Menstrual Hemocidin HbB115?146 Is an Acidophilic Antibacterial Peptide Potentiating the Activity of...

Menstrual Hemocidin HbB115–146 Is an AcidophilicAntibacterial Peptide Potentiating the Activity of HumanDefensins, Cathelicidin and LysozymePawel Mak1, Michal Siwek1, Jan Pohl2, Adam Dubin1

1Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland;2Microchemical and Proteomics Facility, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA

Introduction

The term ‘hemocidins’ was proposed by Mak et al.1

for a group of antimicrobial peptides derived from

hemoglobin (Hb) and other heme-binging proteins.

These proteins do not have distinct antimicrobial

domains and the killing activity of hemocidins is

instead related to the abundance of amphipathic

helices which mimic the structure and topology of

classical, specialized gene-encoded alpha-helical anti-

microbial peptides.2,3 Hemocidins can be easily

obtained in vitro by chemical or proteolytical diges-

tion of precursor proteins.1,4–8 The growing number

of studies describe hemocidins generation also in the

Keywords

Alpha/beta-defensins, cathelicidins,

hemoglobin, lysozyme, urogenital tract

Correspondence

Paweł Mak, Faculty of Biochemistry,

Biophysics and Biotechnology, Jagiellonian

University, 7 Gronostajowa St., 30-387

Krakow, Poland.

E-mail: [email protected]

Submitted September 27, 2006;

accepted November 7, 2006.

Citation

Mak P, Siwek M, Pohl J, Dubin A. Menstrual

hemocidin HbB115–146 is an acidophilic

antibacterial peptide potentiating the activity

of human defensins, cathelicidin and

lysozyme. Am J Reprod Immunol 2007;

57:81–91

doi:10.1111/j.1600-0897.2006.00456.x

Problem

Our recent studies proved that menstrual discharge is exceptionally rich

in bactericidal hemoglobin peptides (hemocidins). Of special interest is

the behavior of hemocidins in low pH of the vagina, in different ionic

strengths, and in the presence of other specialized antibacterial mole-

cules acting in this organ.

Methods of study

We studied the activity of a model representative of menstrual hemoci-

din: the peptide from human beta-globin, spanning residues 115–146

(HbB115–146). The bactericidal action of this peptide in different physic-

ochemical conditions, as well as the evaluation of the synergistic effect

of the peptide with mixtures of neutrophil alpha-defensin HNP-1, epi-

thelial beta-defensin HBD-1, cathelicidin LL-37 and lysozyme were stud-

ied using the bacterial membrane permeability test.

Results

The HbB115–146 is a salt-resistant antibiotic molecule strongly potenti-

ating its activity in acidic conditions (pH 4.4–5.0), characteristic for the

vagina. Hemocidin HbB115–146 is also an effective factor stimulating

the activity of other antibacterial polypeptides present in the female uro-

genital tract. The observed synergistic effect is preserved or enhanced at

lower pH and, with the exception of HBD-1, is observed also at physio-

logical salt concentrations.

Conclusion

The results of this study clearly demonstrate that hemocidins are import-

ant polypeptide factors involved in maintaining vaginal immunity dur-

ing normal menstrual bleedings.

ORIGINAL ARTICLE

American Journal of Reproductive Immunology 57 (2007) 81–91 ª 2007 The Authors

Journal compilation ª 2007 Blackwell Munksgaard 81

living organisms, where they act as antimicrobial

effectors in different tissues.9–14 The early findings

suggested that these peptides emerge mainly in

erythrocytes as a result of natural degradation of he-

moglobin15 but the recent work of Ivanov et al.16

demonstrated that intra-erythrocyte hemoglobin

fragments cannot be precursors of hemoglobin pep-

tides present in tissues.

Our latest studies showed that the female urogeni-

tal tract is an unique organ involved in effective

generation of hemoglobin bactericidal peptides.12–14

Proteolysis of hemoglobin during vaginal bleedings

can effectively support other known factors that con-

tribute to vaginal innate immunity, including epithe-

lium, mucus, low pH, hydrogen peroxide, comensal

microflora, antibodies, lymphocytes, macrophages,

and bactericidal proteins and peptides.

The production of hemocidins is favored by speci-

fic physicochemical conditions in the vagina. Low

pH is responsible for both hemoglobin release from

erythrocytes and its partial denaturation, facilitating

digestion by proteinases present in menstrual secre-

tions (matrix metalloproteinases and leukocyte pro-

teases). In effect, the menstrual discharge is

exceptionally rich in hemoglobin peptides, demon-

strating pronounced antibacterial activities, directed

mainly toward gram-negative bacteria. Moreover,

our studies on puerperal uterine secretions collected

during different stages of labor showed that genera-

tion of hemocidins occurs also during the birth and

begins together with the clinical symptoms of

labor.17

However, although generation of hemocidins dur-

ing bleedings in the female urogenital tract is evi-

dent, and the spectrum of antimicrobial activity of

emerged peptides is well characterized, there is still

little known about the biochemical and physico-

chemical factors that determine bactericidal activities

of these peptides in vivo. Of special interest is the be-

havior of hemocidins in low pH of the vagina, in dif-

ferent ionic strengths, and in the presence of other

specialized antibacterial molecules.

The present study examines the action of a model

menstrual hemocidin, derived from beta-globin,

spanning residues 115–146 (HbB115–146, AH-

HFGKEFTPPVQAAYQKVVAGVANALAHKYH). This

peptide was found to be one of the most active anti-

bacterial hemoglobin fragments present in menstrual

discharge,12 showing minimal inhibitory concentra-

tion (MIC) values in the range of tens of lm. The

presented results describe the action of HbB115–146

peptide in different pH conditions, salt and divalent

cations concentrations, as well as demonstrate the

synergistic effect of this hemocidin with lysozyme

and other peptide effectors responsible for killing

bacteria in the vagina.

Materials and methods

Materials and Chemicals

If not otherwise stated, all chemicals and materials,

including chromatographic columns, were purchased

from Sigma–Aldrich–Fluka–Supelco Co. (St Louis,

MO, USA).

Peptides and Proteins

The homogeneity of all peptides and proteins used

was checked by analytical reversed-phase high-per-

formance liquid chromatography (RP-HPLC) using

Dionex P680 system (Dionex, Sunnyvale, CA,

USA), LC-18-DB 4.6 · 250 mm column (or Kroma-

sil C4 4.6 · 250 mm column in case of cathelicidin

LL-37) and water/acetonitrile/trifluoroacetic acid

(TFA) solvents set. All final peptide and protein

preparations used had 95–99% degree of purity.

The identity of the compounds was confirmed by

mass spectrometry performed on Bruker Reflex IV

MALDI-TOF instrument (Bruker Daltonics, Bremen,

Germany) and by sequencing of the first five resi-

dues on an automatic gas-phase protein sequencer

ABI model 491 (Applied Biosystems, Foster City,

CA, USA).

Human neutrophil defensin 1 (HNP-1) was pur-

chased from Biocentrum Ltd (Krakow, Poland). The

stock solutions of the peptide were prepared in water

and the final concentrations of the preparations

were measured spectrophotometrically, assuming the

absorbance coefficient according to Harwig et al.18

Human recombinant beta-defensin 1 (HBD-1) was

purchased from Peptides International Inc. (Louis-

ville, KY, USA) as a precise weighted amount. The

stock solutions of the peptide were prepared in water

assuming concentrations based on manufacturer’s

data.

Human synthetic cathelicidin LL-37 and HbB 115–

146 peptide were synthesized by the Fmoc (9-fluore-

nylmethoxycarbonyl) chemistry on a solid-phase

support using Advanced ChemTech Model 90

semiautomatic peptide synthesizer (Advanced Chem-

Tech, Lousville, KY, USA). The crude peptides were

MAK ET AL.


82 Journal compilation ª 2007 Blackwell Munksgaard

dissolved in 0.1% TFA (v/v) and purified by RP-

HPLC on a Kromasil C4 column (in case of LL-37) or

LC-18-DB column (in case of HbB115–146 peptide).

The stock solutions of the peptides were prepared in

water and the final concentrations of the prepara-

tions were determined by amino acid analysis.

Briefly, the peptide sample was hydrolyzed in gas

phase using 6 m HCl at 115�C for 24 h. The liberated

amino acids were converted into phenylthiocarbamyl

derivatives and analyzed by RP-HPLC on a PicoTag

3.9 · 150 mm column (Waters, Milford, MA, USA).

Human neutrophil lysozyme was a courteous gift

from Prof. W. Watorek (Wroclaw University,

Poland). The stock solutions of the enzyme were

prepared in water and the final concentrations of

the preparations were measured spectrophotometri-

cally, assuming the absorbtion coefficient from Fass-

man.19 Enzymatic inactivation of lysozyme by heat

denaturation was performed according to Colombie

et al.20 by heating of protein solution in 70�C for

30 min. After heating, the inactivated enzyme was

immediately chilled on an ice-bath to prevent aggre-

gation.

Bacteria Culture, Bacterial Membrane

Permeabilization Assay and Synergistic Activity

Test

Escherichia coli JM83 strain containing plasmid

pCH110 (Pharmacia-Amersham, Piscatway, NJ, USA)

encoding beta-galactosidase and ampicilin-resistance

gene was grown on Luria broth medium containing

1.25 lg/mL ampicilin. All assays were performed on

fresh daily-made mid-logarithmic phase bacteria

inoculated from overnight culture.

The studies on bacterial membrane permeabiliza-

tion by HbB115–146 peptide in different physico-

chemical conditions were performed in several

buffer systems. The pH-dependence was determined

in 20 mm citrate-phosphate buffers pH 4.4, 5.0, 5.6,

6.2 and 6.8. The salt-resistance was assayed in

20 mm phosphate buffer containing 0, 0.05, 0.08,

0.12, 0.15, 0.25 and 0.5 m NaCl. The effect of diva-

lent cations on HbB115–146 activity was measured

in 20 mm MES (2-(N-morpholino)-ethane sulfonic

acid) buffer pH 6.8 containing 0, 0.01, 0.1, 1 and

10 mm MgCl2 or CaCl2.

The aliquots of the appropriate buffer (30 lL) were

pipetted into the wells of 96-well round-bottom poly-

propylene microtiter plates and an appropriate

amounts (2 lL maximum) of HbB115–146 peptide

solution was added, followed by addition of 2 lL of

bacteria suspension containing 5 · 105 CFU. The

final concentration of HbB115–146 was 16 lm for the

pH-dependence experiments and 16.7 lm in the case

of salt-resistance and divalent cations experiments.

The plate was then vortexed and incubated 15 min

in 37�C to allow action of the peptide toward bac-

teria. After incubation, aliquots of 70 lL of 200 mm

Tris-HCl buffer pH 8.0 were added to each well, and

the mixtures were individually transferred to the

wells of a new, flat-bottom clear polyethylene 96-

well plate containing 5 lL of the beta-galactosidase

colorimetric substrate (50 mm aqueous solution of p-

nitrophenyl-beta-d-galactopyranoside). The plate was

again vortexed and after 30 min incubation at 37�C

the absorbance at 405 nm, proportional to the

amount of the released beta-galactosidase, was meas-

ured using PowerWave SelectX microplate reader

(Bio-Tek Instruments, Vinooski, VT, USA). The con-

trol samples for 0% perforation were the bacteria

incubated without the peptide, while the control

samples for 100% of perforation were the bacteria

disintegrated by sonication prior to the assay. All

assays were performed in triplicates.

The studies on synergistic action of HbB115–146

in mixtures with HNP-1, HBD-1, LL-37 and inacti-

vated lysozyme were performed in a slightly differ-

ent manner. Three buffers were used: (a) 20 mm

phosphate buffer pH 6.8, (b) 20 mm acetate buffer

pH 5.0, and (c) 20 mm acetate buffer pH 5.0 contain-

ing 0.15 m NaCl. The aliquots of the appropriate buf-

fer (30 lL) were pipetted into the wells of 96-well

round-bottom polypropylene microtiter plates and

two aliquots (maximum of 2 lL each) of the peptide

solutions were added. The first portion contained dif-

ferent amounts of HbB115–146 and the second one

contained constant amount of HNP-1, HBD-1, LL-37

or inactivated lysozyme solution. The HbB115–146

was diluted previously to maintain the molar ratio of

1:0.02, 1:0.1, 1:1, 1:10 and 1:50 toward other anti-

bacterial factors, and the final concentration range of

this peptide varied from 0.04 to 99 lm for HNP-1,

from 0.05 to 129 lm for HBD-1, from 0.03 to 72 lm

for LL-37, and from 0.03 to 79 lm for inactivated

lysozyme. The mixtures of the antibacterial factors

and HbB115–146 peptide were then vortexed, prein-

cubated 15 min at 37�C and the 2 lL aliquots of the

bacterial suspension containing 5 · 105 CFU cells

were added to each well. The remaining steps of the

protocol were identical to those described above dur-

ing physicochemical assays.

ACTIVITY OF MENSTRUAL HEMOCIDIN



Assay of Lysozyme Enzymatic Activity

Lysozyme activity was determined using the turbidi-

metric assay. The mixtures of the human lysozyme

with HbB115–146 peptide (molar ratios of 1:0.1,

1:0.2, 1:1, 1:5, 1:10, final concentrations of peptide

ranged from 0.05 to 4.8 lm) were preincubated in

the wells of 96-well round-bottom polypropylene

microtiter plates for 15 min at 37�C in 50 lL of the

appropriate buffer (20 mm phosphate buffer pH 6.8,

20 mm acetate buffer pH 4.0 or 20 mm acetate buf-

fer pH 4.0 containing 0.15 m NaCl). After preincu-

bation, the mixtures were transferred to the wells

of a new, flat-bottom clear polyethylene 96-well

plate containing 100 lL of suspension of lyophilized

Micrococcus luteus (ATCC 4689, final concentration

0.2 mg/mL). The respective amount of lysozyme

incubated without hemocidin was used as a control

of 100% of activity. The rate of absorbance decrease

at 450 nm was measured using PowerWave SelectX

microplate reader. All assays were performed in

triplicates.

Results and discussion

HbB115–146 as a Model Hemocidin Peptide

The HbB115–146 peptide was isolated from the nor-

mal menstrual discharge of a healthy young

woman.12 The peptide was chosen for synthesis and

further detailed characterization because of its dis-

tinctive cationic character (pI ¼ 9.41), typical for

many specialized antibacterial peptides, and because

of its high activity toward bacteria, especially E. coli,

a common opportunistic pathogen of the urogenital

tract. Moreover, HbB115–146 is very similar to the

HbB111–146 peptide, originally found by Liepke

et al.11 in placenta.

Influence of Physicochemical Conditions on

Activity of HbB115–146

The bacterial membrane permeability test utilizing

the beta-galactosidase-transformed E. coli cells is a

broadly used and convenient assay, ideal for study-

ing bactericidal peptides able to kill by destruction of

phospholipid bilayers integrity. As we demonstrated

in our previous work,2 this mechanism of action is

probably typical for all hemocidins.

The results of the measurements of the HbB115–

146 peptide activity toward E. coli under different

physicochemical conditions were collected in Fig. 1.

The most significant result is demonstrated in Fig. 1a

and concerns very strong dependence on the pH

milieu. The change of pH from 4.4 to 5.0 reduced

activity of the peptide by 80%. Analogously, the up

to 18-fold difference in the perforation potential of

the peptide was observed between the lowest pH 4.4

(a safe minimal limit preventing from beta-galactosi-

dase inactivation) and the highest pH 6.8 tested (this

4,4 5 5,6 6,20

20

40

60

80

100

Pe

rce

nt

of

pe

rfo

ratio

n

0

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40

60

Pe

rce

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nP

erc

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Pe

rce

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of

pe

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ratio

n

pH

0 0.01 0.1 1 100

20

40

60

80

Log Mg++ (mM)

0 0,01 0,1 1 100

20

40

60

80

Log Ca++ (mM)

(a)(b)

(c) (d)

0 0,05 0,08 0,12 0.15 0.25 0.50

NaCl (mol/L)

Fig. 1 Lytic activity of HbB115–146 hemoci-

din toward Escherichia coli cells measured in

different physicochemical conditions: (a) effect

of pH of the medium, (b) effect of salt concen-

trations, (c) effect of magnesium ions concen-

trations, and (d) effect of calcium ions

concentrations. All measurements were per-

formed using cytoplasmic beta-galactosidase

release assay.

MAK ET AL.



is a safe maximal limit; above this value the peptide

had tendency to precipitation). These data demon-

strate that the HbB115–146 peptide is one of the

most effective antimicrobials present just in the

vagina, where the normal, physiologic pH ranges

from 4.0 to 4.5. The observed behavior of hemocidin

is typical for the other known cationic antimicrobial

peptides,21–23 and is interpreted as a consequence of

the electrostatic forces during the early stages of pep-

tide interaction with the negatively charged bacterial

membrane.

Similar and expected results concerned also the

inhibitory effect of magnesium and calcium ions on

activity of HbB115–146 peptide (Fig. 1c,d). This

effect is also commonly known for most cationic

antimicrobial peptides,24 and is generally related to

the competition of divalent cations with the posi-

tively charged peptides for negatively charged bind-

ing sites on the bacterial membrane. The observed

inhibitory effect on the action of HbB115–146 is sig-

nificant: about 95% inhibition was observed with

1 mm Mg2+ or Ca2+. This is a meaningful observa-

tion, especially taking into consideration the fact

that, e.g. the level of the magnesium ions in normal

serum is in the range of 1.8–2.5 mm. Similar values

were also reported in the vaginal fluids.25,26 How-

ever, these values concern the total amount of mag-

nesium, including the ions forming complexes with

other proteins. The effective level of free ions that

are able to really inhibit bactericidal peptides chan-

ges in a broad range and is very difficult for reliable

estimation.

A different situation was observed in the case of

the studies on the influence of NaCl concentration

(Fig. 1b). The HbB115–146 peptide demonstrated

only slight changes of lytic activity in the buffers

containing up to 0.25 m NaCl. This observation is

not typical of other known cationic antibacterial

peptides, which exhibit a generally significant

decrease of activity just in physiological (0.15 m) salt

concentrations.21–23 The molecular basis of this

unique behavior awaits further examination. How-

ever, hemocidin appears to be the only salt-resistant

antibacterial peptide acting in the vaginal fluid dur-

ing menstruation.

Synergistic Effect of Mixtures of HbB115–146 with

HNP-1, HBD-1, and LL-37

According to our previous works, the total concen-

tration of all hemocidins in menstrual discharge of

healthy women ranged from 38 to 64 lm, which is

equivalent to about 150–250 lg/mL.12 However,

the analyzed discharge contains a heterogeneous

population of hemocidins and their individual con-

centrations are probably much lower, reaching

only several micrograms per mL and falling under

the estimated effective bactericidal doses (in range

of about 30 lm or about 100 lg/mL). Interestingly,

the above-estimated effective concentrations of

individual hemocidins are comparable with those

of other antibacterial peptides in physiological flu-

ids (e.g. in the range of several lg/mL for alpha-

defensins in vaginal fluids27 or LL-37 in mucosal

and gastric secretions28,29) These quantitative con-

siderations led us to examine a potential synergis-

tic effect of vaginal hemocidins for the action of

other bactericidal peptides acting in the urogenital

tract. The synergy causes the effective lethal doses

of different peptide mixtures to be much lower

than those of the individual compounds, constitu-

ting a well-documented and important feature of

classical cationic antibacterial peptides. Moreover,

this phenomenon was recently especially well des-

cribed for human alpha- and beta-defensins and

cathelicidins.21,30,31

As the specific physicochemical conditions (espe-

cially low pH) present in the vagina complicate

the studies, and to thoroughly examine the syn-

ergy phenomenon in this physiologic environment,

we decided to perform the assays in three condi-

tions: neutral, acidic/low ionic strength, and in the

presence of physiological salt concentrations. More-

over, the possible synergistic effect was noted for

several different molar proportions of HbB115–146

peptide with the other assayed compounds. Three

common antibacterial peptides were chosen for the

experiments: (i) epithelial beta-defensin 1 (HBD-1)

(the most common antibiotic peptide of the lung,

gastrointestinal and urogenital mucosal secre-

tions32), (ii) cathelicidin LL-37 (also a frequent

antibacterial peptide of epithelia29), and (iii) neutro-

phil defensin 1 (HNP-1) (one of the principal

microbicidal effectors of the phagocytic cells33).

The results obtained for synergistic experiments

are summarized in Figs 2–4. The white bars repre-

sent the activity of different amounts of HbB115–

146 and the specific concentration of the second

antibacterial compound. The gray bars demonstrate

the observed lytic activity of a mixtures of peptides.

These bars have additionally a lower black section

which shows the calculated sum of activities of both




peptides (i.e. the value expected without the syner-

gistic effect). All gray bars are additionally denoted

by the calculated P value: the data denoted as ‘A’

had the highest statistical significance (P < 0.05)(i.e.

the obtained lytic activity was statistically signifi-

cantly greater than the sum of activities of individual

compounds), the bars denoted as ‘B’ had medium

statistical importance (0.1 > P > 0.05), while the

results with the ‘C’ sign were statistically insignifi-

cant.

The most general conclusion from analysis of the

data presented in Figs 2–4 is that low or equimolar

ratios of the HbB115–146 peptide do not show any

synergistic effect in mixtures with other tested anti-

bacterial peptides, and that the measured perforative

potential of the mixtures is approximately equivalent

to the simple sum of activities. In contrast, in high

molar excess (10- or 50-fold) of HbB115–146 over

other antibacterial peptides, the mixture showed sy-

nergistic activity toward the membrane when com-

pared with the one calculated from the individual

sums of activities. This synergistic effect is most pro-

nounced in the case of HNP-1 (Fig. 4), where the

mixture of the 50-fold molar excess of hemocidin

over defensin is almost three times as active as the

sum of activities of the individual compounds under

all conditions tested. This synergistic effect is slightly

lower at high molar proportions of the studied pep-

tides (about twofold degree of activity amplification)

in case of HBD-1 in neutral as well as in acidic pH.

However, beta-defensin completely lost the activity

in the presence of salt, the entire membrane lytic

potential being derived, in this case, from HbB115–

146 peptide (Fig. 2). Similarly, a high synergistic

effect (about twofold amplification) was found for

hemocidin and LL-37 at low pH in the presence of

physiological salt concentration (Fig. 3). On the

other hand, the neutral milieu and the acidic buffer

without salt relatively lowered the synergistic effect.

However, there is an interesting observation that the

synergistic effect is already evident at equimolar

ratios.

From the structural point of view, among all tes-

ted antibacterial peptides, the alpha-helical cathelici-

din LL-37 is most similar to hemocidin HbB115–

146.34 Moreover, both of them kill bacteria more

effectively in the acidic conditions. These reasons,

and a similar mode of action, can explain cooper-

ation and synergy of these compounds at low molar

ratios. On the other hand, one can speculate that

the weaker synergistic effect at neutral pH can be in

some extent an effect of competition of these pep-

tides in neutral pH, where the activity of both pep-

tides is lower.

Fig. 2 Synergistic effect of mixtures of HbB115–146 and human HBD-

1 defensin. HDB-1 was preincubated with hemocidin at different molar

ratios and subjected to Escherichia coli lytic assay. Individual peptides

were used as a control. The white bars represent activity of individual

compounds, the gray bars represent the obtained lytic activity of a

peptides mixtures. The black sections under grey bars shows the cal-

culated sum of activities of both peptides (i.e. the value expected

without the synergistic effect). All experiments were performed inde-

pendently in three different buffers: neutral, pH 6.8; acidic, pH 5.0;

and acidic, pH 5.0 with addition of 0.15 M NaCl. The gray bars are

denoted by the calculated P value: (a) P < 0.05, (b) 0.1 > P > 0.05,

(c) P > 0.1. All results are a mean values from three independent

measurements ±S.D.

MAK ET AL.



A different behavior was observed in the case of

both defensins. First of all, HBD-1 is an exceptionally

salt-susceptible antibiotic (75 mm of NaCl lower the

activity of this peptide by 50%)35 and this fact com-

pletely explains the above-mentioned loss of both

additive and synergistic effect in the acidic buffer

with salt (Fig. 2). The second characteristic observa-

tion in the case of mixtures of hemocidin and defen-

sins is the evident synergism only at high molar

excess of the HbB115–146 peptide. In our opinion,

this phenomenon can be explained by completely

different structures and modes of action of the hem-

ocidin and both defensins. Hemocidin, an alpha-heli-

cal peptide acting on bacterial membranes by the so

called carpet-like mechanism, has a disordered struc-

ture in aqueous solutions and folds gradually after

contact with the phospholipid bilayers, forming su-

pramolecular aggregates (plaques or carpets) that are

able to disrupt membrane in a detergent-like man-

ner.2 The mechanism of action of the phylogeneti-

cally more advanced and specialized beta-sheet

antimicrobial peptides, defensins, is different and

based mainly on the formation of ion-permeable

voltage-dependent channels.33 In effect, the concen-

tration of hemocidins on a membrane surface must

be much higher than in case of defensins, and, in

consequence, the synergistic effect will be noted

only in an excess of hemocidins molecules.

Pe

rfo

ratio

n p

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0/0.02 0/0.1 0/1 0/10 0/50 1/0 1/0.02 1/0.1 1/1 1/10 1/50

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0/0.02 0/0.1 0/1 0/10 0/50 1/0 1/0.02 1/0.1 1/1 1/10 1/50

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0/0.02 0/0.1 0/1 0/10 0/50 1/0 1/0.02 1/0.1 1/1 1/10 1/50

pH 6.8

pH 5.0

pH 5.0 + 0.15M NaCl

A

C A C C

A

A

A

C C

A A

A

C C

LL-37 / HbB115-146 molar ratio



Fig. 3 Synergistic effect of mixtures of HbB115–146 hemocidin and

human LL-37 cathelicidin (see legend to Fig. 2 for details).

HNP-1 / HbB115-146 molar ratio

tn

ecr

ep

noit

arofr

eP

tn

ecr

ep

noit

arofr

eP

tn

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ep

noit

arofr

eP

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0/0.02 0/0.1 0/1 0/10 0/50 1/0 1/0.02 1/0.1 1/1 1/10 1/50

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0/0.02 0/0.1 0/1 0/10 0/50 1/0 1/0.02 1/0.1 1/1 1/10 1/50

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0/0.02 0/0.1 0/1 0/10 0/50 1/0 1/0.02 1/0.1 1/1 1/10 1/50

pH 6.8

pH 5.0

pH 5.0 + 0.15M NaCl

A

A

C C C

A

A

A C

C

A

C C C A




human HNP-1 defensin (see legend to Fig. 2 for details).




Synergistic Effect of Mixtures of HbB115–146 and

Lysozyme

Lysozyme is an exceptionally widespread and uni-

versal bactericidal protein present in almost all body

fluids and tissues and is able to kill bacteria in enzy-

matic and non-enzymatic way. As a muramidase,

lysozyme degrades the cell wall of a gram-positive

bacteria leading to lysis and death. The other kind of

bactericidal activity, directed mainly toward gram-

negative bacteria, is characteristic for the enzymati-

cally inactive (by partial heat denaturation or by

genetic knock-out) forms of lysozyme as well as its

selected peptide fragments. The mechanism of killing

is based on insertion of this polycationic protein or

its peptide fragments into the membrane and its dis-

ruption or formation of ion channels.36–38

We evaluated the synergistic effect of HbB115–146

on both enzymatic and non-enzymatic action of

lysozyme. In the first case we applied the turbidi-

metric assay which measures the rate of hydrolysis

of a suspension of lyophilized bacteria cell walls.

Surprisingly, we obtained small but statistically signi-

ficant increase of muramidase activity by the

HbB115–146 peptide (Fig. 5). In contrast to the

above results for defensins and cathelicidin, the

effect of hemocidin is measurable at equimolar ratios

of HbB115–146 and lysozyme, significantly increas-

ing the muramidase activity of the enzyme. The

level of amplification is over 40% in acidic buffers

without salt, by 20% in neutral buffers without salt,

and by 15% in acidic buffers containing 0.15 m

NaCl.

These results are somewhat difficult to discuss

because there are no data on the specific peptide

activators of lysozyme muramidase activity. All pre-

vious research focused on the influence of the other

peptide or proteinaceous factors on total potential of

lysozyme toward lysis or killing of bacteria, without

distinguishing between enzymatic and non-enzymat-

ic effect.21,39,40 Thus, our data on potentiation of

muramidase activity of lysozyme by HbB115–146

hemocidin are interesting and worthy of further

detailed exploration.

1/0,1 1/0,2 1/1 1/5 1/10

1/0,1 1/0,2 1/1 1/5 1/10

1/0,1 1/0,2 1/1 1/5 1/10

0

20

40

60

80

100

120

t n

e

c

r e

p

n

o

i t a

r o

f r e

P

Lysozyme / HbB115-146 molar ratio

0

20

40

60

80

100

120

140

160

t n

e

c

r e

p

n

o

i t a

r o

f r e

P


0

20

40

60

80

100

120

t n

e

c

r e

p

n

o

i t a

r o

f r e

P


pH 6.8

pH 4.0

pH 4.0 + 0.15 M NaCl

C C A A B

C B A A A

C C A B A

Fig. 5 Effect of HbB115–146 hemocidin on muramidase activity of

human lysozyme. Lysozyme was preicubated with hemocidin at differ-

ent molar ratios and subjected to muramidase turbidimetric assay

using lyophilized Micrococcus lysodeikticus suspension. The respective

amount of lysozyme incubated without hemocidin was used as a con-

trol of 100% of activity. All experiments were performed independently

in three different buffers: neutral, pH 6.8; acidic, pH 4.0; and acidic,

pH 4.0 with addition of 0.15 M NaCl. The bars are denoted by the cal-

culated P-value: (a) P < 0.05, (b) 0.1 > P > 0.05, (c) P > 0.1. All results

are a mean values from three independent measurements ± S.D.

MAK ET AL.



A more clear situation concerned experiments on

the synergistic effect of HbB115–146 peptide on the

lytic activity of heat-denatured lysozyme (Fig. 6).

The synergistic effect of hemocidin and lysozyme

was greatest in the acidic buffer without salt (from

four- to eightfold increase of activity) and lower in

the low pH buffer containing salt (increase of activity

by two to three times). In the case of the neutral

buffer without salt the degree of amplification was

about 30–40%. The presented results agree well with

the other studies concerning influence of cationic

antibacterial peptides on lysozyme activity.21,39,40 Of

special interest is that the influence of hemocidin,

we showed, was remarkably high, and, interestingly,

a very significant effect was noted just at very low

molar ratio (1 molecule of the peptide per 10 mole-

cules of lysozyme).

It is worth reminding that the synergistic effect at

low molar proportions was also noted in the case of

cathelicidin LL-37. This polypeptide, like the lyso-

zyme and hemocidin HbB115–146, belongs to the

cationic alpha-helical peptides, acting on a bacterial

membrane in a similar way. This may help to

explain the observed cooperation of these com-

pounds, especially in the acidic pH and low salt con-

centration.

Conclusions

Hemocidins, the bactericidal peptide fragments of he-

moglobin, are the newly discovered factors involved

in maintaining vaginal immunity during normal

menstrual bleedings. In this study, we demonstrate

that one of these peptides, hemocidin HbB115–146,

is a relatively salt-resistant antibiotic molecule acting

especially well in the acidic environment typical for

vaginal fluid. Moreover, the peptide is effective in

potentiating the activity of several other antibacterial

peptides present in the female urogenital tract such

as HNP-1, HBD-1, LL-37, as well as lysozymes. The

observed synergistic effect is preserved or enhanced

in lower pH and at physiologic salt concentrations.

Acknowledgments

The work was supported in part by the grant No. 2

PO4B 023 27 from the Ministry of Science and

Higher Education, Poland. Drs Tomasz Dylag and Pa-

vel Svoboda are acknowledged for expert assistance

in peptide synthesis. We thank also Lindsay Feldman

for help in manuscript preparation and Dr P. Suder

0

10

20

30

40

50

60

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90

100

0/0.02 0/0.1 0/1 0/10 0/50 1/0 1/0.02 1/0.1 1/1 1/10 1/50

0

10

20

30

40

50

60

70

80

90

100

0/0.02 0/0.1 0/1 0/10 0/50 1/0 1/0.02 1/0.1 1/1 1/10 1/50

0

10

20

30

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60

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100

0/0.02 0/0.1 0/1 0/10 0/50 1/0 1/0.02 1/0.1 1/1 1/10 1/50




tn

ecr

ep

noit

arofr

eP

tn

ecr

ep

noit

arofr

eP

tn

ecr

ep

noit

arofr

eP

pH 6.8

pH 5.0

pH 5.0 + 0.15M NaCl

A

A B A A

A A

A A

A

A

A

C A A


human heat-denatured lysozyme. Lysozyme was preincubated with

hemocidin at different molar ratios (ranging from 1/0.02 to 1/50 mol/

mol, respectively) and subjected to Escherichia coli lytic assay using

cytoplasmic beta-galactosidase release test. Individual compounds

were assayed separately as a control. The white bars represent activ-

ity of the different amounts of individual compounds, the gray bars

demonstrate the obtained lytic activity of the mixtures. The black sec-

tion under a gray bars shows the calculated sum of activities of both

compounds assuming no synergistic effect. All experiments were per-

formed independently in three different buffers: neutral, pH 6.8; aci-

dic, pH 5.0; and acidic, pH 5.0 with addition of 0.15 M NaCl. The gray

bars are denoted by the calculated P-value: (a) P < 0.05, (b)

0.1 > P > 0.05, (c) P > 0.1. All results are a mean values from three

independent measurements ± S.D.




for assistance in mass spectrometry measurements.

P. Mak was supported by the scholarship from Jagi-

ellonian University.

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