Chapter 19 Aqueous Solution Properties of Bacterial Poly-γ-D-glutamate 1
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V.Crescenzi ,M. D'Alagni , M.Dentini ,and B. Mattei 1
Department of Chemistry, University La Sapienza, P. le A. Moro 5, 00185 Rome, Italy Centro di Studio per la Chimica dei Recettori e delle Molecole Biologicamente Attive, CNR, 00168 Rome, Italy 2
A preliminary physico-chemical characterization of a bacterial poly-γglutamate sample (96% D-glutamic acid content), γ-D-PGA, in dilute aqueous solutions has been carried out by means of potentiometric, viscosimetric, infrared and chiroptical spectroscopic experiments. The biopolymer exhibits properties strikingly dependent on a number of parameters, mainly: polymer concentration, pH, ionic strength, and nature of added salt. In dilute solutions (polymer concentration around 0.1% w/V) and for pH > 7, γ-D-PGA chains assume elongated, stiff conformations while upon protonation (pH < 3) globular states would prevail. Addition of divalent counterions (Ca(II)) also leads to compact γ-D-PGA conformations.
Poly-y-glutamates (γ-PGA) of different stereoregularity and molecular weight can be produced in very good yields mainly from species of the genus Bacillus (1). These natural polyamides, which consist of glutamic acid units linked between the γ-carboxylic and the α - a m i n o functionalities (Fig.l), have been known since the pioneering fundamental studies on macromolecules of natural origin, particularly those from microbial sources, carried out in the early twenties (2). Recently, a revival in interest in these peculiar biopolymers has resulted in a number of publications dealing with their biosynthesis, the resulting stereochemistry of the chains, as well as with the preparation of derivatives with potential as novel biomaterials ( 3-4 ). However, relatively little unambiguous data is available on the conformation dependent solution properties of these poly^-glutamates, for which the
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relationships between structure and physico-chemical properties are still a matter of controversy (5-6). Thus, the potentially large number of applications that these water soluble, biocompatible polymeric materials may exhibit remain essentially unexploited. In our laboratories a detailed physico-chemical characterization of series of poly-y-glutamate samples all produced by Bacillus licheniformis strains but differing in D/L glutamic acid content is in progress. Results obtained so far on the properties of γ-D-PGA (96% D-glutamic acid content, from B. licheniformis ATCC 9945-a) in dilute aqueous solution are herein reported and discussed. Experimental Polymer samples have been obtained ( Bacillus Licheniformis ATCC 9945-a) and purified following procedures reported in the literature ( 1 ). The sample used in the present investigation (sodium salt) had a D-glutamic acid content of 96% . Optical activity measurements were performed with a PerkinElmer 241 polarimeter using a 10 cm path-length, the temperature was controlled by means of a Lauda circulating-water bath . Viscosity measurements for the γ-D-PGA samples were performed at 25°C using a Shott-Geraete automatic viscosimeter equipped with a water thermostat. A range of ionic strengths ( or pH ) were investigated; these strengths ( or pH ) were controlled by varying the level of NaCl ( or H C I O 4 ) added . Circular dichroism (CD) spectra were recorded at 25°C using a Jasco J500-A CD apparatus (calibrated with androsteron) and quartz cells of 1.0, 0.5, and 0.1 cm optical paths. During the experiments the apparatus was fluxed with high purity nitrogen. Infrared spectra of γ-D-PGA solutions in D 2 O were recorded at room temperature using a BIO-RAD, FTS-40 A spectrometer and BaF2 cells of 0.2 mm optical path. The calcium salt form of γ-D-PGA was prepared by addition of an excess of Ca(C104)2 to a solution of the sodium salt form of the biopolymer, followed by extensive dialysis against distilled water and final freeze-drying of the product. Results and Discussion Data to be illustrated in what follows have been collected using γD-PGA concentrations less than 0.3% w/V. At higher polymer
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concentrations the solutions in water are exceedingly viscous and, in addition, weak gel formation and/or precipitation may be observed particularly at low pH values. The physico-chemical properties of γ-D-PGA under acidic and basic conditions differ significantly. Data pertaining to Na+^-D-PGA in water at neutral or basic pH are considered first. Potentiometric measurements of the "activity coefficient" , γ+ , of the sodium counterions were carried out at 25°C as a function of polyelectrolyte concentration ( Fig. 2 ). Variation of the latter results in little change in the value of γ*" (from about 0.42 to 0.49). Such behavior is typically found for relatively highly charged polyelectrolytes. The observed values of γ+ are close to those predicted from Manning's equation (7): ln γ+ = - 0.5 - ln χ in which the linear charge density parameter, χ , is equal to 0.716/b where b ( nm ) is the distance projected on the chain axis between neighboring fixed charges ( the carboxylate groups ) . For a fully extended γ-PGA chain, b is approximately 0.6 nm, and thus χ = 1.19 and γ+= 0.5. These results suggest that the γ-PGA chains in water assume a rather expanded conformation . Addition of NaCl to aqueous Na+-γ-PGA does quite naturally promote a reduction in average chain dimensions, as shown in a concise manner by the set of intrinsic viscosity data reported in Fig. 3 as a function of the inverse square root of the ionic strength I. These data exibit a regular trend and permit estimation of the parameter B, defined as ( 8-9 ) : B=S/([h]0.1 )l-3 (The slope S of the linear plot of Fig. 3 is normalized by the intrinsic viscosity measured for 1=0.1). The results lead to a value of B=0.19. Comparison of various Β values for different natural and synthetic polyelectrolytes, from the flexible polyacrylate to very rigid DNA and Xanthan (11), suggests that the γ-D-PGA chains might be considered of "intermediate stiffness". Similarly, the change in Na ^D-PGA optical activity with increasing ionic strength ( NaCl, 25°C, Fig. 4 ) although quite large shows a regular trend and would reflect the gradually more compact conformation assumed by γ-D-PGA chains in response to the screening effect exerted by the additional Na ions (NaCl) on intrachain electrostatic repulsions . +
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POLYMERS
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Bacterial Poly- η-D-glutamate
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Figure 4. Dependence of poly-y-D-glutamate optical activity on ionic strength (NaCl, moles/L). Polymer concentration 0.1% w/v (6.6 mmoles/L).
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The behaviour of aqueous γ-D-PGA under acidic conditions suggests significantly different conformational behaviour from that observed for the polypeptide under basic conditions. The circular dichroism (CD) spectra of γ-D-PGA at two limiting pH values in water are reported in Fig. 5,a) while in Fig. 5,b) the ellipticity values recorded at 207 and 215 nm (from a number of additional spectra) are plotted as a function of pH. The change in chiroptical properties of γD-PGA with protonation is marked and features a trend which suggests a conformational change in the polypeptide chains with a midpoint around pH 5. Interestingly, upon protonation the viscosity of γ-D-PGA aqueous solutions begins to drop dramatically around pH 5 (Fig. 6). A nearly obvious interpretation of such a marked drop is that fully protonated g-PGA chains assume a globular structure which is quite compact. At high pH where the side chains are negatively charged, the polyions would assume a more extended structure. Very compact conformations would be assumed by γ-D-PGA chains also in the presence of added Ca(II) salts (neutral pH) as clearly demostrated by the viscosity data reported in Fig. 7. In fact from Fig. 7 it is seen that the relative viscosity (25°C) of a 0.08 % (w/V) solution of γ - D-PGA in water is pratically reduced to unity at a Ca(C104)2 concentration as low as 30 mM. In Fig. 8 the CD spectrum is reported of the Ca(II) salt form of γD-PGA (neutral pH) is reported: the spectral features are quite close to those exibited by the sodium salt of γ-D-PGA at high pH. In view of the different conformational states populated by γ-DPGA chains in the two salt forms and pH conditions, a possible deduction is that the CD characteristics of γ-D-PGA are mainly dependent on the ionization state of the carboxylate chromophores. Similar considerations might apply also to IR spectral data given in Fig. 9 showing a marked shift in the characteristic amide band frequencies with changing pH. In fact, also in this case the spectra of the sodium salt form and of the calcium salt form of γ-D-PGA result almost superimposable. In the context, it is interesting to mention that production of γ-DPGA by fermentation is normally carried out at relatively high ionic strengths (about 0.15 M) and with added divalent cations concentration (Ca(II) and Mn(II) of the order of 20mM. In these conditions, according to our data, the viscosity of the fermentation broths should be quite low: this is a factor that contributes to making it possible to reach γ-D-PGA yields as high as 50-60 g/L, a production practically
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HYDROGELS AND BIODEGRADABLE POLYMERS F O R BIOAPPLICATIONS
impossible in the case of other microbial biopolymers characterized by stiff chains, of average dimensions poorly sensitive to added salt, like many polysaccharides. In conclusion, γ-D-PGA chains in dilute aqueous solution exhibit basically two different, limiting shapes, i.e. highly stretched and tightly globular, which interconvert rather sharply in response to minor changes in pH or added salt concentration (Ca(II) salts) within critical intervals. This stimulates additional research aimed at a better understanding of the nature of such limiting conformations and at the exploitation of the associated sharp changes in chains average dimensions in, for example, the formulation of pH/Ca(II) sensitive hydrogels. Studies are in progress in our laboratories along these lines. Acknowledgements This work has been carried out with financial support of the Italian Ministry for University and Scientific and Technological Research (MURST, Rome), "60%-Ateneo" funds. The selection and fermentation of highly mucoid cellular culture of B. Licheniformis (ATCC 9945-a) were carried out in the laboratory of Prof. C. Palleschi ( Department of Cellular Biology & Development, University "La Sapienza", Rome), whose expert collaboration is sincerely appreciated. We are grateful also to Dr. B. Sambuco for a number of spectroscopic measurements. References 1. Gross, R. Α.; Birrer, G. Α.; Cromwick, A. M.; Giannos, S.A.; Mc Carthy, S.P.; Biotecnologycal Polymers:Medical,Pharmaceutical and Industrial Applications, Gebelein, C.G., Ed., Technomic Publ. Co.,1993, Part III, pp 200-214. 2. Lemogne, M.: Ann. Inst. Pasteur (Paris) 1925, 39, 144. 3. Giannos, S.A.; Shah, D.; Gross, R. Α.; Kaplan, D. L.; Mayer, J. M.; Novel Biodegradable Microbial Polymers, Dawes, Ε. Α., Ed., Series Ε, Applied Sciences, Vol. 186, Kluver Academic Publishers, Netherlands, 1990, pp 457460. 4. Borbely, M . ; Nagasaki, Y.; Borbely, J.; Fan, K.; Bhogle, A.,;Sevolan, M . ; Polymer Bull. 1994, 32, 127. 5. Malborough, D. I.; Biopolymers 1973, 12, 1083. 6. Balasubramian, D., Kalita, C.C., Kovacs, J. Biopolymers 1973, 12, 1089. 7. Manning, G. S.: Ann. Rev. Phys. Chem. 1972, 23, 117. 8. Fixmann, M.; J. Chem. Phys. 1964, 41, 3772. 9. Smidsrod, O.; Haug, Α.; Biopolym. 1971, 10, 1213. R E C E I V E D January 3,
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