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Title. SUPRAMOLECULAR MODELS OF SOLID PROTEINS. V.V. Gorbatchuk , M.A.Ziganshin, N.A.Mironov, I.S.Antipin, B.N.Solomonov, A.I.Konovalov Department of Chemistry, Kazan State University, Kazan, Russia. Problems :

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  1. Title SUPRAMOLECULAR MODELS OF SOLID PROTEINS V.V. Gorbatchuk, M.A.Ziganshin, N.A.Mironov, I.S.Antipin, B.N.Solomonov, A.I.Konovalov Department of Chemistry, Kazan State University, Kazan, Russia

  2. Problems: - Thermodynamic description of the role of water in the protein-substrate binding - Thermodynamic criteria of biocompatibility Strategy: Comparison of the structure - binding property relationships for proteins and more simple objects

  3. Choice of protein models Property ? Structure Zeolites Polymers Crystalline organic hosts Solutions ? + Proteins

  4. Object Gas-phase enzymatic reactors Yang F. and Russell A. J., Biotechnol. Bioeng., 49 (1996) 709. Enzymatic reactions in nonaqueous solvents Enzymatic reactions in solid phase Khurgin Yu. I., MaksarevaE. Yu,Bioorg. Khim 17(1991) 76-80 P.J. Halling, Enzyme Microb. Technol. 16 (1994) 178-206 S S S substrate vapor Applications solid protein H2O H2O H2O Protein + + Effect of substrate dehydration masks the molecular recognition n H2O H2O H2O H2O Receptor properties of enzymes and antibodies in water solutions and in solid state are similar A.M.Klibanov, Trends Biotechnol. (2000) 18, 85 enzymes S.S.Setford, Trends Anal. Chem. (2000) 19, 330-339antibodies

  5. Objects Sorbates/substrates/guests t-BuOAc CCl4 CH3OH CHCl3 Casein b-Lactoglobulin c-Hexane n-Heptane CH3CN 1,4-Dioxane o-Xylene n-Hexane EtOH CH3NO2 PrCN EtCN Pyridine Pinacolone C6H5Et Invertase Albumin Trypsin 2,2’-(9-hydroxy-9-fluorenyl)biphenyl Collagen (CH3)2CO n-Octane Toluene C6H6 n-PrOH C6H5OCH3 n-Nonane n poly(acrylic acid 6-aminohexylamide) b-cyclodextrin tert-butylthiacalix[4]arene

  6. ELECTROPNEUMATIC VALVE SPLITTER FUSED SILICA COLUMN VIAL WITH SAMPLE THERMOSTATE DEVICE OF STATIC HEADSPACE GAS CHROMATOGRAPHIC ANALYSIS Electropneumatic dosing B. Kolb, P. Pospisil, T. Borath and M. Auer, J. High Res. Chromatogr. & Chromatogr. Commun., 1979, 2, 283. Activity determination A. Hussam and P. W. Carr, Anal. Chem., 1985, 57, 793. Data examples J. H. Park, A. Hussam, P. Couasnon, D. Fritz and P. W. Carr, Anal. Chem., 1987, 59, 1970. solid phase binding of vapors in comparable conditions

  7. hydration in g H2O/g protein Dried proteins Casein (0.06 h) Pure protein: Trypsin (0.05 h) Effect of lactose (~70%) V.V.Gorbatchuk, M.A.Ziganshin, N.A.Mironov, B.N.Solomonov,Biophys. Biochim. Acta. 1545(2001) 326-338 high binding More selective than zeolites low binding V.V.Gorbatchuk, M.A.Ziganshin, B.N.Solomonov, M.D.Borisover, J.Phys.Org.Chem.,10 (1997) 901. - albumin Conclusion: solid proteins are nonporous objects Protein Isotherms

  8. Phase transition Trypsin (0.05 h) clathrate formation &phase transition V. V. Gorbatchuk, A. G. Tsifarkin, I. S. Antipin, B. N. Solomonov, A. I. Konovalov, P.Lhotak, I.Stibor J. Phys. Chem.B. , 2002, 106(23); 5845-5851 C6H6 Contents of lactose is ~70% No binding without protein V. V. Gorbatchuk, A. G. Tsifarkin, I. S. Antipin, B. N. Solomonov, A. I. Konovalov, J. Seidel, F. Baitalov, J. Chem.Soc.Perkin Trans. 2, 2000, N11, P.2287-2294 V.V.Gorbatchuk, M.A.Ziganshin, N.A.Mironov, B.N.Solomonov, Biophys. Biochim. Acta. 1545(2001) 326-338

  9. Solid proteins in low-water conditions Gas-phase reactors: enzymatic reactions Enthalpies of protein immersion in water-organic mixtures Yang F. and Russell A. J., Biotechnol. Bioeng., 49 (1996) 709. V.A.Sirotkin, M.D. Borisover, B.N.Solomonov, Biophys. Chem. 69 (1997) 239-248. . • Protein suspensions in nonaqueous solvents: • water sorption • enzymatic reactions Enzymatic reactions in solid phase Khurgin Yu. I., MaksarevaE. Yu,Bioorg. Khim 17(1991) 76-80 P.J. Halling, Enzyme Microb. Technol. 16 (1994) 178-206 Cooperative hydration effects rate ofenzymatic reaction What is the hydration effect on the initial state? protein hydration

  10. Cooperative hydration effect 200 Benzene Dioxane 150 l/g EtOAc ), 0 100 /(P/P S V 50 0 0.00 0.05 0.10 0.15 0.20 h , g H O / g protein 2 Hydration threshold Casein Albumin V.V. Gorbatchuk, M.A. Ziganshin, B.N. Solomonov, Biophys. Chem. 81 (1999) 107-123. protein • water •substrate clathrates are formed ---- this process defines the activity of enzymes and binding ability of antibodies

  11. Hydrophobic hydration EtOH n Effect of hydrophobic hydration is a sufficient thermodynamic evidence of biocompatibility h,gH2O/g polymer or HSA

  12. Higher temperature is necessary for protein to function in low-water conditions high effect for hydrophobic compounds No effect for hydrophilic compound opposite effect for water G. Brausse et al. JACS. 1968. V.72. P.3098-3105 b-Lactoglobulin

  13. Acknowledgements • Participants: • Dr. Ziganshin M.A. • Dr. Tsifarkin A.G. • Mironov N.A. • Prof. Antipin I.S. • Prof. Solomonov B.N. • Prof. Konovalov A.I. Cooperation with Dr. W.Habicher TU Dresden, Germany Dr. Th.Haertle LEIMA INRA, Nantes, France Dr. M.D.Borisover Inst. Soil & Water, Bet Dagan, Israel Grants URBR (015.05.01.016), BRHE (REC-007) ANT (7-7.1-60.2001)

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