28
SYNTHESIS AND BIOLOGICAl ACTIVITIES OF PUTATIVE AMPHIPHILIC a-HELICAL SEGMENTS OF
SYNTHESIS AND BIOLOGICAl
ACTIVITIES OF PUTATIVE AMPHIPHILIC
a-HELICAL SEGMENTS OF SEMINALPLASM~N
95
INTRODUCTION
Seminalplasmin (SPLN) consists of a single polypeptide chain of 47 amino acids
(Sitaram et al., 1986; Chapter 2). It was shown to permeabilize the inner membrane of E.
coli (Sitaram et al., 1992; Chapter 3). Secondary structure analysis of the sequence of
SPLN revealed the presence of at least three a-helical segments between amino residues
1-11, 14-26 and 28-40. Two of these helical segments 14-26 and 28-40 are
amphiphilic and map near the 'SURFACE' region on the 'Hydrophobic Moment' plot. One of
these regions also happens to be the most hydrophobic segment of SPLN (Chapter 2). In
order to evaluate the importance of these segments in the biological activity of SPLN, two
peptides of 13 residues each, corresponding to regions 14-26 (SLSR) and 28-40 (SPF)
were synthesised and further characterized for their biological activities.
4.1 MATERIALS AND METHODS
Synthesis of the peptides
Sources of chemicals : All L-amino acids, Boc-ON, t-butyl carbazate,
dicyclohexylcarbodiimide (DCC), diisopropylethylamine (OlEA), triethyl amine (TEA)
and hydrobenzotriazole (HOST) were all purchased from Sigma Chemical Company, USA.
Cesium carbonate, 2-chlorobenzoxycarbonyloxy succinamide and thioanisole were
purchased from Fluka AG, Switzerland. Dichloromethane (DCM), glacial acetic acid,
methanol and silica gel 60 were purchased from Merck, India. HPLC grade and UV 4
spectroscopy grade solvents [acetonitrile, methanol, DCM, N,N dimethylformamide (DMF),
and trifluoroacetic acid (TFA)]were purchased from Spectrochem Pvt. Ltd., Bombay, India.
Purification of solvents
The following solvents were purified as described by Vogel{1978). Dry benzene
was obtained by storing benzene over metallic sodium. OlEA and TEA were each distilled
.96
over ninhydrin. Ethanol was twice distilled. DCM was distilled over P205. All other
solvents were used as supplied by the manufacturers.
Preparation of amino acid derivatives
Soc-azide
Soc-azide was prepared as described by Carpino et al.,(1959). A solution of 30g t
butylcarbazate in 27 ml glacial acetic acid was prepared and 27.7 ml water was added to it
This solution was cooled to ooc with constant stirring and 17.4 g of sodium nitrite added
slowly over 15 min. This was stirred for 90 min and the oily layer formed separated,
using a separating funnel. The aqueous layer was washed with ether and the ether wash
added to the previous oily layer. This layer was then washed with water, saturated solution
of sodium bicarbonate and again water repeatedly till the aqueous layer was clear. The oily
layer was dried over sodium sulfate and the residual ether evaporated under reduced
pressure. The golden yellow liquid of Boc-azide so obtained was stored at 4oC.Yield was
about 90%.
Boc amino acids
Boc amino acids were prepared either by Schnabel's (1967) or Boc ON (ltoh et al.,
1975) method.
Schanbel's method
To a solution of1 0 mmol of amino acid in 1 0 ml of 1 :1 mixture of dioxane and
water 1.55 ml Boc azide was added. The pH of the solution was raised to 9.0 by adding 4N
NaOH and stirred for 24 h maintaining the pH at 9.0. Then, 15 ml of water was added to
the solution and a couple of ether washes done. The aqueous layer was acidified to pH 2.0
with 6N HCI and then extracted with ethyl acetate several times (in case of Boc-Leu and
Boc-Lys, ether was used for the extraction). Ethyl acetate was dried over sodium sulfate
and then evaporated and the residual oil triturated with petroleum ether. In the cases of
97
Boc-Giy, Boc-Leu, Boc-Aia and Boc-Pro, the resulting solids were stored at room
temperature. The following protected derivatives were, however, stored as DCM solutions
at ooc : Boc-lle: Boc-Vai,Boc-Thr,Boc-Phe and Boc-Lys(2-CIZ). All Bee-amino acids
were prepared with over 80% yield.
Boc ON method
To 10 mmol of amino acid taken in a round bottom flask,11 mmol Boc ON, 11 mmol
TEA and 12 ml of 1 :1 mixture of dioxane and water was added, and stirred for 12 hrs at
room temperature. The reaction mixture was acidified with 6N HCI and extracted with
ethyl acetate rapidly a few times. The ethyl acetate extract was dried over sodium sulfate
and rotoevaporated to get the Boc amino acid.
Lysine(2-CIZ)
Lysine(2-CIZ) was prepared as described by Erickson and Merrifield (1973). To
3.6g of L-lysine dissolved in 20 ml of water, a hot solution of 3.6 g of copper carbonate
in 120 ml water was added. Both the solutions were mixed and boiled for 45 min and the
resulting copper complex of lysine was filtered. To this 2 gm of sodium bicarbonate was
added. A 10 ml solution of 20 mmol (5.67 g) of 2-chlorobenzoxy carbonyloxy succinamide
in DMF was added to the above solution, with the help of a dropper, in 30 min, keeping
the pH between 7-8. The suspension was stirred overnight and filtered. The residue was
washed thoroughly with water, methanol and ether (three times each). The dry solution
was dissolved in warm 2N HCI to get a greenish solution and to this, 11.1 g of EDTA
dissolved in hot water was added . The pH was adjusted to 7.0 with 4N NaOH. After
completing the precipitation by storing at ooc for two hours, it was brought to room
temperature, filtered and the precipitate washed with water, methanol and ether three
times with each solvent. The yield was about 80%.
98
Preparation of N-formyl tryptophan
N-formyl tryptophan was made according to Previero et al.,{1967). A solution of
4g of L-tryptophan {20 mmol) in 30 ml of 98-1 00% formic acid was prepared. HCI
generated by the action of sulfuric acid on sodium chloride was bubbled through H2S04 and
the dry HCI so generated was passed through the above solution of tryptophan in formic acid
for 1 hr, in a hood with manual shaking from time to time. The light brown solution slowly
turned purple. The saturated reaction mixture was stoppered and left at room temperature
for 2 hrs. The reaction mixture was then transferred to a petri dish and left for half an
hour for the HCI to evaporate, then transferred to a round bottomed flask and dried on a
rotary evaporator. Excess of diethyl ether was added to the residual blue precipitate and
thoroughly washed to remove the residual formic acid. The residue was dissolved in
ethanol and kept overnight at 4°C. The blue precipipate formed was dried and weighed. The
yield was 4.46 g.
Purity of Boc amino acids :
This was checked by TLC on silica gel plates using either solvent systems CHCI3 :
AcOH {95:5) or CHCI3 : MeOH : AcOH {85 : 10 : 5).
Synthesis of SPF
The peptide SPF corresponding to the sequence PKLLETFLSKWIG was synthesized by
the solid phase method manually, esse~tially as described by Reddy and Nagaraj (1986 and
1989).
Incorporation of the first Boc amino acid to the resin
This was accomplished by the cesium salt method of Gisin {Gisin, 1973).Two nmol
of Boc glycine was dissolved in 1 0 ml ethanol and 2 ml of water in a round bottom flask. A
cesium carbonate solution (800 mg/ml) was added till pH 8.0 was achieved. Ethanol was
99
evaporated and benzene added to form an azeotrope with water. This was evaporated and the
benzene treatment repeated several times till a dry solid was obtained.
The cesium salt of Soc-glycine was dissolved in 10 ml of DMF (stored for several
days over 4.!. molecular sieves) and added to 1 g of Merrifield resin [Chloromethylated
polystyrene Co. 1% divinyl benzene, 200-400 mesh, (Sigma Chemical Co, USA)). The
suspension was gently stirred for 48 hrs at 5ooc in an oil bath. Thereafter it was filtered
using a G-2 funnel and washed with DMF, DMF-water, MeOH and DCM very thoroughly.
After drying under mild vacuum, the derivatized resin was weighed (1.15g) and the
increase in weight (0.15g) was noted. The degree of incorporation of the first amino acid
was also estimated by the picric acid method ( Gisin, 1972) described below.
Picric acid titration of resins
About 4 mg of accurately weighed deprotected or protected resin was placed in a
5ml sintered funnel. If the resin was protected it was subjected to 30% TFNDCM
treatment for half an hour, washed thoroughly with DCM (Thereafter the two resins are
treated identically). The resin was treated with 0.1 M picric acid/DCM for 5 min and all
unbound picric acid was then washed off with multiple DCM washings. The
stoichiometrically bound picrate was then eluted with 5% DIENDCM, till the eluate was
clear. 0.2 ml of the eluate was diluted to 2 ml with 95% ethanoi/DCM and the absorbance
at 358 nm determined. Using the value of e = 14,500 M-1 cm-1 for picric acid, the
degree of substitution was calculated. This was found to be within 0.1 nmol of the
substitution, as calculated from peptide (crude) recovery after the synthesis.
Synthesis of the peptide, SPF
1 . The derivatized resin was swollen in DCM in a reaction vessel. After a couple of
washes with DCM, it was subjected to 5% TFNDCM treatment for 5 min (all steps
were performed on the basis of 15 ml solvent per gm resin). This prewash was
100
followed by 30 min of 30% TFNDCM treatment followed by six 2 min washes with
DCM.
2. The free amino group of the peptide was then neutralized using a prewash of 5%
TENDCM for 2 min followed by a 10 min wash with 5% DIENDCM. This was then
washed off with six 2 min washes of DCM.
3. A three fold excess of next Boc amino acid was added (in 1 0 ml DCM per gm of
resin) and allowed to mix thoroughly with the resin for about 5 min. An equivalent
amount of DCC dissolved in 5 ml of DCM was then added and the reaction allowed to
proceed for half an hour.
4. The reaction bye product DCU was washed off with three 2 min washes of 33%
EtOH/DCM followed by three DCM washes. Steps 3 and 4 were repeated a second
time in case of all amino acids to enable double couplings. Completion of couplings
was checked by the picric acid test.
5. After the addition of all the 13 amino acids, the resin was washed with DCM
thoroughly and dried. The weight of the resulting dried resin was also recorded.
The final weight of the resin was 2.0g.
Cleavage of peptide from the resin
Cleavage and deprotection of the peptide was effected by treatment with
trifluoroacetic acid (TFA) thioanisole (TA) : metacresol (MC) : ethanedithiol (EDT) in the
ratio of 10:1 :1 :1 v/v at room temperature for 12 h. After removal of the TFA by
evaporation, the residue was triturated with ether to yield the peptide. The peptide was
washed extensively with ether.
101
Synthesis of SLSR peptide
The SLSR peptide having the sequence of SLSRYAKLANRLA was synthesised by solid
phase method in collaboration with Dr Mathew Chandy of M.G. University, Kottayam,
Kerala.
Purification of peptides
SPF peptide was purified by HPLC on a reverse phase ultrapore RPMC 5 11m C-8
column [4.6 x 75 mm] from Beckman Instruments. The solvent system consisted of (A)
10% isopropanol in 0.1% TFA and (B) 50% isopropanol in 0.1% TFA. A linear gradient of
0-100% of B in A in 30 min was run at a flow rate of 1 ml/min. The peak eluting around
15 mins gave the correct amino acid analysis and this was further confirmed by sequencing
on a. gas phase sequencer (Applied Biosystems Model 470A connected to an online PTH
analyser 120A).
SLSR peptide was also purified by reverse phase HPLC on a 11Bondapak C18 column
(3.9 x 300 mm) from Waters, using a solvent system of (A) 0.1% aqueous TFA and (B)
0.1% TFA in acetonitrile. The gradient consisted of 0-25% B in A from 0-10 min, and
25-70% of B in A from 10 to 80 min. The peak eluting at 29.5 min gave the correct
amino acid composition and was further characterized by sequencing as above.
Amino acid analysis.
Amino acid analysis was carried out as described in Chapter 2.
Antibacterial assays
The antibacterial assays were done as described in Chapter 3.
Bacteriolytic assays
Bacteriolytic activity on E. coli and inner membrane permeability experiments
were also done as described in Chapter 3. For determining the effect of membrane
depolarization on the activity of SPLN and SPF, ONPG influx experiments were done in the
presence of carbonyl cyanide -m-chlorophenylhydrazide (CCCP).
Hemolytic assays
Rat erythrocytes were isolated by centrifugation of freshly collected blood followed
by three washes with 5 mM phosphate buffer (pH 7.4) containing 150 mM NaCI to remove
the buffy coat. Erythrocytes (0.5% v/v) were incubated at 37°C in the same buffer with
different concentrations of the peptide for different time periods, centrifuged and
absorbance in the supernatant was measured at 540 nm. The lysis obtained by treatment of
erythrocytes with 1% triton X-1 00 was taken as 1 00%.
Osmotic protection
Erythrocytes were suspended in 0.135 M NaCI, 5 mM phosphate buffer (pH 7.4) in
30 mM solution of one of the following substances D-Mannitol, sucrose, raffinose PEGs of
molecular weights 600, 1540, 3000 or 4000. Then the peptide was added and hemolysis
was determined as above, after incubating for 30 min at 37oc.
0
The molecular diameters of the substances used were taken as mannitol, 7A; 0 0 0 0 0
sucrose, 9A; raffinose, 11 A; PEG 600, 16A ; PEG 1540, 24A; PEG 3000, 30 A; and PEG 0
4000, 38A (Scherrer and Gerhardt, 1971 ).
103
4.2 RESULTS
Characterization of peptides
The sequences of SPLN, SLSR and SPF are shown in Fig. 4.1. The HPLC profile of
crude SLSR peptide is shown in Fig. 4.2(a). The peak eluting at 29.5 min gave the correct
amino acid composition. D, 1.00(1.0); S, 1.7 (2.0); A, 3.2 (3); L, 2.8 (3); Y, 0.9 (1);
K, 1.1 (1) and R, 1.8 (2). (The values indicated in brackets are the theoretical values).
The peak on sequencing gave the correct sequence of SLSRYAKLANRLA. The rerun of the
collected peak is shown in Fig. 4.2(b).
Fig. 4.3(a) shows the HPLC profile of the crude SPF preparation. The peak eluting
at around 15 min gave the correct amino acid composition T, 0.99 (1); S , 0.86 (1); E ,1
(1); P ,1 (1); G ,1.05 (1); I, 1.0 (1); L, 3.1 (3); and K, 2.1 (2). W could not be
estimated due to its destruction during acid hydrolysis. The collected peak gave the correct
sequence of PKLLETFLSKWIG. The rerun of the collected peak on HPLC is shown in Fig.
4.3(b).
HPLC purified peaks of the peptides were used for further studies.
Antibacterial activity
The percentage inhibition of growth of E. coli W160-37 cells in logarithmic phase
of growth, on incubation with synthetic peptides corresponding to the two putative
amphiphilic a-helical regions of SPLN ,was compared with the effect of SPLN. The results
are summarised in Table 4.1. The minimal inhibitory concentrations (MICs) of SLSR and
SPF are 60 Jlg/ml and 50 Jlg/ml respectively as compared to 30 Jlg/ml of SPLN. The
fragment SPF which was comparatively more active was chosen for further
characterization. The antibacterial activity of SPLN has been shown to arise from its
ability to alter the peremability properties of the bacterial inner membrane (Chapter 3).
Hence the bacteriolytic activity of SPF and its effect on the membrane permeability of the
SPLN:
SDEKASPDKHHRFSLSRYAKLANRLANPKLLETFLSKWIGDRGNRSV 10 20 30 40 +1
SLSR:
SLSRYAKLANRLA 14 26
SPF:
PKLLETFLSKWIG 28 40
Fig 4.1 Sequences of SPLN, SLSR and SPF peptides
b
0 10 20 30 40
TIME {min)
Fig. 4.2 HPLC profile of SLSR peptide (a) crude peptide preparation. The peak eluting at
29.50 min gave the correct amino acid composition and sequence. This peak was -0 (JI
collected and rerun, (b) purified SLSR peptide.
a.
b
0 7·5 15 22·5 30
TIME (min)
Fig. 4.3 HPLC profile of SPF peptide (a) crude peptide preparation. The peak eluting at
- 15 min gave the correct amino acid composition and sequence. This peak was
collected and rerun, (b) purified SPF peptide. -0 (J")
107
Table 4.1
Antibacterial activity of synthetic peptides against E. coli W160-37
Concentration % inhibition of growth
of peptide Jlg/ml
SLS SPF SPLN
1 0 0 0 15
2 0 1 5 25 95
30 50 60 1 0 0
4 0 75 90 1 0 0
50 9 0 100 1 0 0
60 100 1 0 0 1 0 0
108
bacterial inner membrane were studied. Incubation of strains of E. coli W 160-37, CSH
57 (lac y-} and C-90 (constitutive for alkaline phosphatase} cells with varying
concentrations of SPF for different intervals of time and analysis of the cell free
supernatant did not reveal the presence of p-galactosidase (a cytoplasmic enzyme} and
alkaline phosphatase (a periplasmic enzyme}. Therefore SPF does not have the ability to
lyse bacteria.
The activity of p-galactosidase in sedimented E. coli W160-37 cells as a function of
time and peptide concentration is shown in Fig. 4.4. At a concentration of 100 J.l.g/ml,
there is a gradual increase in the activity of p-galactosidase until about 30 min, followed
by a rapid rise in activity approaching the value of the enzyme activity when the inner
membrane of E. coli is permeabilised completely by a few drops of chloroform and SDS. At
a peptide concentration of 200 J.l.g/ml, a very rapid increase in enzyme activity is observed
even at 30 min. The enhanced activity of p-galactosidase in sedimented E. coli cells in
presence of SPF as compared to the control suggests the presence of an additional pathway
for influx of ONPG through the bacterial inner membrane, in addition to the transporter
protein lac permease. In order to confirm that SPF permeabilizes the inner membrane of
E. coli, thereby providing an additional pathway for influx of ONPG, experiments were done
in a strain of E. coli which lacks lac permease. Fig. 4.5 shows the activity of p
galactosidase in sedimented E. coli CSH 57 (lac Y} cells in which p-galactosidase is
preinduced by isopropylthiogalactoside IPTG}. As a result of incubation with SPF,
considerable p-galactosidase is detected, confirming the ability of the peptide SPF to
permeabilise the inner membrane of E. coli.
The effect of depolarising the bacterial inner membrane on the ability of SPLN and
SPF to alter the permeability character of bacterial inner membrane was checked by
performing the experiment in the presence of CCCP. As is evident from Table 4.2 there is
no apparent change in the effect. Hence the effect of these two peptides on the inner
membrane of E. coli is not dependent on the metabolic state of the cell.
109
100
>-..... 80 ->
..... (.)
<t _J
60 <t ..... 0 ..... ~ 40 0
20
. o~----~------_.------~-------30 60 90 120
Tl ME (Min.)
Fig. 4.4 Effect of SPF on the influx of ONPG in E. coli W160-37. .1, control (no SPF);
0, 100 J.l.g/ml SPF; andCJ, 200 11g/ml SPF. The cells were grown in minimal A
medium containing 0.4% (w/v) lactose to a A600 of 0.6 and incubated with SPF
at 37°C for the stated period. The cells were centrifuged and the influx of ONPG
through the inner membrane of the cells was estimated by the activity of p-
galactosidase in the cytoplasm. Total p-galactosidase was determined by treating
the cells with a few drops of 0.1% SDS/chloroform and this activity was taken
as 100%. The values in the Y -axis are percentages of the total activity and are
taken as an indicator of the influx of ONPG.
Fig. 4.5
>t-> tu <!
_J
~ 0 t-~ 0
110
100
30 60 90
TIME (Min)
Effect of SPF on the influx of ONPG into E. coli CSH 57 (lac Y) !'!., control (no
SPF; O; 100 ~-tQ/ml SPF;O, 200 ~-tQ/ml of SPF: t, 400 1-1g/ml of SPF. Other
details are as described in the legend of Fig. 4.4
111
Table 4.2
Effect of SPLN and SPF on the influx of ONPG into E. coli W160-37 in the presence and absence of CCCP
% ONPG influx in the presence of
Time Control
(min) CCCP SPLN SPLN SPF SPF +
100 ~M 150 ~g I +CCCP 200 ~g/ CCCP
ml 100 ~M ml 100 ~M
1. 0 1 5. 75 1 5. 5 51.8 60 18.7 16.5
1 5 1 7 1 8 69.5 80 21.8 33.2
30 1 7 2 2 87 87 33 44.7
112
Hemolytic activity of the peptides
The hemolysis of rat erythrocytes by SPLN, SLSR and SPF peptides was investigated
as a function of time and peptide concentration . Both SPLN and SLSR peptides did not show
any hemolysis upto 30 J.lM concentration whereas SPF peptide was hemolytic. Fig. 4.6
shows the hemolysis of erythrocytes as a function of the concentration of SPF. Upto 6 J.lM,
very little lysis is observed and 100% lysis is observed at 30 J.lM. The time course of
lysis of erythrocytes by the peptide is shown in Fig. 4.7. It is evident that hemolysis
occurs gradually and is not a rapid process. In order to determine whether the lysis was
due to a colloid- osmotic process and also to determine the size of the membrane lesion,
hemolysis of the red blood cells was studied in the presence of peptide and various
osmoprotectants. The lysis data is presented in Fig. 4.8. Protection to lysis is observed to
some extent in the presence of PEG 3000 and to a large extent in the presence of PEG 4000,
0
indicating that the lesions produced by the peptide are 36-40 A in diameter. The osmotic
protection at various concentrations of the peptide was investigated next. The data shown in
Fig. 4.9 indicates that even at a peptide concentration of 8 11M when - 40% lysis is
observed in the absence of any osmoprotectant, lysis is prevented only by PEG 3000 and
PEG 4000. The perturbation of the red blood cell membrane did not, however, result in the
release of membrane fragments into the supernatant as no phospholipid was detected by
lipid estimation assays, in the lysis experiments. The membrane perturbing abilities of
SPF were completely abolished on preincubation of the peptide with trypsin, showing that
shorter fragments of SPF generated by trypsin do not possess membrane perturbing
ability.
In order to gain an insight into the structural basis of the membrane perturbing
ability of SLSR and SPF, the circular dichroism spectra of the peptides were examined in
micelles of SDS (Fig 4.10 & Fig 4. 11 ). Two minima, approximately at 205 and 222 nM
and a cross over at - 200 nm were observed showing that the peptides are predominantly
a.-helical.
100
-~ 0 -(f)
(f)
>-...J 0 50 ~ w J:
Fig. 4.6
6 12 18 24 30
PEPTIDE CONCENTRATION (jJ M)
Hemolysis of rat erythrocytes as a function of SPF
concentration. Erythrocytes (0.5% v/v) were incubated in
phosphate buffered (1 0 mM) isotonic saline containing various
concentrations of SPF for 20 min.
--t.:l
-~ 0 -en en > ~ 0 :E laJ :t:
Fig. 4.7
114
100
80
60
40
20
10 20
TIME (Min)
Hemolysis of rat erythrocytes of SPF as a function of time. A) 12 j..iM; e,)18 j..iM; 0, 24 j..iM. Experimental conditions were
identical to that of Fig. 4.6 except that hemolysis was
determined at different times of incubated with different
concentrations of SPF.
Fig. 4.8.
-'ft. 100 -en -en ~ 0 ~ UJ J:
50
0 0 0 ~ (0 I()
C) C)
-PEG. 3000
10 20 30 40 •
DIAMETER (A )
115
SPF induced hemolysis in the presence of various osmoprotectants. The
erythrocytes (0.5% v/v) were suspended in 0.135 mM NaCI, 5 mM phosphate
buffer (pH 7.4) and 30 mM protectant. Subsequently, SPF (final concentration
30 JlM) was added and hemolysis was determined after incubation for 30 min at
-~ 0 -(/) -(/)
~ 0 ~ llJ :X:
Fig. 4.9
•
20
116
8 16 24
PEPTIDE ( jJ M )
Protection of hemolysis by osmoprotectants at various concentrations of SPF. 0,
PEG 600; ~. PEG 1540; 0 , PEG 3000; e, PEG 4000. Assay conditions were
same as in Fig. 4.8 except that the concentration of SPF was varied.
·-0 E
X
~ I <I>' ~ -24
Fig. 4.10
A 8
)dnm) R24 A22
L15
Circular dichroism spectrum (CD) and helical wheel projection of SLSR peptide.
(A} CD spectrum in micelles of sodium dodecyl sulphate (30 mM)., Peptide
concentration = 0.05 mg/ml. Spectrum was recorded in a Jobin Yvon
Dichrograph V Spectropolarimeter at 25°C in cells of 1 mm path length. e
values are mean residue ellipicitities. (B) helical wheel projection of SLSR
peptide. Polar residues are underlined.
---.J
·-0 E
"'0
E 0
0\ CD
"'0
tC') ...
•o
Fig. 4.11
A
24 ,.....----------....,
12
A (nm) 20~ 21!5 22!5 23!5 24!5
CD spectrum and helical wheel projection of SPF peptide. (A) CD spectrum in
micelles of sodium dodecyi sulphate (30 mM). Peptide concentration = 0.05
mg/ml. Recording conditions and other details were same as in Fig. 4.1 0. (B)
helical wheel projection of SPF peptide. Polar residues are underlined.
L35 P28 139
B
E32
536
K29
K37
--00
119
4.3 DISCUSSION
Amphiphilic secondary structures play an important role in the activity of
antimicrobial peptides, cytolytic peptides and apolipoproteins (Kaiser and Kezdy, 1984;
1987). Many of these peptides bind to phospholipid surfaces by adopting an amphiphilic
a.-helical conformation. Detection of potential amphiphilic a.-helical regions in a protein
is easily done by constructing helical-wheel diagrams (Schiffer and Edmundson, 1967).
(The possibility of an amphiphilic p strands is detected much more easily by observing a
regular alternation of hydrophobic and polar amino acid residues in a sequence). However,
helical-wheel diagram gives only a qualitative picture of amphiphilicity of a sequence.
Hydrophobic moment <J.l.H> (Discussed in detail in earlier Chapters) on the other hand gives
a quantitative picture of asymmetry of distribution of polar residues. 'Hydrophobic
moment' plot (Eisenberg, . 1984) goes one step further in classifying helices into
'globular' 'transmembrane' and 'surface seeking' helices. While the success rate of
'hydrophobic moment' plots in detecting transmembrane segments in a protein is not high,
it is fairly successful in detecting 'surface seeking' or 'membrane binding' domains in
cytolytic/antimicrobial peptides (Eisenberg and Wesson, 1990). However, it does not
throw any light on the other structural attributes of the peptide like the role of the
flanking regions or the length of the peptides in their activities. Hence, it would become
necessary to synthesize these segments to study their importance in determining the
activity of the parent peptide.
Very few cytolytic/antimicrobial peptides are known which are 13 to 14 residues
in length, whose entire sequences form amphiphilic secondary structures. These include
the mastoparans and crabrolin (Argiolas and Pisano, 1985) which form a.-helical
structures and a dodecapeptide containing disulfide bridges from bovine neutrophils
(Romeo et al., 1988). Recently, a series of cationic antimicrobial peptides, with
amphiphilic p structure and having disulfide bridges (17-18 residues in length) from
hemocytes of horseshoe crabs named Tachyplesins and polyphemusins have been discovered
120
(Miyata et al., 1990). Many other peptides like magainins (Zasloff, 1987) and cecropins
(Boman et al., 1991) are considerably longer and only a part of them will be amphiphilic
and plot in the surface region of the hydrophobic moment plot. Cecropins intact have two
amphiphilic a-helical segments separated by a hinge region. Similarly SPLN also has two
putative amphiphilic segments between residues 14-26 with a sequence of
SLSRYAKLANRLA (SLSR peptide) and 29-40 with a sequence of KLLETFLSKWIG separated
by a probable hinge region consisting of a dipeptide NP. To independently evaluate the
importance of these regions, they were synthesized and characterized in this study .
Both synthetic peptides corresponding to the two amphiphilic regions designated as
SLSR and SPF respectively show antibacterial activity. SPF is comparatively more active
than SLSR and hence was further characterized. Both the peptides did not inhibit the in
vitro transcription of E. coli RNA polymerase unlike SPLN. SPLN has also been shown to
alter the inner membrane permeability of E. coli and subsequently kill the cells (Chapter
3). Hence the inner membrane permeability of E. coli was assessed in the presence of SPF.
The normal cytoplasmic membrane acts as a selective permeability barrier protecting the
cell from the external environment. The modification of permeability can be readily
recognised by the release of small cytoplasmic metabolites from the cells like K+,
phosphate, amino acids and sugars, and of even macromolecules which cannot normally leak
out. The appearance of these molecules outside the cell, measures the extent of membrane
damage. At the same time internal enzymes may be rendered accessible to substrates which
normally do not pass through the membrane. Depending on the membrane damage and the
type of the cell, a slight change in membrane permeability may have more or less serious
consequences. If the cell has little capacity for membrane repair it will undergo lysis.
SPF peptide does not cause the release of p-galactosidase into the medium from E.
coli cells. Its antibacterial activity appears to stem from its ability to insert into the
inner membrane of E. coli and render it permeable to substrates like ONPG which cannot
cross the membrane without the presence of a protein transporter as discussed in Chapter
3. The breakdown of this permeability barrier eventually results in inhibition of growth
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of E. coli. Defensins kill only metabolically active cells and are protected by membrane
depolarising agents such as CCCP and DNP (Lehrer et al., 1989) . Both SPLN and SPF are
capable of bringing about permeability changes in the bacterial inner membrane even in
the presence of CCCP and hence their activities do not depend on the metabolic activity of
the cell.
Antimicrobial peptides with potent eukaryotic cell-lytic or cytotoxic effect, will
be unsuitable for any eventual therapeutic use. Cytolytic activity is usually tested on
erythrocytes as they provide particularly suitable experimental material. This is because
of (i) the simplicity of the cell compared with others and (ii) the chemical composition of
the bilayer leaflet being well understood. The estimation of the degree of lysis of
erythrocytes is simple and involves the measurement of hemoglobin released under defined
conditions. Both SPLN and SLSR peptide did not show any hemolytic activity. SPF,
however, lysed erythrocytes. The gradual release of hemoglobin during lysis as well as the
lysis protection experiments indicate that a colloid-osmotic process( discussed in detail in
Chapter 5) is involved in hemolysis. Although both SLSR and SPF peptides are comprised
of only 13 amino acid residues each, circular dichroism studies indicate that they adopt a
helical structure particularly in hydrophobic environment. Helical-wheel projections of
these segments(Fig 4.1 0&4.11) indicate that both are amphiphilic in nature. The polar
face of SLSR peptide comprises of R, K, Sand N residues and the apolar face of L, A andY.
In the case of SPF, the polar face comprises of E, S and K and the apolar face of I, P, L, W
and F residues. SLSR, despite being hydrophilic, has a high hydrophobic moment while SPF
has average hydrophobicity (0.24) and hydrophobic moment (0.41 ). As mentioned
earlier, these peptides represent two of very few peptides of 13 residues having
antibacterial activity. Even though both peptides show antibacterial activity, individually
they are both only half as potent as SPLN. Thus, higher antibacterial activity of SPLN could
arise due to the additive effects of both the segments. SPF shows hemolytic activity also
whereas SPLN is not hemolytic. The activity of this segment of SPLN thus appears to be
modulated by the flanking regions.
