Microcalorimetric Study of Iodized and Noniodized Cells and C

Institute of Physics of the Georgian Academy of Sciences, Tbilisi 380077, Georgia. Biomacromolecules , 2002, 3 (3), pp 415–420. DOI: 10.1021/bm01559...
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Biomacromolecules 2002, 3, 415-420

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Articles Microcalorimetric Study of Iodized and Noniodized Cells and C-Phycocyanin of Spirulina platensis Levan S. Topchishvili, Shota I. Barbakadze, Anna I. Khizanishvili, George V. Majagaladze, and Jamlet R. Monaselidze* Institute of Physics of the Georgian Academy of Sciences, Tbilisi 380077, Georgia Received August 1, 2001; Revised Manuscript Received November 22, 2001

It was shown that eight stages of transition are observed in the heating process of Spirulina platensis cells in temperature range 5-140 °C. The first stage covers the temperature range 5-53 °C with maximum ∼45 °C. The heat evolved in this temperature range is equal to 380 ( 20 J/g of dry biomass, it does not change at scanning rate lower than 0.083 °C/min and belongs, mainly, to cell respiration in a stationary regime, in the dark. It was shown that endotherm ∼66 °C belongs to denaturation of C-phycocyanin which denaturates in solutions with Td ) 64.2 °C, ∆Hd ) 34.7 ( 2.1 J/g and for it ∆Hdcal/∆HV.H is equal to 10.8 ( 1.2. The endotherms with Td equal to 58 and 88 °C are connected with denaturation of phycobilisome proteins and endotherm with Td ) 48 °C and ∆Hd ) 4.2J/g of dry biomassswith denaturation of protein which, apparently, is connected with cell respiration. The studies of the last 20 years have shown that the method of highly sensitive differential scanning microcalorimetry (DSC) for investigation of complex biological systems is one of the leading methods for study of thermostability of subcellular structures in situ.1-5 The importance of these studies is that the structural transformations of biopolymers in dilute solutions do not always coincide with the same changes in situ and in vivo. And it is not surprising because many biopolymers function in living cells as supramolecular complexes. Lately a great amount of attention has been paid to the study of the physiology, biology, and biotechnology of Spirulina platensis.6 This interest is connected with the fact that Spirulina pl. and its protein complexes are widely used not only in the food and pharmacological industries but also in medicine. Therefore, a great deal of attention is paid to creation of preparations and immunostimulators on the basis of Spirulina pl. and its main protein component C-phycocyanin (C-PC) with various additions, which increase the influence of preparation of the organism. Among these additions, iodine compounds take one of the first places. The consequences caused by iodine deficiency and excess in human organisms are well-known in the medical practice. That is why it is necessary to know the influence of iodine on the subcellular structure of Spirulina pl. cells and on their survival. According to the data,7,8 there is a strict correlation between survival of cells or growth and heat production. The growth of cells and thermal effects are usually studied by isothermic calorimetry.7-9 On the basis of these classic * To whom correspondence may be addressed. E-mail: mon@ iph.hepi.edu.ge.

works7-9 very important results explaining many physicochemical processes proceeding in living cell both in procaryote and eucaryote and in aerobic and nonaerobic conditions were obtained. But as far as we know, there are no data on heat production of Spirulina pl. and there are also practically no data on thermostability of subcellular structures.10 The last has a decisive significance for maintenance of cell vital capacity. In this paper we tried, using differential scanning microcalorimetry, to investigate (1) the thermal characteristics of iodized and noniodized Spirulina pl. cells in wide temperature range (5-140 °C) using low rates of heating, and (2) the thermodynamic parameters of denaturation of the main protein component of Spirulina pl., C-PC, in dilute solution at various concentrations of added KI. Materials and Methods The measurements were carried out with a differential scanning microcalorimeter with a sensitivity of 10-7 W11 created12 for the study of complex biological systems. The volume of the measuring vessel was 0.30 cm3, the heating rate was 0.016-1.16 °C/min, and the temperature range of measurements was 5-140 °C. The exactness of temperature measurements was not less than 0.05 K. The errors in determination of heat evolution (-Q) and heat absorption (Qd) of Spirulina pl. cells were not more than 10%. The error in determination of C-PC ∆Hd was not more than 6%. The Institute of Plant Physiology, Russian Academy of Sciences, kindly granted strain IPPA B256. Spirulina pl. cultivation was carried out on a special photo bioreactor

10.1021/bm0155928 CCC: $22.00 © 2002 American Chemical Society Published on Web 03/20/2002

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Figure 2. Microcalorimetric record of the thermal effect observed in the process of heating of Spirulina pl. cells in Zarrouk’s medium in stationary regime, in the dark and in a closed vessel (pH 9.85): there was 291.0 mg of cell suspension; quantity of dry biomass, 1.62 mg; scanning rate, 0.42 °C/min.

Figure 1. (A) Polyacrylamide gel electrophoresis of Spirulina.pl cells and C-PC: 1, iodized cells (R ) 1 mg of KI/g of dry biomass); 2, noniodized cells; 3, C-PC (r ) 1.85); 4, C-PC (r ) 3.5); 5, molecular weight markers calibrated in base pairs. (B) Absorption spectra of CBPC with various degrees of purification in 100 mM Na-K phosphate buffer (pH 6.0): 3, r ) 1.85; 4, r ) 3.5.

created at the Institute of Physics, Georgian Academy of Sciences.13 Spirulina pl. was mixed for 30 min every hour at 30-34° C, under 2-4 lux lighting.14 Spirulina pl. was grown in Zarrouk’s nutrient medium.15 A 7-day culture of Spirulina pl. was used in experiments. C-PC preparations were obtained by the method given in ref 16. The purity of preparation was estimated from the ratio of absorption peak intensity at 620 and 280 nm and was equal to 1.85 and 3.5 (A620/A280 ) r) (Figure 1b). The weight content of admixture proteins (nonpigment) (m0) in C-PC preparations r ) 1.85 and 3.5 was determined by dry weight according to the elementary formula m0 ) m - (m1 + m2) where m, the integral mass of preparation, was determined at 105 °C, m1, the ash mass of preparation, was determined at 400 °C, and m2 was the C-PC mass (a molar coefficient of extinction Eλ)615 ) 27900 cm-1 was used for determining of C-PC concentration). The spectra of C-PC absorption were done on a UNICAM 1800 (Great Britain) spectrophotometer in the range 240-700 nm. The content for r ) 1.85 was equal to 18 ( 4% and for r ) 3.5 was equal to 3 ( 2%. The concentration of total protein was determined according to the method of Lowry (described in ref 17) on the seventh day, when cells were in stationary phase (pH medium 9.95 ( 0.1) and equal to 67.0 ( 5%. C-PC concentration

was determined according to quantity of its isolation from Spirulina pl.16 also on the seventh day and was equal to 13.5 ( 1.5%. Electrophoresis of iodized and noniodized Spirulina pl. cells and C-PC in 10% polyacrylamid gel PAGE was carried out in a Leammly system18 (Figure 1a). A mix of marker proteins with molecular weights from 25000 to 67000 was used. Preparations of firm “SIGMA” were used for electrophoresis. Dry weight of Spirulina pl. biomass, dried out directly in a measuring vessel at 105 °C, was determined by weighing with an exactness of no more than 2.0%. The quantity of nonorganic compounds (ash) in samples dried at 105 °C was determined by weighing after their annealing at 400 °C. The exactness of the weighing was no more than 1.0%. The quantity of iodine determined by the method of nuclear activation analysis was 1 µg of iodine/mg of dry biomass of Spirulina pl. cells. It should be noted that DSC, which is used in our experiments, is equipped with all programs needed for determination of the thermodynamic parameters of the protein denaturation process in solutions and deconvolution of calorimetric curves. Results The microcalorimetric record of the denaturation process of Spirulina pl. cell culture in Zarrouk’s medium is given in Figure 2. It is seen that curve profiles have complex character covering the temperature range 5-130 °C. An intensive asymmetric peak of heat evolution with maximum about 47.1 °C and with expressed shoulders from the low-temperature side about 25 and 40 °C is observed in the temperature range 5-53 °C and is equal to 63.5 ( 5 J/g. Four heat absorption maxima are observed above 53 °C; their temperature maxima are about 66, 75, 87, and 108 °C. Q integral absorbed in the range 50-130 °C is equal to 24.6 J/g. As seen (Figures 3 and 4), the decrease of heat rate for two times (from 0.25 to 0.125 °C/min) shifts the maximum of heat evolution peak to low temperatures only by 1.2 °C both for native (closed circles) and iodized Spirulina pl. (closed triangles) while heat evolved by native (open circles)

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Figure 3. The dependences of heat-evolution value (-Q) and heatevolution peak maxima temperature (T) on heating rate, Zarrouk’s medium (conditions as in Figure 1): O and b, native Spirulina pl.; 4 and 2, iodized Spirulina pl.; R ) 1 mg of KI/g of dry biomass.

Figure 5. Heat absorption curves as a function of temperature (dQ/ dT, J g-1 K-1) of Spirulina pl. cells in Zarrouk’s medium (conditions as in Figure 2): (a) cells were kept in dark for 48 h at 15 °C in closed calorimetric vessel; (b) cells were preliminary heated to 51 °C and cooled for 30 min up to 5 °C; (c) cells were preliminary heated to 60 °C and cooled for 30 min up to 5 °C; (d) cells were preliminary heated to 100 °C and cooled for 40 min up to 5 °C.

Figure 4. Heat absorption curves as a function of temperature (dQ/ dT, J g-1 K-1) of Spirulina pl. cells in Zarrouk’s medium (conditions as in Figure 1): 1, 0.25 °C/min; 2, 0.166 °C/min; 3, 0.125 °C/min.

and iodized (open triangles) Spirulina pl. increased four times in the temperature range 5-53 °C. The further decrease of heating rate insignificantly decreases maximum temperature of heat evolution peak, and it does not practically change the value of heat absorbed in the temperature range 5-53 °C (Figure 3) (in this range of temperatures the profile of the heat evolution curve does not also change (Figure 4)). Extrapolation of dependence curves Q ) ×a6(V) and T ) ×a6(V) to zero heating rate (V0) gives values -Q0 ) 380.0 ( 20 J/g and T0 ) 43.8 ( 0.5. Similar measurements were carried out for iodized Spirulina pl. cells. As seen from Figure 1a, electrophoresis does not show any changes in polypeptide profile of iodized and noniodized cells. The only difference, which was observed, is the shift by 1.0 °C of heat evolution peak to low temperatures (Figure 3). A different picture is observed when a Spirulina pl. suspension was kept in dark for 48 h at 15 °C in a closed cell (Figure 5a). In this case, the heat evolution in the temperature range 5-53 °C is absent. The repeated scanning of iodized and native Spirulina pl. cellular suspensions preliminary heated to 51 °C and cooled for 0.5 h to 5 °C showed that in this case also the heat evolution observed in the temperature range 5-53 °C completely disappeared (Figure 5b).

However, in the temperature range 30-50 °C a heat absorption peak with a maximum about 41 ( 1.0 °C, which was not seen at the first heating, is registered. Other heat absorption stages remained practically invariable at high temperatures. Deconvolution of curve on Gaussian constituents gives eight stages of transition (parts a and b of Figure 5). Scanning of Spirulina pl. suspension preliminarily heated to 60 °C and then cooled for 1 h to 5 °C showed that the peak at 41 °C is not restored and a deconvolution peak with Td at 58 °C is restored insignificantly. The rest stages of heat absorption remain invariable. At scanning of preliminarily heated suspension up to 100 °C, a peak at 109 °C and shoulder at 118 °C are observed on the calorimetric curve (Figure 5d). It was interesting to find out which stages of heat absorption of dependence curves of excess heat capacity on temperature (Figures 2 and 5) correspond to denaturation of the Spirulina pl. main protein C-PC, and how iodine ions influence on this protein stability in vitro. For this purpose, we carried out a microcalorimetric study of C-PC solutions with various degrees of purification and C-PC solutions in the presence of various concentrations of added KI. These results are presented in Figures 6-9. As Figures 6a and 7a show, the denaturation of C-PC, the purification degree of which is not high (r ) 1.85), and the preparation contains also other bilisomes and membrane proteins (Figure 1a) (see also ref 18), covers the temperature range 42-100 °C with a clear and intensive maximum at 65.2 ( 0.5 °C and diffusive peaks about 54 and 86 °C. Total ∆Hd is 31.7 J/g, ∆CND ) 0.64 J g- 1 Κ-1 (∆CND is the change of heat capacity due to denaturation). The denaturation process of purified C-PC (Figures 6b and 7b) has one stage, and only the deconvolution of the curve shows the second maximum (Figure 6b). The denaturation parameters are Td ) 64.2, ∆Hd ) 34.7 J g-1, ∆CND ) 0.60 J g-1 K-1. The addition of small doses of KI into C-PC dilute solution does

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Figure 8. Heat absorption curves as a function of temperature (dQ/ dT, J g-1 K-1) of C-phycocyanin solutions at different concentrations of added KI: 1 ) 0; 2 ) 11 mol of KI/mol of protein; 3 ) 30 mol of KI/mol of protein (100 mM Na-K phosphate, pH 6.0). Protein concentration: 1, 0.42%; 2, 0.40%; 3, 0.38%. The quantity of solutions in all cases was 280.0 g.

Figure 6. Heat absorption curves as a function of temperature (dQ/ dT J g-1 K-1) of C-phycocyanin solutions with various degree of purification (100 mM Na-K phosphate, pH 6.0): (a) A620/A280 ) 1.85, protein concentration was 0.33 mg mL-1, the quantity of solution was 280.0 mg; (b) A620/A280 ) 3.5, protein concentration was 0.38 mg mL-1, the quantity of solution was 281.0 mg.

Figure 9. Dependences of denaturation temperature, Td, denaturation range width, ∆Td, determined at half-hight of heat absorption peak and denaturation enthalpy, ∆Hd, of C-phycocyanin solution at various KI concentrations (R); R ) mol of KI/mol of protein (conditions as in Figure 8).

Figure 7. (a) Differential scanning calorimetric recording of Cphycocyanin solution in 100 mM Na-K phosphate (pH 6.0): heating rate 0.42 K/min; protein concentration 0.57 mg/mL, the quantity of solution 282.0 mg, r ) 1.85. (b) Differential scanning calorimetric recording of C-phycocyanin solution in 100 mM Na-K phosphate (pH 6.0), heating rate 0.42 K/min; protein concentration 0.35 mg/mL, the quantity of solution 281.5 mg, r ) 3.5.

not influence the protein stability (Figures 8 and 9), but the change of denaturation parameters is observed (∆Hd, Td, ∆Td), when the concentration of KI is higher than 5.0 mol of KI/mol of protein. Discussion According to numerous data19,20 the process connected with photosynthesis and respiration proceeds in Spirulina pl. cells and in many cyanobacteria in the temperature range 5-50

°C. Both of these reactions influence each other and involve some supramolecular complexes, each of these complexes was isolated from various strains of Spirulina pl. and was characterized.20 The processes of photosynthesis and respiration are also characterized in detail.19,20,22 In the presented experiments, the influence of photosynthesis on thermal characteristics of Spirulina pl. cells is minimum. This is connected with the specificity of DSC measurements. Cell suspension in the process of heating is in a closed titan vessel; therefore, we think that the observed effects connected with heat evolution belong, mainly, to respiration of Spirulina pl. cells. It is shown21 that the rate curve of oxygen absorption by Spirulina pl. cells in the dark has an asymmetric profile with a maximum about 45 °C and clearly expressed shoulders about 25 and 38 °C. In these experiments, the oxygen absorption rate is minimum and it is close to zero at 10 and 52 °C. The comparison of peak profiles of heat evolution in the temperature range 5-53 °C (Figures 2 and 4) with the above-mentioned data20 shows a surprising likeness of curve forms. Besides, the direct correlation between heat evolution and rate of oxygen

Microcalorimetric Study of Spirulina pl. Cells

absorption for bacteria, yeast, and mold A-riger has been shown.23 On the basis of literature19-23 and our experimental data, we concluded that total heat evolved in the temperature range 5-52 °C at a heating rate less than 0.083 °C/min equal to 0.380 kJ g-1 dry biomass (Figures 3 and 4) is the heat of vital capacity maintenance (it includes respiration, mobility, osmotic work of cells, opposition to inhibition influences, etc.) of Spirulina. pl. cells which are in a stationary state, in the dark, and in “nonaerobic” conditions. A sharp increase of the heat evolution value in the range of scanning rates 0.25 °C/min and higher and the constancy of -Q at V < 0.083 °C/min (Figures 2-4) evidently indicate that the biochemical processes proceeding in living cells and maintaining its vital capacity, under a given set of experimental conditions, proceed with strictly appointed rates and in a strictly appointed range of temperatures. When the scanning rate is higher than the rate of these reactions, the reaction itself has a temperature optimum20 and then, because of rapid raising of temperature, these reactions may be partially or completely inhibited, as observed at high rates of heating (0.083 °C/min and higher). According to ref 20, the maximum rate of oxygen absorption by Spirulina pl. cells is reached at 45 °C, and only a temperature increase of several degrees causes it to drop to zero. This effect22 is explained by supposition about denaturation of proteins responsible for respiration. Our experimental data confirm this supposition. Indeed, as it is seen (Figure 5b) a preliminary heating of suspension up to 51 °C reveals a heat absorption peak with Td1 ) 41 ( 1 °C, ∆Td1 ) 6.1°, and Qd1 ) 2.1 J g-1 of dry biomass, which is compensated by a heat evolution peak at 47.1 °C at the first scanning (Figure 2). Figure 5b clearly shows that Qd1 of preliminarily heated Spirulina. pl. up to 51 °C is by 50% less than Qd1 of native Spirulina pl. incubated for 48 h in the dark at 15 °C under unaerobic conditions (Figure 5a). It indicates that the only 50% renaturation of the protein structure, whose denaturation happens in the temperature range 30-65 °C (Td1 ) 48 °C) (Figure 5a), occurs at the repeated heating of a suspension preliminary heated to 51 °C. It is known that globular protein C-PC exists in solution as a monomer or polymer consisting of 3, 6, or 12 subunits and the dissociation constant depends on pH and ionic forces of solution.24 About 68% of the protein is in a hexamer form, the molecular weight of which is 264000, at pH 6.0 and 0.1 M phosphate buffer.24 It was shown on the basis of fundamental studies of small globular proteins and proteins consisting of subunits,25,26,27 that ∆Hv.H and ∆Hcal may be obtained from one experiment carried out by the method of scanning microcalorimetry.25,26 Simple calculations show that ∆H

v.H

) 4RT

2

∆Cdmax ∆Hcal

where ∆Cdmax is heat capacity increase at T ) Tmax. According to ideas developed in these works, if a considered denaturation process really represents a two-stage transition, then ∆v.H must be equal to ∆Hcal. Consequently, the denaturating structure of a macromolecule represents a single

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cooperative system and a denaturation process may be considered as a transition between two various macroscopic states with the absence of intermediate forms. When ∆Hv.H/ ∆Hcal > 2, it means that a stable intermediate state occurs in the process of transition and a tertiary structure of protein contains not less than two cooperative units. Putting the obtained values of ∆Hd, ∆Cdmax, and Td for C-PC (Figure 7) in the equation we get 10.8 ( 1.2, and this means that C-phycocyanin at pH 6.0 in 100 mM phosphate buffer exists as a polymer consisting of 10-12 cooperative regions which denaturate as independent units. The values of ∆Hd and ∆CND(dQ/dT)ND obtained for C-PC with purification degree (r ) 3.5) equal to 34.7 ( 2.1 J/g and 0.60 J g-1 K-1 accordingly (Figure 7) are very close to the values of such compact globular proteins as lisocim (∆Hd ) 32.5 J/g) and mioglobin (∆CND ) 0.65 J g-1 K-1),28,29,31 but differ from globular proteins with lower molecular mass: eglin C (8.1 kDa); ∆CND ) 0.43 J K-1 g-1;30 and RNase T1(11.1 kDa) ∆CND ) 0.45 J K-1 g-1.31 Deconvolution of the dependence curve dQ/dT ) ×a6(T) on Gaussian constituents gives two peaks with transition parameters ∆Hd1 ) 11.43 J/g, Td1 ) 60.8 °C, ∆Td1 ) 13.3 °C, and ∆Hd2 ) 23.2 J/g, Td2 ) 64.1 °C, and ∆Td2 ) 4.5 °C (Figure 6). The calculations show that ∆Hd1 is equal to ∼33% of the total enthalpy and ∆Hd2 is ∼67%. If we assume that ∆Hd2 corresponds to denaturation of hexamers (Rβ)6 and ∆Hd1 corresponds to denaturation of trimers (Rβ)3, then we obtain a good coincidence with early obtained data based on study of sedimentation rate of C-phycocyanin as a function of pH, ionic strength, and temperature.24 The comparison of data given in Figure 5a with data of Figures 6 and 7 shows that Td of the third peak coincides with Td of C-PC in dilute solutions. If we take into consideration that the first six peaks of heat absorption (Figure 5a) correspond to denaturation of proteins and their integral value is equal to 22.1 J/g, then a heat coming to peak 3, with Td ) 64.5 °C, equal to 5.90 ( 0.8 J/g total protein calculated per gram of C-PC (content of C-PC in Spirulina pl. is 13.5%), gives a value equal to 43.2 ( 6.0 J/g. This value with exactness of experimental error coincides with ∆H of C-PC with r ) 3.5. Hence, it may be affirmed that C-PC in Spirulina pl. composition denaturates with Td ) 64.5 °C, and diffusion peaks with Td equal to 56 and 86 °C, obviously, are connected with denaturation of membrane proteins and high molecular proteins of phycobilisoms (see Figure 1) whose concentration, according of our calculation, is equal to 18 ( 4% (see also ref 18). It is known that C-PC is not a potassium binding protein and it does not play any significant role in cellular processes (such as cell differention, transformation, and cell damage). Therefore, we think that in the given conditions of experiment, the change of thermodynamic parameters of denaturation is connected with influence of iodine ions on protein structure. But as C-PC exists in dilute solution (pH 6.0, 100 mM phosphate) in various aggregative states: as trimers (7S), hexamers (11S), and dodecamers (18S) with predominance of hexamer forms (∼68%),24 we cannot affirm that the decrease of protein stability and broadening of denaturation range is connected with losening of monomer tertiary

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structure or with partial decondensation of various aggregative forms of C-PC. These data will be presented in following publications on the basis of C-PC studies at different values of pH in the presence of KI salts. As for the peak with Td about 108 °C, we have not determined it yet. But carrying out the comparison of these data with results of refs 32 and 33 in which the cells and nuclei of eucaryot were studied by the method of scanning microcalorimetry, we can say that the endotherm at 108 °C corresponds to DNA denaturation, and the endotherm at 118 °C from DSC studies of E. colli cells34 may be also attributed to DNA denaturation. Acknowledgment. The work was supported by ISTC Grant G-342. References and Notes (1) Monaselidze, J.; Chanchalashvili, Z.; Majagaladze, G.; Mgeladze, G. J. Polym. Sci. 1981, 69, 17-20. (2) Touchette, N.; Cole, D. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 26422646. (3) Balbi, C.; Abelmoschi, M.; Parodi, S.; Barboro, P.; Carazza, R.; Patrone, E. Biochemistry 1989, 28, 3220. (4) Monaselidze, J. Kalandadze, Ya.; Khachidze, D. J. Therm. Anal. 1996, 46, 431-440. (5) Monaselidze, J.; Kalandadze, Ya.; Topuridze, I.; Gadabadze, M. High Temp.-High Pressures 1997, 29, 677-681. (6) Spirulina platensis (Arthrospira). Physiology, Cell Biology and Biotechnology; Vonshak, Avigad, Ed.; Taylor and Francis Ltd.: Stockholm, 1997; Chapters 1-8. (7) Monk, P.; Wadso, I. J. Appl. Bacteriol. 1975, 38, 71-74. (8) Wadso, I. Thermal and Energetic Studies of Cellular Biological System; James, A. M., Ed.; John Wright and Sons: Bristol, U.K., 1987, Chapter 3, pp 35-67. (9) Johanssen, P.; Wadso, I. J. Therm. Anal. Calorim. 1999, 57, 275281. (10) Topchishvili, L.; Majagaladze, G.; Tananashvili, D.; Monaselidze, J. Abstracts, 5th Symposium/Workshops on Pharmacy and Thermal Analysis, Basel, Switserland, September 19-21, 2000. (11) Majagaladze, G.; Monaselidze, J.; Chikvashvili, R. Author’s Certificate No.1267175 USSR, 1986.

Topchishvili et al. (12) Privalov, P.; Monaselidze, J. Experiment DeVices 1967, 6, 174 (in Russian). (13) Topchishvili, L.; Tananashvili, D.; Kikvilashvili, Z. Device for Microorganizm Cultivation. Certificate No. 1323042, Georgia, 1997. (14) Topchishvili, L.; Tananashvili, D.; Kikvilashvili, Z.; Naskidashvili, A.; Kvirilashvili, T. Methods of Creation of Iodized Spirulina platensis. Certificate No. 624, Georgia, 2000. (15) Zarrouk, C. PhD Thesis, University of Paris, 1966. (16) Teale, F. W. J.; Dale, R. E. Biochem. J. 1976, 116, 161. (17) Filipovich, Yu. B.; Egorova, T. A.; Sevastianova, G. A. In Practical work on general Biochemistry; Enlightenment: Moscow, 1982; Vol. 75 (in Russian). (18) Leammly, U. C. Nature 1970, 223, 680. (19) Mohanty, P.; Srivastava, M.; Bala Krishna, K. In Spirulina platensis (Arthrospira). Physiology, Cell Biology and Biotechnology; Vonshak, Avigad, Ed.; Taylor and Francis Ltd.: Stockholm, 1997; Chapter 2, pp 19-40. (20) Vonshak, Avigad In Spirulina platensis (Arthrospira). Physiology, Cell Biology and Biotechnology;, Taylor and Francis Ltd.: Stockholm, 1997; Chapter 3, pp 43-45. (21) Nanba, J.; Saton, K. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 109. (22) Vonshak, Avigad; Gay, K.; Poplawski, P.; Ohad, I. Plant Cell Physiol. 1988, 29, 721. (23) Cooney, C. L.; Wang, D. I.; Mateles, R. Biotechnol. Bioeng. 1968, 11, 269-281. (24) Scott, E.; Berns, D. S. Biochemistry 1965, 4, No. 12. (25) Privalov, P. L.; Khechinashvili, N. N. J. Mol. Biol. 1974, 86, 665684. (26) Privalov, P. L. AdV. Protein Chem. 1979, 33, 167-241. (27) Privalov, P. L. AdV. Protein Chem. 1982, 35, 1-104. (28) Privalov, P. L.; Makhatadze, G. I. J. Mol. Biol. 1990, 213, 385391. (29) Makhatadze, G. I.; Privalov, P. L. AdV. Protein Chem. 1995, 47, 307-425. (30) Bae, S. J.; Sturtevant, J. M. Biophys. Chem. 1995, 55, 247-252. (31) Yu, Y.; Makhatadze, G. I.; Pace, C. N.; Privalov, P. L. Biochemistry 1994, 33, 3312-3319. (32) Tauhette, N.; Cole Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 2642. (33) Balbi, C.; Abelmoshi, M.; Gogiso, L.; Parodi, S.; Barboro, P.; Cavazza, B.; Patrone, E. Biochemistry 1989, 28, 3220. (34) Monaselidze, J. In CooperatiVe Changes of Biopolymers in Solution; Tbilisi, 1975; p 151 (in Russian).

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