- Email: [email protected]
Detection of lipopolysaccharide binding peptides by the use of a lipopolysaccharide-coated piezoelectric crystal biosensor
ANALYTICA CHIMICA ACTA ELSEVIER
Analytica
Chimica
Acta 340
(1997)49-54
Detection of lipopolysaccharide binding peptides by the use of a lipopolysaccharide-coated piezoelectric crystal biosensor Hsien-Chang aInstitute bDepartment
Chang”, Chaur-Chin
Yang”, Trai-Ming Yehb’*
of Biomedical Engineering, National Cheng Kung University, Tainan 70101, Taiwan
of Medical Technology, Medical College, National Cheng Kung Universir), No. I University Road, Tainan 70101, Taiwan Received 2 August
1996; accepted
25 October
1996
Abstract A method has been developed to monitor the interaction between a lipopolysaccharide (LPS) and LPS-binding peptides using a piezoelectric quartz crystal (PQC). Different pH conditions were evaluated to coat LPS onto AT-cut crystals that had been sputtered with gold and carboxylated with a 4,4-dithiodi(n-butyric acid). The optimal pH for LPS coating onto the crystal was 4-5. Synthetic peptides that represent different regions of human bactericidal/permeability-increasing protein, BP1 (BP1 85-99, BP1 90-101, BP1 157-167) and polymyxin B (PmB) as well as negative control peptide (HBsAg 139-147) were utilized to compare their binding ability to this LPS-coated PQC sensor. The results showed that PmB gave the greatest decrease to the resonant frequency indicating greatest binding ability. BP1 85-99, considered the main part of the LPS binding domain of BPI, was the next greatest, while BP1 157-167 and HBsAg 139-147 showed little response. In addition, BP1 90-101 and PmBmimicking peptide showed intermediate LPS-binding ability, which was less than that of BP1 85-99, but was higher than that of BP1 157-167. These results suggest the PQC biosensor is potentially useful for the detection and comparison of the LPSbinding ability of different peptides by using an LPS-coated piezoelectric crystal. Keywords:
Sensors; Piezoelectric
quartz crystal sensor; Lipopolysaccharide
1. Introduction Lipopolysaccharide (LPS), also known as an endotoxin, is the major outer-membrane constituent of gram-negative bacteria which is composed of three distinct regions: the lipid A, core oligosaccharide, and the O-specific antigen [ 11. Lipid A is responsible for many of the patho-physiological effects associated with gram-negative bacterial infection. These *Corresponding 886-6-236-3956.
author. Tel: 886-6-235-3535
0003-2670/97/$17.00 Copyright PIf SOOO3-2670(96)00520-X
5794 (ext.); fax:
effects include septic shock, pyrogenicity, and disseminated intravascular coagulation [2]. Therefore, to find a drug which can bind to LPS and neutralize its toxicity is very important for clinical therapy. Polymyxin B (PmB), an antibiotic produced by Bacillus polymyxa, is an amphipathic, cationic cyclic peptide [3] which can bind to lipid A and neutralize its toxicity [4]. However, due to its toxicity, PmB is not suitable for systemic use. PmB has been described as a model for designing synthetic peptides which can mimic the structure of PmB in their interaction with LPS [5]. The LPS-binding affinity of
‘$1 1997 Elsevier Science B.V. All rights reserved.
50
H.-C. Chang et al./Analytica Chimica Acia 340 (1997) 49-54
the peptide was estimated by direct competition of the peptide with PmB for LPS, using liquid chromatography (LC). Bactericidal/permeability-increasing protein (BPI), on the other hand, is a 55-60 kDa cationic glycoprotein from the azurophilic granules of polymorphonuclear leucocytes [6]. BP1 can also neutralize the toxic effects of LPS [7]. The LPS-binding domain of BP1 has recently been studied using synthetic peptides representing different regions of the BPI. The strongly basic and helical amphiphilic regions of BP1 amino acids, 85-99 and 90-301, are important for BP1 to bind to the negatively charged LPS [8,9]. However, all these studies were done by LC or an LPS neutralization test to detect the interaction of synthetic peptides and LPS, which is very timeconsuming and difficult to perform [5,8,9]. In order to speed up and simplify the detection method, we developed a system using an LPS-coated PQC to detect the LPS-binding ability of different synthetic peptides. It is well known that the resonant frequency of an oscillating piezoelectric (PZ) crystal can be affected by a minute change in mass, at the crystal surface [lo]. The relationship between surficial mass change (AM) and resonant-frequency shift (AF) of a crystal, vibrating at the fundamental frequency F, was given by the following equation [I I]:
this system was found to be similar to data, previously reported by LC and an LPS neutralization test [5,8,9].
2. Experimental 2.1. Chemicals
and synthetic peptides
2-(N-Morpholino)ethanesulfonic acid (MES) and polymyxin B (PmB) were purchased from Sigma Chemical Company (St. Louis, MO). 4,bDithiodi(nbutyric acid) (DTBA) was obtained from Tokyo Kasei Organic Chemicals (Tokyo). LPS, purified from Escherichia coli 011 l:B4, was obtained from Difco (Detroit, MI). Three different peptides corresponding to different regions of the BP1 [6] were synthesized by the Peptide Synthesis Center, National Science Council, Taipei, Taiwan. The sequences of these three peptides, together with a PmB-mimicking peptide (mimic-PmB) are shown in Fig. 1. An additional peptide corresponding to amino acids 139-147 of hepatitis-B virus surface-antigen (HBsAg) [15] was also synthesized to serve as a negative control. The purity of all of these peptides was confirmed by LC and amino-acid analysis. The solutions used in this study were all prepared using water from a Millipore Milli-Q purification system.
AF = -F2AM/A(~q&0.5 where A is the crystal electrode area, ps is the density of quartz and ,LL~is the shear modulus. The technology has been applied to detect biomedically interesting substances, such as immunoglobulin G, using protein A-modified crystal [ 121; different pathogens, such as the bacteria [ 12,131, and human herpes viruses [14] are detected using a specific antibody-modified crystal. In this study, different synthetic peptides which represent different regions of a BP1 were compared with polymyxin B for their LPS-binding ability, using an LPS-coated PQC probe. The AT-cut quartz crystal, with a basic resonant frequency of 10 MHz, was coated with LPS. The resulting LPS-coated crystal was positioned inside a home-made system and the resonant frequency shift resulting from the binding of peptides to the LPS-coated crystal was measured. The LPS-binding ability of different peptides detected by
BP1 90-101 Lys-Trp-Lys-Ala-Gin-Lys-Arg-Phe-Leu-Lys-ble%Ser
BP1 85-99 Ile-Lys-lle-Ser-Gly-Lys-Trp-Lys-Ala-Gln-Lvs-Ar~-Phe-Leu-LvS
BP1 157-167 Leu-Phe-His-Lys-Lys-Ile-Glu-Ser-Ala-Leu-Arg
Mimic-PmB Ile-Lys-Thr-Lys-Lys-Phe-Leu-Lys-Lys-Thr
HBV (HBsAg
139-147)
Cys-Thr-Lys-Pro-Thr-Asp-Gly-Asn-Cys-Tyr Fig. 1. Sequences
sequence lined.
between
of peptides used in this study: the homologous BPIs and PmB-mimicking peptide are under-
H.-C. Chang et al. /Analytica
2.2. Quartz crystal microbalance
system
The PZ crystals used in this work were AT-cut with a basic resonant frequency of 10 MHz. Each side of the crystal was gold-sputtered to a diameter of 3 mm. The home-made system is shown schematically in Fig. 2A; the oscillator circuit was connected to a Hewlett-Packard 53 13 1A universal counter; the digital output of the frequency counter was interfaced to a 486 personal computer by the IEEE-488 interface and operated by HP-VEE software. The PZ probe was set in the measuring cell as shown in Fig. 2(B); one of the gold electrodes was connected to the side of crystal which was immersed in the test solution. The other gold electrode was connected to the other side of the crystal which was exposed to air. 2.3. LPS immobilization
on the crystal
The gold-sputtered crystal was first immersed in a water, ethanol (1 : 1 v/v) solution containing 0.05 mol DTBA for 1 h, and washed sufficiently
(4
Chimica
Acta 340 (1997) 49-54
51
with distilled water. Next, the carboxyl groupmodified electrode was set in the cell described above (Fig. 2B) and 4 ml of the measuring solution (phosphate buffer or MES buffer) was added to the cell chamber. After the crystal resonant frequency reached to a constant value, 0.2 ml of the LPS solution (1 mg ml-‘, pH 7.4 in phosphate buffer) was added and mixed. The real-time change in the frequency was recorded for 10 min. To evaluate the optimal pH condition for LPS immobilization on the surface of the carboxyl group-modified electrode, pH values were adjusted in the range 3.5-10 in a series of measuring solutions. 2.4. Detection of peptide binding to the LPS coated PQC LPS was bound to the crystal by adding 0.15 ml of LPS solution (1 mg ml-‘) to the cell containing the crystal and 4 ml of 0.05 mol MES buffer (pH 4.5). After the crystal resonant frequency had decreased to a constant value, the solution was replaced with fresh MES solution three times, and finally filled with 4 ml of MES solution (pH 4.5) for testing the bindingability of different peptides. 5 1.11of peptide solution (1 mg ml-‘, pH 7.4 in phosphate buffer) was added five times successively and the decrease in frequency was recorded.
3. Results and discussion Buffer Solution 3.1. Effect oj’pH on LPS coating of the crystal
Gold Electrode
Cell
\ ‘Quartz
Plate
Fig. 2. (A) Schematic diagram of the PQC measurement set-up; and (B) Cross-section of the measuring cell showing the arrangement in detail.
When the gold-sputtered crystal was immersed in DTBA solution, the adsorption of organosulfuric compounds on the gold surfaces by chemisorption via. the thiol groups, leaving the carboxyl groups, were exposed on the crystal-electrode surface [ 161. LPS was added to the DTBA-modified PQC at various pH values (3.0-7.0). The frequency-time response for the process of LPS immobilization on the PQC is shown in Fig. 3. From the frequency changes, it was found that the optimal pH for LPScoating of the carboxyl group-modified surface was 4-5 (Fig. 4). Since the pK, value of the carboxyl group is also 4-5, it is suspected that LPS binds electrostatically to the DTBA-modified electrode.
H.-C. Chang et aL/Analytica
Time (SK)
250
R E $
200
fn 150 2‘ 5 loo 5 e! IA
50
;
0 400
800
1200
looa
1400
Time (set)
(b)
Fig. 3. Relationship between reaction time and steady resonantfrequency shift after LPS modification of the electrode: (A) MES buffer (pH 4.5, 4 ml); (B) phosphate buffer (pH 7.4, 4 ml). LPS solution (I mg ml-‘) was added successively in volumes of: (a) 20, (b) 20, (c) 20, (d) 20, (e) 20, (f) 50 and (g) 50 ul.
G 1200
h
3 IOOO800600400 -
/
200OI
02
\ 4
6
8
10
12
pH value Fig. 4 Effect of pH on LPS immobilization concentration of LPS is 50 ug ml-‘.
Chimica Acta 340 (1997) 49-54
showed that, as expected, PmB had the greatest binding ability, and BP1 85-99, the functional domain of BPI, also had very good ability to bind to the LPScoated crystal. The frequency change was proportional to the amount of peptides added, with the correlation coefficient (r’) 0.995 for PmB and 0.968 for BP1 85-99 (n=5). BP1 157-167 and HBV peptide, on the other hand, showed only slight nonspecific binding ability compared to PmB and BP1 85-99 (Fig. 5). In our previous study, we noticed that BP1 90-101 also contained the LPS binding and antibacterial activities. Therefore, the frequency shift of these different peptides was compared; the results are shown in Fig. 6. The binding affinity of these peptides was in the order: PmB>BPI 85-99>BPI 90IOl=mimic-PmB>BPI 157-167=HBV peptide. These results are consistent with other groups’ data [5,9]. In addition, by comparing the sequence BP1 8599, BP1 90-101 and PmB-mimicking peptide, the five amino acid sequence, Lys-Lys-Phe-Leu-Lys, is strikingly similar among these peptides (Fig. 1). One discrepancy is that the second Lys in human BP1 is replaced by Arg, which, however, will not make any change on the net charge (positive) in this segment of the peptide. Therefore, this structure may enable the peptides to bind to the negatively charged lipid A of LPS. 500
B
400
K r a z?
300
E 1
200
8 li 100
on the PQC. The
0
3.2. Detemination of LPS-binding different peptides
ability of
The LPS-coated PQC was used to detect the LPSbinding ability of different synthetic peptides. Fig. 5
3
Peptide concentration &g/ml) Fig. 5. Effect of different peptides on the resonant-frequency shift of an LPS-modified PQC. Data represent the mean of three samples&SD.
H.-C. Chang et al./Analytica
Chimica Acta 340 (1997) 49-54
53
property will allow us to develop a flow injection analysis system which can be reused several times.
4. Conclusions
Peptides Fig. 6. Frequency changes induced by different peptides 7.5 pg ml ~I. Data represent the mean of three samples+SD.
3.3. Regeneration
of an LPS-cooted
at
crystal
From previous results, we know that LPS binding to a DTBA-modified crystal is pH dependent. Therefore, we can regenerate the probe after finishing the test by washing it with an acidic buffer, such as pH 2.5 glycine buffer. The frequency of the crystal after LPS coating decreased by about 210 Hz (Fig. 7a) and reverted to the base-line level after glycine-buffer washing for 3 min (Fig. 7b). The washed crystal can be remodified with LPS and used again without significant change in the frequency (Fig. 7~). This
0
400
800
1200
1600
2000
To deduce the toxicity of LPS, we need a drug which can bind and neutralize LPS before it stimulates the cells. The present study demonstrates that an LPS-coated PQC was useful for the detection and comparison of the LPS-binding ability of different synthetic peptides. The results show great similarity with previously reported results using LC and LPS neutralization tests. The advantage of this method to detect the LPS-binding ability of different peptjdes is that it is much easier and quicker than these other tests. Therefore, the LPS-coated PQC model could provide an attractive alternative method for estimating the LPS-binding ability of new drugs.
2400
2800
Time (set) Fig. 7. Real-time frequency change of crystal after LPS coating and regeneration: (a) LPS was coated onto the crystal by adding 100 pi of LPS solution (1 mg ml-‘) as described in the text; (b) this LPS-coated crystal was then washed with 3 ml of glycine buffer (pH 2.5) for 3 min; and (c) After changing to 3 ml fresh MES solution, the crystal was re-coated with LPS.
Acknowledgements This work was supported by grants NSC 84-22 14E-006-001 and NSC 82-0412-B-006-083 from the National Science Council, Taiwan.
References [I] C.R.H. Raetz, Rev. Biochem., 59 (1990) 129. [2] D.C. Morrison and R.J. Ulevitch. Am. J. Pathol., 93 ( 1978) 527. [3] D.R. Storm, K.S. Rosenthal and P.E. Swanson, Ann. Rev. Biochem., 46 (1977) 723. [4] H.S. Warren, S.F. Amato, C. Fitting, K.M. Black, P.M. Loiselle, M.S. Pastemack and J. Cavaillon, J. Exp. Med., 177 (1993) 89. IS] A. Rustici. M. Velucchi, R. Faggioni, M. Sironi. P. Ghezzi, S. Quataert, 8. Green and M. Porro, Science, 259 (1993) 361. [6] P.W. Gray, G. Flaggs, S.R. Leong, R.J. Gumina, J. Weiss, C.E. Ooi and P. Elsbach, J. Biol. Chem., 264 (1989) 9505. (71 M.N. Marra, C.G. Wilde, MS. Collins. J.L. Snable. M.B. Thornton and R.W. Scott, J. Immunol., 148 (1992) 532. [S] T.M. Yeh, SC. Chao and H.C. Chang, Chinese J. Microbial. Immunol., 27 (1994) 120. [9] R.G. Little, D.N. Kelner, E. Lim, D.J. Burke and PJ. Conlon, J. Biol. Chem., 269 (1994) 1865. [IO] M. Minunni, M. Mascini, G.G. Guilbault and B. Hock, Anal. Len., 28 ( 1995) 749.
54
H.-C. Chang et al./Analytica
[ll] G.Z. Sauerbrey, Physik, 1.55 (1959) 206. [12] H. Muramatsu, J.M. Dicks, E. Tamiya and I. Karube, Anal. Chem., 59 (1987) 2760. [ 131 P. Michael, G.G. George and H. Bertold, Enzyme Microbial. Technol., 14 (1992) 230.
Chimica Acta 340 (1997) 49-54 [14] B. Konig and M. Gratzel, Anal. Chem., 66 (1994) 341. [IS] Y.M. Thanavala, S.E. Brown, C.R. Howard, 1.M. Roitt and M.W. Steward, J. Exp. Med., 164 (1986) 227. [ 161 R.G. Nuzzo, B.R. Zegarki and L.H. Dubois, J. Am. Chem. sot., 109 (1987) 733.
Analytica
Chimica
Acta 340
(1997)49-54
Detection of lipopolysaccharide binding peptides by the use of a lipopolysaccharide-coated piezoelectric crystal biosensor Hsien-Chang aInstitute bDepartment
Chang”, Chaur-Chin
Yang”, Trai-Ming Yehb’*
of Biomedical Engineering, National Cheng Kung University, Tainan 70101, Taiwan
of Medical Technology, Medical College, National Cheng Kung Universir), No. I University Road, Tainan 70101, Taiwan Received 2 August
1996; accepted
25 October
1996
Abstract A method has been developed to monitor the interaction between a lipopolysaccharide (LPS) and LPS-binding peptides using a piezoelectric quartz crystal (PQC). Different pH conditions were evaluated to coat LPS onto AT-cut crystals that had been sputtered with gold and carboxylated with a 4,4-dithiodi(n-butyric acid). The optimal pH for LPS coating onto the crystal was 4-5. Synthetic peptides that represent different regions of human bactericidal/permeability-increasing protein, BP1 (BP1 85-99, BP1 90-101, BP1 157-167) and polymyxin B (PmB) as well as negative control peptide (HBsAg 139-147) were utilized to compare their binding ability to this LPS-coated PQC sensor. The results showed that PmB gave the greatest decrease to the resonant frequency indicating greatest binding ability. BP1 85-99, considered the main part of the LPS binding domain of BPI, was the next greatest, while BP1 157-167 and HBsAg 139-147 showed little response. In addition, BP1 90-101 and PmBmimicking peptide showed intermediate LPS-binding ability, which was less than that of BP1 85-99, but was higher than that of BP1 157-167. These results suggest the PQC biosensor is potentially useful for the detection and comparison of the LPSbinding ability of different peptides by using an LPS-coated piezoelectric crystal. Keywords:
Sensors; Piezoelectric
quartz crystal sensor; Lipopolysaccharide
1. Introduction Lipopolysaccharide (LPS), also known as an endotoxin, is the major outer-membrane constituent of gram-negative bacteria which is composed of three distinct regions: the lipid A, core oligosaccharide, and the O-specific antigen [ 11. Lipid A is responsible for many of the patho-physiological effects associated with gram-negative bacterial infection. These *Corresponding 886-6-236-3956.
author. Tel: 886-6-235-3535
0003-2670/97/$17.00 Copyright PIf SOOO3-2670(96)00520-X
5794 (ext.); fax:
effects include septic shock, pyrogenicity, and disseminated intravascular coagulation [2]. Therefore, to find a drug which can bind to LPS and neutralize its toxicity is very important for clinical therapy. Polymyxin B (PmB), an antibiotic produced by Bacillus polymyxa, is an amphipathic, cationic cyclic peptide [3] which can bind to lipid A and neutralize its toxicity [4]. However, due to its toxicity, PmB is not suitable for systemic use. PmB has been described as a model for designing synthetic peptides which can mimic the structure of PmB in their interaction with LPS [5]. The LPS-binding affinity of
‘$1 1997 Elsevier Science B.V. All rights reserved.
50
H.-C. Chang et al./Analytica Chimica Acia 340 (1997) 49-54
the peptide was estimated by direct competition of the peptide with PmB for LPS, using liquid chromatography (LC). Bactericidal/permeability-increasing protein (BPI), on the other hand, is a 55-60 kDa cationic glycoprotein from the azurophilic granules of polymorphonuclear leucocytes [6]. BP1 can also neutralize the toxic effects of LPS [7]. The LPS-binding domain of BP1 has recently been studied using synthetic peptides representing different regions of the BPI. The strongly basic and helical amphiphilic regions of BP1 amino acids, 85-99 and 90-301, are important for BP1 to bind to the negatively charged LPS [8,9]. However, all these studies were done by LC or an LPS neutralization test to detect the interaction of synthetic peptides and LPS, which is very timeconsuming and difficult to perform [5,8,9]. In order to speed up and simplify the detection method, we developed a system using an LPS-coated PQC to detect the LPS-binding ability of different synthetic peptides. It is well known that the resonant frequency of an oscillating piezoelectric (PZ) crystal can be affected by a minute change in mass, at the crystal surface [lo]. The relationship between surficial mass change (AM) and resonant-frequency shift (AF) of a crystal, vibrating at the fundamental frequency F, was given by the following equation [I I]:
this system was found to be similar to data, previously reported by LC and an LPS neutralization test [5,8,9].
2. Experimental 2.1. Chemicals
and synthetic peptides
2-(N-Morpholino)ethanesulfonic acid (MES) and polymyxin B (PmB) were purchased from Sigma Chemical Company (St. Louis, MO). 4,bDithiodi(nbutyric acid) (DTBA) was obtained from Tokyo Kasei Organic Chemicals (Tokyo). LPS, purified from Escherichia coli 011 l:B4, was obtained from Difco (Detroit, MI). Three different peptides corresponding to different regions of the BP1 [6] were synthesized by the Peptide Synthesis Center, National Science Council, Taipei, Taiwan. The sequences of these three peptides, together with a PmB-mimicking peptide (mimic-PmB) are shown in Fig. 1. An additional peptide corresponding to amino acids 139-147 of hepatitis-B virus surface-antigen (HBsAg) [15] was also synthesized to serve as a negative control. The purity of all of these peptides was confirmed by LC and amino-acid analysis. The solutions used in this study were all prepared using water from a Millipore Milli-Q purification system.
AF = -F2AM/A(~q&0.5 where A is the crystal electrode area, ps is the density of quartz and ,LL~is the shear modulus. The technology has been applied to detect biomedically interesting substances, such as immunoglobulin G, using protein A-modified crystal [ 121; different pathogens, such as the bacteria [ 12,131, and human herpes viruses [14] are detected using a specific antibody-modified crystal. In this study, different synthetic peptides which represent different regions of a BP1 were compared with polymyxin B for their LPS-binding ability, using an LPS-coated PQC probe. The AT-cut quartz crystal, with a basic resonant frequency of 10 MHz, was coated with LPS. The resulting LPS-coated crystal was positioned inside a home-made system and the resonant frequency shift resulting from the binding of peptides to the LPS-coated crystal was measured. The LPS-binding ability of different peptides detected by
BP1 90-101 Lys-Trp-Lys-Ala-Gin-Lys-Arg-Phe-Leu-Lys-ble%Ser
BP1 85-99 Ile-Lys-lle-Ser-Gly-Lys-Trp-Lys-Ala-Gln-Lvs-Ar~-Phe-Leu-LvS
BP1 157-167 Leu-Phe-His-Lys-Lys-Ile-Glu-Ser-Ala-Leu-Arg
Mimic-PmB Ile-Lys-Thr-Lys-Lys-Phe-Leu-Lys-Lys-Thr
HBV (HBsAg
139-147)
Cys-Thr-Lys-Pro-Thr-Asp-Gly-Asn-Cys-Tyr Fig. 1. Sequences
sequence lined.
between
of peptides used in this study: the homologous BPIs and PmB-mimicking peptide are under-
H.-C. Chang et al. /Analytica
2.2. Quartz crystal microbalance
system
The PZ crystals used in this work were AT-cut with a basic resonant frequency of 10 MHz. Each side of the crystal was gold-sputtered to a diameter of 3 mm. The home-made system is shown schematically in Fig. 2A; the oscillator circuit was connected to a Hewlett-Packard 53 13 1A universal counter; the digital output of the frequency counter was interfaced to a 486 personal computer by the IEEE-488 interface and operated by HP-VEE software. The PZ probe was set in the measuring cell as shown in Fig. 2(B); one of the gold electrodes was connected to the side of crystal which was immersed in the test solution. The other gold electrode was connected to the other side of the crystal which was exposed to air. 2.3. LPS immobilization
on the crystal
The gold-sputtered crystal was first immersed in a water, ethanol (1 : 1 v/v) solution containing 0.05 mol DTBA for 1 h, and washed sufficiently
(4
Chimica
Acta 340 (1997) 49-54
51
with distilled water. Next, the carboxyl groupmodified electrode was set in the cell described above (Fig. 2B) and 4 ml of the measuring solution (phosphate buffer or MES buffer) was added to the cell chamber. After the crystal resonant frequency reached to a constant value, 0.2 ml of the LPS solution (1 mg ml-‘, pH 7.4 in phosphate buffer) was added and mixed. The real-time change in the frequency was recorded for 10 min. To evaluate the optimal pH condition for LPS immobilization on the surface of the carboxyl group-modified electrode, pH values were adjusted in the range 3.5-10 in a series of measuring solutions. 2.4. Detection of peptide binding to the LPS coated PQC LPS was bound to the crystal by adding 0.15 ml of LPS solution (1 mg ml-‘) to the cell containing the crystal and 4 ml of 0.05 mol MES buffer (pH 4.5). After the crystal resonant frequency had decreased to a constant value, the solution was replaced with fresh MES solution three times, and finally filled with 4 ml of MES solution (pH 4.5) for testing the bindingability of different peptides. 5 1.11of peptide solution (1 mg ml-‘, pH 7.4 in phosphate buffer) was added five times successively and the decrease in frequency was recorded.
3. Results and discussion Buffer Solution 3.1. Effect oj’pH on LPS coating of the crystal
Gold Electrode
Cell
\ ‘Quartz
Plate
Fig. 2. (A) Schematic diagram of the PQC measurement set-up; and (B) Cross-section of the measuring cell showing the arrangement in detail.
When the gold-sputtered crystal was immersed in DTBA solution, the adsorption of organosulfuric compounds on the gold surfaces by chemisorption via. the thiol groups, leaving the carboxyl groups, were exposed on the crystal-electrode surface [ 161. LPS was added to the DTBA-modified PQC at various pH values (3.0-7.0). The frequency-time response for the process of LPS immobilization on the PQC is shown in Fig. 3. From the frequency changes, it was found that the optimal pH for LPScoating of the carboxyl group-modified surface was 4-5 (Fig. 4). Since the pK, value of the carboxyl group is also 4-5, it is suspected that LPS binds electrostatically to the DTBA-modified electrode.
H.-C. Chang et aL/Analytica
Time (SK)
250
R E $
200
fn 150 2‘ 5 loo 5 e! IA
50
;
0 400
800
1200
looa
1400
Time (set)
(b)
Fig. 3. Relationship between reaction time and steady resonantfrequency shift after LPS modification of the electrode: (A) MES buffer (pH 4.5, 4 ml); (B) phosphate buffer (pH 7.4, 4 ml). LPS solution (I mg ml-‘) was added successively in volumes of: (a) 20, (b) 20, (c) 20, (d) 20, (e) 20, (f) 50 and (g) 50 ul.
G 1200
h
3 IOOO800600400 -
/
200OI
02
\ 4
6
8
10
12
pH value Fig. 4 Effect of pH on LPS immobilization concentration of LPS is 50 ug ml-‘.
Chimica Acta 340 (1997) 49-54
showed that, as expected, PmB had the greatest binding ability, and BP1 85-99, the functional domain of BPI, also had very good ability to bind to the LPScoated crystal. The frequency change was proportional to the amount of peptides added, with the correlation coefficient (r’) 0.995 for PmB and 0.968 for BP1 85-99 (n=5). BP1 157-167 and HBV peptide, on the other hand, showed only slight nonspecific binding ability compared to PmB and BP1 85-99 (Fig. 5). In our previous study, we noticed that BP1 90-101 also contained the LPS binding and antibacterial activities. Therefore, the frequency shift of these different peptides was compared; the results are shown in Fig. 6. The binding affinity of these peptides was in the order: PmB>BPI 85-99>BPI 90IOl=mimic-PmB>BPI 157-167=HBV peptide. These results are consistent with other groups’ data [5,9]. In addition, by comparing the sequence BP1 8599, BP1 90-101 and PmB-mimicking peptide, the five amino acid sequence, Lys-Lys-Phe-Leu-Lys, is strikingly similar among these peptides (Fig. 1). One discrepancy is that the second Lys in human BP1 is replaced by Arg, which, however, will not make any change on the net charge (positive) in this segment of the peptide. Therefore, this structure may enable the peptides to bind to the negatively charged lipid A of LPS. 500
B
400
K r a z?
300
E 1
200
8 li 100
on the PQC. The
0
3.2. Detemination of LPS-binding different peptides
ability of
The LPS-coated PQC was used to detect the LPSbinding ability of different synthetic peptides. Fig. 5
3
Peptide concentration &g/ml) Fig. 5. Effect of different peptides on the resonant-frequency shift of an LPS-modified PQC. Data represent the mean of three samples&SD.
H.-C. Chang et al./Analytica
Chimica Acta 340 (1997) 49-54
53
property will allow us to develop a flow injection analysis system which can be reused several times.
4. Conclusions
Peptides Fig. 6. Frequency changes induced by different peptides 7.5 pg ml ~I. Data represent the mean of three samples+SD.
3.3. Regeneration
of an LPS-cooted
at
crystal
From previous results, we know that LPS binding to a DTBA-modified crystal is pH dependent. Therefore, we can regenerate the probe after finishing the test by washing it with an acidic buffer, such as pH 2.5 glycine buffer. The frequency of the crystal after LPS coating decreased by about 210 Hz (Fig. 7a) and reverted to the base-line level after glycine-buffer washing for 3 min (Fig. 7b). The washed crystal can be remodified with LPS and used again without significant change in the frequency (Fig. 7~). This
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To deduce the toxicity of LPS, we need a drug which can bind and neutralize LPS before it stimulates the cells. The present study demonstrates that an LPS-coated PQC was useful for the detection and comparison of the LPS-binding ability of different synthetic peptides. The results show great similarity with previously reported results using LC and LPS neutralization tests. The advantage of this method to detect the LPS-binding ability of different peptjdes is that it is much easier and quicker than these other tests. Therefore, the LPS-coated PQC model could provide an attractive alternative method for estimating the LPS-binding ability of new drugs.
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Time (set) Fig. 7. Real-time frequency change of crystal after LPS coating and regeneration: (a) LPS was coated onto the crystal by adding 100 pi of LPS solution (1 mg ml-‘) as described in the text; (b) this LPS-coated crystal was then washed with 3 ml of glycine buffer (pH 2.5) for 3 min; and (c) After changing to 3 ml fresh MES solution, the crystal was re-coated with LPS.
Acknowledgements This work was supported by grants NSC 84-22 14E-006-001 and NSC 82-0412-B-006-083 from the National Science Council, Taiwan.
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