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Aggregation of polyene antibiotics as revealed by absorption spectroscopy
Colloidsand Surfaces A: Physicochemicaland EngineeringAspects, 70 (1993) 2 13-217 Elsevier Science Publishers
213
B.V., Amsterdam
Aggregation of polyene antibiotics as revealed by absorption spectroscopy Zameer Shervani Liposome Research Centre, New Delhi-l 10021, India
Department
of Biochemistry,
University of Delhi South Campus,
(Received 30 March 1992; accepted 6 October 1992) Abstract Methanolic solutions of the heptaene macrolide antibiotics, namely amphotericin B and hamycin, are molecular dispersions. These polyenes were found to be self-aggregated in neutral aqueous solution. The aggregates of hamycin dissociate in acidic or basic aqueous solutions. The smaller chain aliphatic alcohols have a higher solvating power for hamycin.
Keywords: Amphotericin
B; hamycin;
polyene
antibiotics;
self-aggregation;
Introduction The heptaene polyene antibiotics amphotericin B [l-4] and hamycin [S], are known to have potent antifungal activity. It is generally accepted that such polyene antibiotics form channels by interacting with sterol-containing membranes, thus inducing permeability changes in the membrane [6]. The mode of the interaction is related to the aggregation of amphotericin B [7,S]. Ergosterol does not react, or reacts extremely slowly, with monomeric amphotericin B in aqueous solutions of propanol. Traces of small aggregates, probably dimers, enable a cooperative reaction. The interaction with ergosterol is hindered when the antibiotic is in the micellar form. Marked differences in toxicity have been noticed among various batches of the polyene antibiotics. Such differences have been attributed not to the particle size, but to different molecular spatial arrangements of the product, which modify their bioavailability [9]. One of the mechanisms of antibiotic action is considered to be through the autoxidation of amphotericin B, which brings about membrane 0927-7757/93/$06.00
0
1993 -
Elsevier Science Publishers
solvent effect.
lipid peroxidation. The kinetics of autoxidation of amphotericin B, responsible for membrane lipid peroxidation, is also found to be dependent on the aggregation of the polyene. Therefore, it is useful to obtain information about the aggregation of these polyenes at the molecular level. Although several articles [7,10- 191 containing information about the aggregation of heptaene macrolide antibiotics have appeared, none considers the effect of the local environment on the aggregation of these antibiotics. A series of aliphatic alcohols, varying with respect to the size of the alkyl chains, and aqueous buffers of varying pH have been selected as bulk solvents to investigate the effect of the local environment on the aggregation of the polyene antibiotics. It has been observed that the size of the alkyl chains of the alcohols and the pH of the aqueous buffers are important parameters in determining the aggregation of the molecules. The heptaene chromophore and the polar head group of the polyene have been found to play an important role in determining the state of the polyene molecules. This work mostly concerns the investigation of hamycin, a member B.V. All rights reserved.
214
2. ShervanijColloids
Surfaces
of the series of aromatic heptaene macrolide antibiotics, which has enhanced biological activity as compared to non-aromatic polyenes against pathogenic yeast-like microorganisms [20-231. The heptaene chromophore of amphotericin B or hamycin, in a dispersive medium, has a characteristic absorption spectrum with four maxima in the ultraviolet-visible region. Any alteration in the state of the molecules, such as aggregation, is reflected by the change in their absorption spectra. Therefore, in the present study, the ratio of absorbances at different wavelengths has been considered to be a “fingerprint” for monitoring the aggregation states of these polyene antibiotics.
A: Physicochem.
r---TTr
Experimental
Amphotericin B and hamycin were obtained from Sigma, St. Louis, MO, and Hindustan Antibiotics Ltd., India, respectively. The hamycin used in the present work had four components, namely A, B, C and D. Hamycin A was the major component [24]. Dimethyl sulfoxide was of spectroscopy grade from SDS, India. The alcohols were of analytical grade, and were dried, distilled and final traces of moisture were removed with the aid of molecular sieve. Tris-acetate buffer, concentration 10 mM was used as an aqueous solution and to carry out the pH-dependent measurements. Amphotericin B and hamycin stock solutions, concentration, 1O-’ M, were prepared daily by dissolving the powders in dimethyl sulfoxide. The samples were prepared by adding stock solutions to the bulk solvents, and fine dispersions of the polyenes were obtained by sonicating the samples in a Branson B- 1200 El sonicator having a working frequency of 47 kHz. The samples thus prepared were never allowed to have a dimethyl sulfoxide concentration exceeding 1%. The absorption spectra were recorded with use of a Shimadzu UV260 spectrophotometer at 25’ C. Results and discussion
Figures 1 and 2 are the absorption spectra of amphotericin B and hamycin, respectively. The
Eng. Aspects 70 (1993) 213-217
WAVELENGTH
,_....’
0
____----
300
(nm)
__--
400
350 WAVELENGTH
(nn)
Fig. I. Electronic absorption spectra of different concentrations of amphotericin B in methanol (MeOH) and in aqueous solution (aq. soln). (a) 10m6M in MeOH (-) and in aq. soln (-.-); 5*10-‘A4 in MeOH (---) and in aq. soln (..,). (b) 10m5M in MeOH (-) and in aq. soln (-.-); 5. 10m6M in MeOH (- - -) and in aq. soln (...).
spectra of amphotericin B and hamycin in methanol exhibit fine vibronic structure and are concentration independent. The fine vibronic structure in the absorption spectrum suggests the presence of the polyene antibiotic as the unit molecule. The spectra of amphotericin B and hamycin in neutral aqueous buffer (10 mM Tris-acetate) are
Z. ShervanilColloids Surfaces A: Physicochem. Eng. Aspects 70 (1993) 213-217
a)
I
I
(b)
0
300
350 WAVELEUGT”
400
4
450
(“In)
Fig. 2. Electronic absorption spectra of different concentrations of hamycin in MeOH and in aq. soln (a) 10e6 M in MeOH (-) and in aq. soln (-.-); 5*10-’ A4 in MeOH (- --) and in aq. soln (...). (b) 10m5A4 in MeOH (-) and in aq. soln (-.-); 5.10-6 M in MeOH (---) and in aq. soln (...).
concentration dependent. Initially, at 5. lo-’ or 10m6 M amphotericin B concentrations the spectra are similar to those obtained in methanol with a hypochromism and a bathochromism of about 3 nm. At a concentration of 5*10e6 M the spectrum of amphotericin B changes in a characteristic manner, with a decrease in the bands at 408,
215
385 and 363 nm, and the appearance of a new band at 339 nm. On increasing the concentration to lo-’ M, the bands at 408,385 and 363 nm show a further decrease in absorbance with a simultaneous increase in absorbance at 339 nm, taking into the account the dilution factor. The absorption band at 339 nm has been attributed to aggregation, as has also been observed in other polyenic molecules [25,26]. The absorptions at 408 nm and 339 nm are characteristic of non-aggregated and aggregated species of amphotericin B, respectively. Therefore, the ratio of the absorbances at 339 and 408 nm has been exploited to monitor the extent of self-aggregation of amphotericin B. In the absence of the band at 339 nm, the band at 345 nm has been considered for obtaining the absorbance ratio. The band at 345 nm is submerged under the 339 nm band during the aggregation. In neutral aqueous buffer, at hamycin concentrations of 5. lo-’ or lop6 M, the submergence of the 341 nm band under the 361 nm band has been observed. With an increase in the concentration to 5*10m6 M, the complete loss of the fine structure has been observed in the form of a single, flattened and blue-shifted absorption maximum at around 361 nm. The development of this band has been attributed to the hamycin aggregation. The absorptions at 404 nm and 361 nm have been identified as representing the non-aggregated and aggregated forms of hamycin, respectively. Therefore, the ratio of the absorbances at 361 and 404 nm will reflect the extent of aggregation of hamycin. The longest absorption band in the spectrum of 1O-5 M hamycin in aqueous solution appears at 417 nm. A similar red shift in the longest wavelength bands in the violaxanthin and neoxanthin carotenoids [25] has been attributed to the aggregation of the carotenoids. Figure 3 is the plot of absorbance ratio vs concentration of amphotericin B and hamycin. In methanol, the absorbance ratio remains constant during the entire concentration range studied for both amphotericin B and hamycin, suggesting the existence of these polyene antibiotics in the monomeric state. In neutral aqueous buffer, an increase
216
2. ShervanilColloids Surfaces A: Physicochem. Eng. Aspects 70 (1993) 213-217
Fig. 3. Effect of concentration on the absorbance ratio. Amphotericin B absorbance ratio (peak 4/peak 1) in MeOH (0) and in aq. soln (0); hamycin absorbance ratio (peak 3/peak 1) in MeOH (0) and in aq. solns of pH 7 ( n ), pH 2.5 (A), pH 8.5 (A), pH IO(V) and pH 12 (V).
in the absorbance ratio was observed for both amphotericin B and hamycin, suggesting that the increase in the concentration induces aggregation. The absorbance ratios of 1.21 and, 1.37 at concentrations of 5 - lo- 7 M and low6 M, respectively, for hamycin, and the absorbance ratios of 0.28 and 0.37 at concentrations of 5. 10e7 M and 10e6 M, respectively, for amphotericin B are characteristic of the non-aggregated state of the polyene antibiotics. The aggregation becomes prominent at a concentration of 5 * low6 M, since at this concentration a considerable increase in the absorbance ratio was seen. The observed absorbance ratios of 2.48 for hamycin and 2.33 for amphotericin B at a concentration of 10m5 M are characteristic of the aggregated form of the polyene antibiotics. Figure 3 shows a general decrease in the aggregation tendency of hamycin in acidic (pH 2.5) and basic (pH 8.5, 10 and 12) solutions as compared to the situation in the neutral solution. However, with increase in concentration a slow aggregation has been observed in basic solutions. The aggregation is least in basic solution of pH 12. The hamycin molecule is amphoteric in neutral, aqueous solution. Hamycin aggregates in neutral, aqueous solution appear to be stabilized by the participation of
water molecules, as the water molecules are hydrogen bonded to the polar head groups of the polyene antibiotics, thus resulting in favourable head group-head group interactions inside the aggregates. In acidic or basic solutions, the binding sites on the polar head groups of hamycin, which are susceptible to hydrogen bonding, are saturated. The saturation of these binding sites results in unfavourable head group-head group interactions and subsequently the aggregate dissociation, thus shifting the equilibrium to the monomolecular state. The primary interactions involved in the selfaggregation of heptaene polyene antibiotics are known to be hydrophobic [I 1,26,27]. The participation of polar head groups of the polyene antibiotics in hydrogen bonding in the process of polyene antibiotic-sterol complexation has also been proposed [28]. The absorption spectra (Fig. 4) of 10m5 M hamycin solutions in a series of aliphatic alcohols were recorded. The absorbance ratios of peak 3 to peak 1 in the spectra of hamycin solutions in all alcohols in the series have been calculated to be 1.1 f 0.1. A decrease in absorbances in the hamycin spectra with increase in the length of the alkyl 0-C
I
I
I
350 WAVELENGTH
400 (nm)
Fig. 4. Electronic absorption spectra of 10m5M hamycin in I-propanol (-), I-pentanol (- - -), I-octanol (-.-), I-decano1 (...) and I-dodecanol (-..-).
Z. S~~uanil~ollo~dsSurfaces A: Physicochem.Eng. Aspects 70 (1993) 213-217
chains of the alcohols has been observed. The decrease in the absorbance value with almost no change in the absorbance ratio suggests that the solubility of hamycin decreases with an increase in the chain length of the alcohols. In other words, aliphatic alcohols with a smaller alkyl chain are associated with higher solvating power for hamycin. An absorbance ratio of 1.1 + 0.1 is characteristic for hamycin in molecular dispersions or true solutions. Conclusion The experimental results presented in this article are consistent with the following findings. Self-aggregation of amphotericin B and hamycin was observed in neutral aqueous solutions at concentrations of or above 5 - 10 -6 M. No aggregation of these polyene antibiotics was noticed in methanol. A decrease in the aggregation tendency of hamycin in acidic or basic aqueous solutions suggests that favourable head group-head group interactions are important for stabilizing the aggregates. The solvating power of aliphatic alcohols for hamycin was found to be inversely related to the length of the alkyl chain of the alcohols. Acknowledgements I am grateful to Professor B.K. Bachhawat for giving me the opportunity of working with him as Research Associate (CSIR) and helping me to carry out this work. I thank the Department of Biotechnology and CSIR, New Delhi, for financial assistance.
11 I2 13 14 15 16 17 18 19 20 21
22 23
24
25 26
Refere~e~ 27
1 1. Ahmed, A.K. Sarkar and B.K. Bachhawat, Indian J. Biothem. Biophys., 26 (1989) 351.
28
211
I. Ahmed, A.K. Sarkar and B.K. Bachhawat, Mol. Cell. Biochem., 91 (1989) 85. I. Ahmed, A.K. Sarkar and B.K. Bachhawat, Biote~hnol. Appl. Biochem., 12 (1990) 550. B.K. Bachhawat, I. Ahmed and A.K. Sarkar, in T. Ozawa (Ed.), Trends in Biological Chemistry, Japan Sci. Sot. Press, Tokyo, and Springer-Verlag, Berlin, 1991, p. 239. M. Moonis, I. Ahmed and B.K. Bachhawat, Indian J. Biothem. Biophys., submitted for publication. 2. Shervani, A.K. Sarkar and B.K. Bachhawat, Indian J. Biochem. Biophys., 28 (1991) 227. 1. Gruda and N. Dussault, Biochem. Cell. Biol., 66 (1988) 171. I. Gruda and J. Bolard, Biochem. Cell. Biol., 65 (1987) 234. G. Ghielmetti, T. Bruzzese, C. Bianchi and F. Recusani, J. Pharm. Sci., 65 (1976) 905. I. Gruda, D. Milette, M. Brother, G.S. Kobayashi, G. Medoff and J. Brajtburg, Antimicrob. Agents Chemother., 35 (1991) 24. C. Ernst, J. Grange, H. Rinnert, G. DuPont and J. Lematre, Biopolymers, 20 (198 1) 1575. J. Mazerski, J. Bolard and E. Borowski, Biochim. Biophys. Acta, 719 (1982) 11. H. Rinnert, C. Thirion, G. DuPont and J. Lematre, Biopolymers, 16 (1977) 2419. G. DuPont, H. Rinnert and J. Lematre, C.R. Acad. Sci., Ser. C, 284 (1977) 853. C. Ernst, G. DuPont, H. Rinnert and J. Lematre, C.R. Acad. Sci., Ser. B, 286 (1978) 175. C. Ernst, J. Lematre, H. Rinnert, G. DuPont and J. Grange, C.R. Acad. Sci., Ser. D, 289 (1979) 1145. J. Lematre and H. Moulki, C.R. Acad. Sci., Ser. C, 280 (1975) 481. M.T. Lamy-Freund, V.F.N. Ferreira and S. Schreier, Biochim. Biophys. Acta, 981 (1989) 207. J. Bolard, M. Seigneuret and G. Boudet, Biochim. Biophys. Acta, 599 (1980) 280. E.F. Gale, J. Gen. Microbial., 80 (1974) 451. B. Cybulska, E. Jabobs, L. Falkowski and E. Borowski, Systemic Fungicides, Lyr-Polter Akademic Verlag, 1974, p. 77. S.M. Hammond, Prog. Med. Chem., 14 (1977) 153. J. Kotler-Brajtburg, G. Medoff, G.S. Kobayashi, S. Boggs, D. Schlessinger, C. Pandey and K.L. Rinehart, Jr, Antimicrob. Agents Chemother., 15 (1979) 716. M.J. Thirumalachar and M.J. Narasimhan, Jr, in R.A. Fromtling (Ed.), Recent Trends in the Discovery, Development and EvaIuation of Antjfungal Agents, J.R. Prous Sci. Publishers, South Africa, 1987, p. 619. A. Hager, Planta, 91 (1970) 38. J. Lematre, B. Maudinas and C. Ernst, Photochem. Photobiol., 31 (1980) 201. I. Gruda, P. Nadeau, J. Brajtburg and G. Medoff, Biochim. Biophys. Acta, 602 (1980) 260. T.E. Andreoli, Ann. N.Y. Acad. Sci., 235 (1974) 448.
213
B.V., Amsterdam
Aggregation of polyene antibiotics as revealed by absorption spectroscopy Zameer Shervani Liposome Research Centre, New Delhi-l 10021, India
Department
of Biochemistry,
University of Delhi South Campus,
(Received 30 March 1992; accepted 6 October 1992) Abstract Methanolic solutions of the heptaene macrolide antibiotics, namely amphotericin B and hamycin, are molecular dispersions. These polyenes were found to be self-aggregated in neutral aqueous solution. The aggregates of hamycin dissociate in acidic or basic aqueous solutions. The smaller chain aliphatic alcohols have a higher solvating power for hamycin.
Keywords: Amphotericin
B; hamycin;
polyene
antibiotics;
self-aggregation;
Introduction The heptaene polyene antibiotics amphotericin B [l-4] and hamycin [S], are known to have potent antifungal activity. It is generally accepted that such polyene antibiotics form channels by interacting with sterol-containing membranes, thus inducing permeability changes in the membrane [6]. The mode of the interaction is related to the aggregation of amphotericin B [7,S]. Ergosterol does not react, or reacts extremely slowly, with monomeric amphotericin B in aqueous solutions of propanol. Traces of small aggregates, probably dimers, enable a cooperative reaction. The interaction with ergosterol is hindered when the antibiotic is in the micellar form. Marked differences in toxicity have been noticed among various batches of the polyene antibiotics. Such differences have been attributed not to the particle size, but to different molecular spatial arrangements of the product, which modify their bioavailability [9]. One of the mechanisms of antibiotic action is considered to be through the autoxidation of amphotericin B, which brings about membrane 0927-7757/93/$06.00
0
1993 -
Elsevier Science Publishers
solvent effect.
lipid peroxidation. The kinetics of autoxidation of amphotericin B, responsible for membrane lipid peroxidation, is also found to be dependent on the aggregation of the polyene. Therefore, it is useful to obtain information about the aggregation of these polyenes at the molecular level. Although several articles [7,10- 191 containing information about the aggregation of heptaene macrolide antibiotics have appeared, none considers the effect of the local environment on the aggregation of these antibiotics. A series of aliphatic alcohols, varying with respect to the size of the alkyl chains, and aqueous buffers of varying pH have been selected as bulk solvents to investigate the effect of the local environment on the aggregation of the polyene antibiotics. It has been observed that the size of the alkyl chains of the alcohols and the pH of the aqueous buffers are important parameters in determining the aggregation of the molecules. The heptaene chromophore and the polar head group of the polyene have been found to play an important role in determining the state of the polyene molecules. This work mostly concerns the investigation of hamycin, a member B.V. All rights reserved.
214
2. ShervanijColloids
Surfaces
of the series of aromatic heptaene macrolide antibiotics, which has enhanced biological activity as compared to non-aromatic polyenes against pathogenic yeast-like microorganisms [20-231. The heptaene chromophore of amphotericin B or hamycin, in a dispersive medium, has a characteristic absorption spectrum with four maxima in the ultraviolet-visible region. Any alteration in the state of the molecules, such as aggregation, is reflected by the change in their absorption spectra. Therefore, in the present study, the ratio of absorbances at different wavelengths has been considered to be a “fingerprint” for monitoring the aggregation states of these polyene antibiotics.
A: Physicochem.
r---TTr
Experimental
Amphotericin B and hamycin were obtained from Sigma, St. Louis, MO, and Hindustan Antibiotics Ltd., India, respectively. The hamycin used in the present work had four components, namely A, B, C and D. Hamycin A was the major component [24]. Dimethyl sulfoxide was of spectroscopy grade from SDS, India. The alcohols were of analytical grade, and were dried, distilled and final traces of moisture were removed with the aid of molecular sieve. Tris-acetate buffer, concentration 10 mM was used as an aqueous solution and to carry out the pH-dependent measurements. Amphotericin B and hamycin stock solutions, concentration, 1O-’ M, were prepared daily by dissolving the powders in dimethyl sulfoxide. The samples were prepared by adding stock solutions to the bulk solvents, and fine dispersions of the polyenes were obtained by sonicating the samples in a Branson B- 1200 El sonicator having a working frequency of 47 kHz. The samples thus prepared were never allowed to have a dimethyl sulfoxide concentration exceeding 1%. The absorption spectra were recorded with use of a Shimadzu UV260 spectrophotometer at 25’ C. Results and discussion
Figures 1 and 2 are the absorption spectra of amphotericin B and hamycin, respectively. The
Eng. Aspects 70 (1993) 213-217
WAVELENGTH
,_....’
0
____----
300
(nm)
__--
400
350 WAVELENGTH
(nn)
Fig. I. Electronic absorption spectra of different concentrations of amphotericin B in methanol (MeOH) and in aqueous solution (aq. soln). (a) 10m6M in MeOH (-) and in aq. soln (-.-); 5*10-‘A4 in MeOH (---) and in aq. soln (..,). (b) 10m5M in MeOH (-) and in aq. soln (-.-); 5. 10m6M in MeOH (- - -) and in aq. soln (...).
spectra of amphotericin B and hamycin in methanol exhibit fine vibronic structure and are concentration independent. The fine vibronic structure in the absorption spectrum suggests the presence of the polyene antibiotic as the unit molecule. The spectra of amphotericin B and hamycin in neutral aqueous buffer (10 mM Tris-acetate) are
Z. ShervanilColloids Surfaces A: Physicochem. Eng. Aspects 70 (1993) 213-217
a)
I
I
(b)
0
300
350 WAVELEUGT”
400
4
450
(“In)
Fig. 2. Electronic absorption spectra of different concentrations of hamycin in MeOH and in aq. soln (a) 10e6 M in MeOH (-) and in aq. soln (-.-); 5*10-’ A4 in MeOH (- --) and in aq. soln (...). (b) 10m5A4 in MeOH (-) and in aq. soln (-.-); 5.10-6 M in MeOH (---) and in aq. soln (...).
concentration dependent. Initially, at 5. lo-’ or 10m6 M amphotericin B concentrations the spectra are similar to those obtained in methanol with a hypochromism and a bathochromism of about 3 nm. At a concentration of 5*10e6 M the spectrum of amphotericin B changes in a characteristic manner, with a decrease in the bands at 408,
215
385 and 363 nm, and the appearance of a new band at 339 nm. On increasing the concentration to lo-’ M, the bands at 408,385 and 363 nm show a further decrease in absorbance with a simultaneous increase in absorbance at 339 nm, taking into the account the dilution factor. The absorption band at 339 nm has been attributed to aggregation, as has also been observed in other polyenic molecules [25,26]. The absorptions at 408 nm and 339 nm are characteristic of non-aggregated and aggregated species of amphotericin B, respectively. Therefore, the ratio of the absorbances at 339 and 408 nm has been exploited to monitor the extent of self-aggregation of amphotericin B. In the absence of the band at 339 nm, the band at 345 nm has been considered for obtaining the absorbance ratio. The band at 345 nm is submerged under the 339 nm band during the aggregation. In neutral aqueous buffer, at hamycin concentrations of 5. lo-’ or lop6 M, the submergence of the 341 nm band under the 361 nm band has been observed. With an increase in the concentration to 5*10m6 M, the complete loss of the fine structure has been observed in the form of a single, flattened and blue-shifted absorption maximum at around 361 nm. The development of this band has been attributed to the hamycin aggregation. The absorptions at 404 nm and 361 nm have been identified as representing the non-aggregated and aggregated forms of hamycin, respectively. Therefore, the ratio of the absorbances at 361 and 404 nm will reflect the extent of aggregation of hamycin. The longest absorption band in the spectrum of 1O-5 M hamycin in aqueous solution appears at 417 nm. A similar red shift in the longest wavelength bands in the violaxanthin and neoxanthin carotenoids [25] has been attributed to the aggregation of the carotenoids. Figure 3 is the plot of absorbance ratio vs concentration of amphotericin B and hamycin. In methanol, the absorbance ratio remains constant during the entire concentration range studied for both amphotericin B and hamycin, suggesting the existence of these polyene antibiotics in the monomeric state. In neutral aqueous buffer, an increase
216
2. ShervanilColloids Surfaces A: Physicochem. Eng. Aspects 70 (1993) 213-217
Fig. 3. Effect of concentration on the absorbance ratio. Amphotericin B absorbance ratio (peak 4/peak 1) in MeOH (0) and in aq. soln (0); hamycin absorbance ratio (peak 3/peak 1) in MeOH (0) and in aq. solns of pH 7 ( n ), pH 2.5 (A), pH 8.5 (A), pH IO(V) and pH 12 (V).
in the absorbance ratio was observed for both amphotericin B and hamycin, suggesting that the increase in the concentration induces aggregation. The absorbance ratios of 1.21 and, 1.37 at concentrations of 5 - lo- 7 M and low6 M, respectively, for hamycin, and the absorbance ratios of 0.28 and 0.37 at concentrations of 5. 10e7 M and 10e6 M, respectively, for amphotericin B are characteristic of the non-aggregated state of the polyene antibiotics. The aggregation becomes prominent at a concentration of 5 * low6 M, since at this concentration a considerable increase in the absorbance ratio was seen. The observed absorbance ratios of 2.48 for hamycin and 2.33 for amphotericin B at a concentration of 10m5 M are characteristic of the aggregated form of the polyene antibiotics. Figure 3 shows a general decrease in the aggregation tendency of hamycin in acidic (pH 2.5) and basic (pH 8.5, 10 and 12) solutions as compared to the situation in the neutral solution. However, with increase in concentration a slow aggregation has been observed in basic solutions. The aggregation is least in basic solution of pH 12. The hamycin molecule is amphoteric in neutral, aqueous solution. Hamycin aggregates in neutral, aqueous solution appear to be stabilized by the participation of
water molecules, as the water molecules are hydrogen bonded to the polar head groups of the polyene antibiotics, thus resulting in favourable head group-head group interactions inside the aggregates. In acidic or basic solutions, the binding sites on the polar head groups of hamycin, which are susceptible to hydrogen bonding, are saturated. The saturation of these binding sites results in unfavourable head group-head group interactions and subsequently the aggregate dissociation, thus shifting the equilibrium to the monomolecular state. The primary interactions involved in the selfaggregation of heptaene polyene antibiotics are known to be hydrophobic [I 1,26,27]. The participation of polar head groups of the polyene antibiotics in hydrogen bonding in the process of polyene antibiotic-sterol complexation has also been proposed [28]. The absorption spectra (Fig. 4) of 10m5 M hamycin solutions in a series of aliphatic alcohols were recorded. The absorbance ratios of peak 3 to peak 1 in the spectra of hamycin solutions in all alcohols in the series have been calculated to be 1.1 f 0.1. A decrease in absorbances in the hamycin spectra with increase in the length of the alkyl 0-C
I
I
I
350 WAVELENGTH
400 (nm)
Fig. 4. Electronic absorption spectra of 10m5M hamycin in I-propanol (-), I-pentanol (- - -), I-octanol (-.-), I-decano1 (...) and I-dodecanol (-..-).
Z. S~~uanil~ollo~dsSurfaces A: Physicochem.Eng. Aspects 70 (1993) 213-217
chains of the alcohols has been observed. The decrease in the absorbance value with almost no change in the absorbance ratio suggests that the solubility of hamycin decreases with an increase in the chain length of the alcohols. In other words, aliphatic alcohols with a smaller alkyl chain are associated with higher solvating power for hamycin. An absorbance ratio of 1.1 + 0.1 is characteristic for hamycin in molecular dispersions or true solutions. Conclusion The experimental results presented in this article are consistent with the following findings. Self-aggregation of amphotericin B and hamycin was observed in neutral aqueous solutions at concentrations of or above 5 - 10 -6 M. No aggregation of these polyene antibiotics was noticed in methanol. A decrease in the aggregation tendency of hamycin in acidic or basic aqueous solutions suggests that favourable head group-head group interactions are important for stabilizing the aggregates. The solvating power of aliphatic alcohols for hamycin was found to be inversely related to the length of the alkyl chain of the alcohols. Acknowledgements I am grateful to Professor B.K. Bachhawat for giving me the opportunity of working with him as Research Associate (CSIR) and helping me to carry out this work. I thank the Department of Biotechnology and CSIR, New Delhi, for financial assistance.
11 I2 13 14 15 16 17 18 19 20 21
22 23
24
25 26
Refere~e~ 27
1 1. Ahmed, A.K. Sarkar and B.K. Bachhawat, Indian J. Biothem. Biophys., 26 (1989) 351.
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