et al. / J. Biomedical Science and Engineering 3 (2010) 1069-1072
1070
was most effective against the biofilm formation of C.
albicans [16]. In this study, we investigated the inhibi-
tory effect of protamine and its hydrolysate on growth of
periodontopathic bacteria.
2. MATERIALS AND METHODS
2.1. Bacteria and Culture Condition
For liquid culture, P. gingivalis ATCC 33277, ATCC53977,
W50 (ATCC, American Type Culture Collection) and P.
intermedia ATCC 25611 were cultured in trypticase soy
broth (Becton Dickinson and Company, Sparks, MD,
USA) supplemented with hemin (5 g L-1; Sigma Chemi-
cal Co., St Louis, MO) and menadione (0.5 g L-1; Wako
Pure Chemical Industries, Osaka, Japan). A. actimomy-
cetemcomitans 310a and Y4 were cultured in Todd Hew-
itt Broth (Becton Dickinson and Company) supple-
mented with Yeast Extract (10 g L-1; Becton Dickinson
and Company). For plate culture, the bacteria were
grown on blood agar plates consisting of Tryptic soy
agar (Becton Dickinson and Company) supplemented
with 10% defibrinated horse blood, hemin (5 g L-1), and
menadione (0.5 g L-1). Cultures were performed at 37°C
in an anaerobic chamber filled with an atmosphere of
80% N2, 10% H2 and 10% CO2.
2.2. Preparation of Protamine and its
Hydrolysates
Protamine (designated as Prot) was obtained from Ma-
ruha Nichiro foods, Inc., Tokyo, Japan. This product is
prepared from the milt of salmon (Oncorhynchus keta),
living in the northern part of Japan. This milt has been
reported to contain four different molecular species [17]
rich in arginine, the primary structures of which were
reported to be as follows:
PRRRRRSSSRPIRRRRRPRASRRRRRGGRRRR,
PRRRRSSRRPVRRRRRPRVSRRRRRRGGRRRR,
PRRRRSSSRPVRRRRRPRVSRRRRRRGGRRRR,
PRRRRASRRIRRRRRPRVSRRRRRGGRRRR.
The Prot consisted of 30 or 32 amino acids in this
study, and the arginine residues constituted 60-70% of
the amino acid sequences. Protamine hydrolysate (des-
ignated as Brom) obtained by digestion with the enzyme
bromelain [18], a cysteine protease, was kindly donated
by Maruha Nichiro foods, Inc.
2.3. Evaluation of Minimum Inhibitory
Concentration.
Agar plates containing Prot or Brom were used to de-
termine minimum inhibitory concentration (MIC). Pep-
tide concentration was adjusted by stepwise dilution.
Using a loop, each bacterial strain was streaked onto an
agar plate. The agar plates were then incubated for 3-7
days in the anaerobic chamber at 37°C. Minimum in-
hibitory concentration was defined as the lowest concen-
tration of peptides that would inhibit the visible growth
of the microorganism after incubation. The values were
expressed as the mean ± SD of four experiments.
2.4. Antibacterial Activity of Protamine against P.
gingivalis ATCC 33277
P. gingivalis ATCC 33277 was anaerobically grown at
37°C to the early-stationary phase in the broth described
above. The harvested cells were washed once in auto-
claved water and then resuspended in freshly autoclaved
water containing adequate concentrations of protamine.
Cell suspensions were incubated at 37°C, and every 30
minutes the samples were examined for cell viability.
Cell viability was determined by ATP-bioluminescent
assay using the BacTiter-Glo Microbial Cell Viability
Assay kit (Promega, Madison, USA). Briefly, a volume
of BacTiter-Glo reagent equal to the volume of each
suspension was added and briefly mixed. The lumines-
cence of the solution was then recorded by using the
AUTO-LUMICOUNTER Model 1422EX (Microtec Co.,
LTD, Funabashi, Japan). The value obtained was ex-
pressed as the ratio to that at the start of incubation. The
results were expressed as the mean ± SD of three ex-
periments.
2.5. Statistical Analysis
Data were analyzed for statistical significance using a
two-way analysis of variance (ANOVA) followed by the
Scheffe test for multiple comparisons.
3. RESULTS AND DISCUSSION
As shown in Figure 1, protamine and its derivative
showed an inihibitory effect on growth of all the perio-
dontopathic bacteria tested, with inhibitory effect great-
est on growth of the P. gingivalis strains. The inhibitory
effect of native protamine was greater than that of its
hydrolysate (p < 0.05). The MIC values for P. gingivalis
ranged from 6.3 × 10-7 to 7.5 × 10-7 g L-1, while those for
A. actinomycetemcomitans and P. intermedia required a
higher concentration, of almost double or more. Recently,
we reported that protamine absorbed onto PMMA or
PMMA treated with oxygen (O2) plasma caused a mar-
ginal decrease in initial attachment of C. albicans [16].
In the case of C. albicans, the initial amount of fungal
attachment to Brom- or O2 plasma-treated PMMA di-
minished slightly in comparison to that treated with na-
tive peptide. These findings suggest that protamine ex-
hibits selective inhibitory action against growth of oral
microorganisms.

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1071
Figure 1. Minimum Inhibitory Concentration of Prot. and Brom. against periodontopathic patho-
gens. Identical letters indicate no significant difference (p > 0.05). 33277, 53977 and W50: P.
gingivalis; 310a, Y4: A. actinomycetemcomitans; 25611: P. intermedia.
Figure 2. Influence of protamine on cell viability of P. gingivalis ATCC33277. Identical letters
indicate no significant difference (p > 0.05).
To further investigate possible mechanisms of inhibi-
tion of P. gingivalis growth, the bactericidal activity of
protamine was assessed. As shown in Figure 2, pro-
tamine and its derivative possessed bactericidal activity
in a dose- and time-dependent manner against P. gin-
givalis. At 6.0 × 10-6 g L-1, which is approximately
ten-fold the value of the MIC (6.0 × 10-7 g L-1), the num-
ber of P. gingivalis cells after two hours incubation was
less than 40% of that at 30 min incubation. A higher
concentration of protamine induced further inhibition of
growth of P. gingivalis. A significant difference was ob-
served in inhibitory action between the two concentra-
tions used (p < 0.01). This indicates that this peptide has
an inhibitory effect on the growth of oral bacteria. In this
study, we found that protamine had an inhibitory effect
on periodontal pathogens, as well as fungi. Further in-
vestigation is necessary to elucidate the properties of this
peptide, for example, using immobilization methods
established at our laboratory. We believe that the appli-
cation of protamine to dental implants would offer ad-

T. Miura et al. / J. Biomedical Science and Engineering 3 (2010) 1069-1072
1072
vantages in the prevention of periodontal diseases such
as peri-implantitis and oral care.
4. ACKNOWLEDGEMENTS
This research was supported by Oral Health Science Center Grant hrc7
from Tokyo Dental College, and by a “High-Tech Research Center”
Project for Private Universities: matching fund subsidy from MEXT
(Ministry of Education, Culture, Sports, Science and Technology) of
Japan, 2006-2010.
The authors would like to thank Associate Professor Jeremy Wil-
liams, Tokyo Dental College, for his assistance with the English of this
manuscript.
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