由於聚乙二醇(polyethylene glycol or PEG)可以抵抗蛋白質的吸附，因此其擁有很好的生物相容性。目前已有相當多種聚乙二醇以及其相關的衍生物，被運用於各種不同的研究與應用的領域。然而聚乙二醇抗蛋白質吸附的作用機制尚未明確。我們認為由於聚乙二醇擁有良好的水合能力，具有從鄰近的水層優先排出其他生物分子的特性，因此可以有效的抵抗蛋白質的吸附。對於此機制在熱力學上的相關資訊並未被提出，因此在本次研究中，我們利用恆溫滴定微卡計在不同的鹽濃度、鹽離子、溫度以及不同PEG分子量等環境下，量測聚乙二醇的稀釋焓以及其與蛋白質之間的吸附焓。藉此來探討不同環境對於聚乙二醇水合能力的影響以及對聚乙二醇與蛋白質之間吸附行為的影響。
在稀釋焓結果方面，所有的稀釋焓都呈現放熱的結果，代表聚乙二醇分子在本研究當中所使用的不同溶液環境(包括不同鹽濃度、鹽種類、溫度及不同PEG分子量)下，均較傾向水合的狀態。其中在鹽濃度、溫度以及PEG分子量效應中，發現聚乙二醇水合能力會隨著溶液中的鹽濃度、溫度或者PEG分子量的提升而降低，從能量觀點上來說，在高鹽、高溫或高PEG分子量環境下，稀釋PEG為較不energy favorable的程序。而在鹽離子效應方面，發現影響PEG水合能力的離子序列符合了Hofmeister series。另外，我們發現Flory-Huggins parameter(χ12)在各種環境下的值均為負值，代表着聚乙二醇與水溶劑分子作用良好。
從恆溫吸附曲線的結果來看，當額外添加鹽類時，則降低了溶菌酶(lysozyme)吸附在Toyopearl Ether-650s上的吸附量以及吸附親合力。推測溶菌酶吸附於Ether-650過程中，除了疏水作用力外，靜電作用力的貢獻也必須考慮。此外，隨著溶液中鹽濃度或溫度的提升，溶菌酶與Ether-650間的吸附親合力有增加的趨勢。主要原因來自於溶菌酶與Ether-650間疏水作用力的提升。而在鹽離子效應方面，其影響溶菌酶吸附程度的序列也符合Hofmeister series。
在吸附焓的研究方面，在不同的鹽濃度、鹽種類及溫度環境下，溶菌酶與Ether-650間的吸附焓大多呈現吸熱，表示在吸附過程中大多為去水合的程序所主導。而在高鹽之1M KCl(25℃)下的溶液環境則呈現放熱，在此環境下溶菌酶的吸附則為焓驅動的非典型的疏水作用。在鹽離子的效應方面，發現添加銨鹽離子的溶液中，吸附焓的吸熱量較添加其他鹽類離子大。此外本研究也利用P.I. Model來計算在添加不同鹽類離子溶液下的吸附過程中，整體系統去水合的量。結果發現：(1)在添加銨鹽的溶液環境下，整體溶液系統去水合的量較鉀鹽的溶液環境下多。(2)對於溶菌酶與聚乙二醇吸附的系統而言，其吸附過程中去水合的量相較於文獻當中，溶菌酶與疏水觸手(如，C4,C8分子)吸附的系統還要來的少。從此點也證明聚乙二醇相較於其他非極性分子而言，其擁有較強大的水合能力，而此特性也是抵抗蛋白質吸附的重大因素之一。

The characteristics of preventing nonspecific adsorption of protein has lead to extensive usage of PEG and its derivatives for biomedical applications. We consider that the interaction of water with the PEG is a major determinant of preventing protein adsorption. However, the thermodynamics aspect of the mechanism has not been well addressed. Therefore, in this study, we described the hydration behavior of PEG by measuring the dilution heat of PEG with various salt concentration, types of salt ions, temperature and molecular weight of PEG. In addition, we measured the isotherms and the interaction enthalpy between protein and Ether-650S with various salt concentrations, salt types and temperature by batch isotherms and ITC.
From the results of dilution heat, we observed that all the dilution heat are exothermic at all condition (i.e. salt conc. and types, temperature, PEG MW). It indicated that the PEG molecule is prefer to hydrate with water than aggregation in the conditions investigated. At high salt concentration, temperature and molecular weight of PEG, the dilution heat of PEG is less exothermic due to the poor hydration of PEG. In thermodynamics, the dilution of PEG is more energy unfavorable at high salt concentration, temperature and molecular weight of PEG. And the extent of salt ions which affect the hydration of PEG is consistent with the Hofmeister series. Besides, we also observed that all the values of Flory-Huggins parameter(χ) are negative at each condition. It also indicated that all the solvent which we used are good solvent for PEG.
From the results of isotherm, the amount of lysozyme adsorb on Ether-650 will decrease with increase the salt concentration. We considered that both of hydrophobic and electrostatic interaction affect the binding affinity of lysozyme.
The enthalpy of lysozyme adsorbed on Ether-650S are almost endothermic. It indicated that the hydrophobic force is the driving force during the adsorption process. However, the enthalpy of adsorption is exothermic at 1M KCl. This lead to the suggestion that the adsorption of lysozyme with the Ether-650 is of the “nonclassical” hydrophobic type interaction at 1M KCl. In this study, we also calculated the number of water molecules released during the adsorption by preferential interaction model. From the results of P.I Model, we can conclude : (1) when we adding
ammonium chloride to the solution, the system released more water molecules than add that of potassium chloride during the binding process.(2)compare with literature data, PEG ligand have stronger capability of hydration than other hydrophobic ligands.

1. Harris, J. M. “Poly(ethylene glycol) chemistry
biotechnical and biomedical applications”, New York
Plenum Press c1992, p1-12
2. Dolan A. K. and Edwards S. F. “The effect of excluded
volume on polymer dispersant action”, Proc. R. Soc.
Lond. A. 343(1975) 427-442
3. Hermans J. “Excluded-volume theory of polymer–protein
interactions based on polymer chain statistics”, J
Chem Phys 77(1982) 2193-2203
4. Queriroz J. A., Garcia F. A. P. and Cabral J. M. S.
“Hydrophobic interaction chromatography of
Chromobacterium viscosum lipase on polyethylene glycol
immobilized on Sepharose”, Journal of chromatography
A. 734(1996) 213-219
5. Fee C. J. and Van Alstine J. M. “PEG-proteins:Reaction
engineering and separation issues”, Chemical
Engineering Science 61(2006) 924-939
6. Nie F. Q., Xu Z. K., Ye P., Wu J. and Seta P. “
Acrylonitrile-based copolymer membranes containing
reactive groups:effects of surface-immobilized poly
(ethylene glycol)s on anti-fouling properties and blood
compatibility”, Polymer 45(2004) 399-407
7. Fan X. W., Lin L. J. and Messersmith P. B. “ Cell
Fouling Resistance of Polymer Brushes Grafted from Ti
Substrates by Surface-Initiated Polymerization:Effect
of Ethylene Glycol Side Chain Length”,
Biomacromolecules 7(2006) 2443-2448
8. Abuchowski A., van Es T., Palczuk N. C. and Davis F. F.
“Alteration of immunological properties of bovine serum
albumin by covalent attachment of polyethylene
glycol”, J. Biol. Chem. 252(1977)3578-3581
9. Roberts M. J., Bentley M. D. and Harris J. M.
“Chemistry for peptide and protein PEGylation”,
Advanced Drug Delivery Reviews 54(2002) 459-476
10. Zhang M. Q., Desai T. and Ferrari M. “Proteins and
cells on PEG immobilized silicon surfaces”,
Biomaterials 19(1998) 953-960
11. Sharma S., Johnson R. W. and Desai T. A. “XPS and AFM
analysis of antifouling PEG interfaces for
microfabricated silicon biosensors” Biosensors and
Bioelectronics 20(2004) 227-239
12. Farruggia B., Nerli B. and Picó G. “Study of the
serum albumin-polyethyleneglycol interaction to
predict the protein partitioning in aqueous two-phase
systems”, Journal of Chromatography B 798(2003) 25-33
13. Halperin A. “Polymer Brushes that Resist Adsorption
of Model Proteins: Design Parameters”, Langmuir 15
(1999) 2525-2533
14. Napper D. H. and Netschey A. “Studies of the steric
stabilization of colloidal particles”, Journal of
Colloid and Interface Science 37(1971)528-535
15. Leckband D., Sheth S. and Halpern A. “Grafted poly
(ethylene oxide) brushes as nonfouling surface
coating”, J. Biomater. Sci. Polymer Edn. 10(1999)
1125-1147
16. Hunter R. “Foundations of Colloid Science”, vol. I,
Oxford Science Publications, New York, 1989.
17. Dill K. A. “Dominant forces in protein folding”,
Biochemistry 29(1990) 7133-7155
18. Makhatadze G. I. and Privalov P. L. “Energetics of
protein-structure”, Adv. Protein Chem. 47(1995) 307-
425
19. Bruinsma G. M., van der Mei H. C. and Busscher H. J.
“Bacterial adhesion to surface hydrophilic and
hydrophobic contact lenses”,Biomaterials 22(2001)
3217.
20. Farruggia B., Garcia G., D''Angelo C. and Picó G.
“Destabilization of human serum albumin by
polyethylene glycols studied by thermodynamical
equilibrium and kinetic approaches”, International
journal of biological Macromoleculaes 20(1997) 43-51
21. Herold D. A., Keil K.and Bruns D. E. “Oxidation of
polyethylene glycols by alcohol dehydrogenase”,
Biochem. Pharm. 38(1989) 73-76.
22. Hribar B., Southall N. T., Vlachy V. and Dill K. A.
“How ions affect the structure of water”, J. Am.
Chem. Soc. 124(2002) 12302-12311
23. Dill K. A., Truskett T. M., Vlachy V. and Hribar-Lee
B. “Modeling water, the hydrophobic effect, and ion
salvation”, Annu. Rev. Biophys. Biomol. Struct. 34
(2005) 173-199
24. Robinson RA and Stokes RH. Electrolyte
Solutions.NewYork:Dover (2002) p571
25. Samoilov O. Y. “A new approach to the study of
hydration of ions in aqueous solutions.”Discuss.
Faraday Soc. 24(1957) 141-146
26. Hofmeister F. “Zur Lehre von der Wirkung der Salze”,
Arch. Exp. Pathol. Pharmakol. 24(1888) 247-260
27. Szlifer I. “Protein Adsorption on Surfaces with
Grafted Polymers:A Theoretical Approach”, Biophys J.
72(1997) 595-612
28. Szlifer I. “Polymers and proteins: interactions at
interfaces”, Curr.Opin. Solid State Mater. Sci. 2
(1997) 337-344
29. Szlifer I. “Protein adsorption on tethered polymer
layers:effect of polymer chain architecture and
composition”, Physica A. 244(1997)370-388
30. Feldman K., Hahner G., Spencer N. D., Harder P. and
Grunze M. “Probing Resistance to Protein Adsorption
of Oligo(ethylene glycol)-Terminated Self-Assembled
Monolayers by Scanning Force Microscopy”, J. Am.
Chem. Soc. 121(1999) 10134-10141.
31. Harder P., Grunze M., Dahint R., Whitesides G. M. and
Laibinis P. E. “Molecular Conformation in Oligo
(ethylene glycol)-Terminated Self- Assembled
Monolayers on Gold and Silver Surfaces Determines
Their Ability To Resist Protein Adsorption”, Phys.
Chem. B. 102(1998) 426-436.
32. Wang, R. L. C., Kreuzer, H. J. and Grunze, M.“
Molecular Conformation and Solvation of Oligo(ethylene
glycol)-Terminated Self-Assembled Monolayers and Their
Resistance to Protein Adsorption”, J. Phys. Chem. B.
101(1997) 9767-9773.
33. Kane R. S., Deschatelets P. and Whitesides G. M.
“Kosmotropes Form the Basis of Protein-Resistant
Surfaces”, Langmuir 19(2003) 2388-2391
34. Yancey P.H., Clark M. E., Hand S. C., Bowlus R. D. and
Somero G. N. “Living with water stress: evolution of
osmolyte systems”, Science 217(1982) 1214-1222.
35. Timasheff S. N. “Control of protein stability and
reactions by weakly interacting cosolvents: the
simplicity of the complicated.” Adv. Protein Chem. 51
(1998) 355-432
36. Shulgin I. L. and Ruckenstein E. “Preferential
hydration and solubility of proteins in aqueous
solutions of polyethylene glycol”, Biophysical
Chemistry 120(2006) 188-198
37. Lazos D., Franzka S. and Ulbricht M. “Size-Selective
Protein Adsorption to Polystyrene Surfaces by Self-
Assembled Grafted Poly(ethylene Glycols) with Varied
Chain Lengths”, Langmuir 21(2005) 8774-8784
38. McPherson T., Kidane A., Szleifer I. and Park K.
“Prevention of Protein Adsorption by Tethered Poly
(ethylene oxide) Layers: Experiments and Single-Chain
Mean-Field Analysis”, Langmuir 14(1998) 176-186
39. Li L. Y., Chen S. F., Zheng J., RatnerB. D. and Jiang
S. “Protein Adsorption on Oligo(ethylene glycol)-
Terminated Alkanethiolate Self-Assembled Monolayers:
The Molecular Basis for Nonfouling Behavior”, J.
Phys. Chem. B 109(2005) 2934-2941
40. Schroen C. G. P. H., Stuart M. A. C., van der Voort
Maarschalk K.,van der Padt A. and van''t Riet K.
“Influence of Preadsorbed Block Copolymers on Protein
Adsorption: Surface Properties, Layer Thickness, and
Surface Coverage”, Langmuir 11(1995) 3068-3074
41. Vermette P. and Meagher L. “Interactions of
phospholipid- and poly(ethylene glycol)-modified
surfaces with biological systems: relation to physico-
chemical properties and mechanisms”, Colloids and
Surfaces B: Biointerfaces 28(2003) 153-198
42. Sofia S. J., Premnath V. and Merrill E. W. “Poly
(ethylene oxide) Grafted to Silicon Surfaces: Grafting
Density and Protein Adsorption”, Macromolecules 31
(1998) 5059-5070
43. Queiroz J. A., Garcia F. A. P., Cabral J. M. S.
“Hydrophobic interaction chromatography of
Chromobacterium viscosum lipase on polyethylene
glycol immobilized on Sepharose”, Journal of
Chromatography A 734 (1996) 213-219
44. Wang R. W., Zhang Y., Ma G. H., Su Z. G. “
Modification of poly(glycidyl methacrylate–
divinylbenzene) porous microspheres with polyethylene
glycol and their adsorption property of protein”,
Colloids and Surfaces B: Biointerfaces 51(2006) 93-99
45. Ladbury J. E. “Application of Isothermal Titration
Calorimetry in the Biological Sciences: Things Are
Heating Up!”, BioTechniques 37(2004) 885-887.
46. Safronov A. P. and Zubarev A. Y. “Flory-Huggins
parameter of interaction in polyelectrolyte solutions
of chitosan and its alkylated derivative”, polymer 43
(2002) 743-748
47. Gedde U. W. “Polymer physics”, London New York
Chapman & Hall 1995, p64
48. Lin F. Y., Chen W. Y., Ruaan R. C. and Huang H. M.
“Microcalorimetric Studies of Interactions between
Proteins and Hydrophobic Ligandsin Hydrophobic
Interaction Chromatography: Effects of Ligand Chain
Length, Density and the Amount of Bound Protein”,
Journal of Chromatography A 872(2000) 37-47
49. Huang H. M., Lin F. Y., Chen W. Y. and Ruaan R. C.
“Isothermal Titration Microcalorimetric Studies of the
Effect of Temperature on Hydrophobic Interaction
between Proteins and Hydrophobic Adsorbents”, Journal
of colloid and Interface Science 229(2000) 600-606
50. Richards E. G. “An Introduction to Physical
Properties of Large Molecules in Solutoin”, Cambridge
University Press: Cambridge,1980
51. Harris J. M. and Zalipsky S. “Poly(ethylene glycol)
Chemistry and Biological Application”, Washington,
DC : American Chemical Society c1997,Chapter 2
52. Mooney J. F., Hunt A. J., McIntosh J. R., Liberko C.
A., Walba D. M. and Rogers C. T. “Patterning of
functional antibodies and other proteins by
photolithography of silane monolayers”, Proc. Natl.
Acad. Sci. USA. 93(1996) 12287-12291
53. Maurya N. S. and Mittal A. K. “Applicability of
Equilibrium Isotherm Models for the Biosorptive
Uptakes in Comparison to Activated Carbon-Based
Adsorption”, Journal of environment engineering. 132
(2006) 1589-1599
54. Shimomura O., Flood P. R., Inouye S., Bryan B. and
Shimomura A. “Isolation and Properties of the
Luciferase Stored in the Ovary of the Scyphozoan
Medusa Periphylla periphylla”, Biol. Bull. 201(2001)
339-347
55. Ceccaroli P., Cardoni P., Buffalini M., De Bellis R.
and Piccoli G. “Separation of hexokinase activity
using different hydrophobic interaction supports”,
Journal of chromatography B. 702(1997) 41-48
56. McHenry C. S., Seville M. and Cull M. G. “A DNA
Polymerase III Holoenzyme-like Subassembly from an
Extreme Thermophilic Eubacterium”, J. Mol. Biol. 272
(1997) 178-189
57. van Oss C. J., Good R. J. and Chaudhury M. K. “Nature
of the antigen-antibody interaction: Primary and
secondary bonds: Optimal conditions for association
and dissociation”, Journal of Chromatography B 376
(1986) 111-119
58. Arnulphi C., Jin L. H., Tricerri M. A. and Jonas A.
“Enthalpy-Driven Apolipoprotein A-I and Lipid Bilayer
Interaction Indicating Protein Penetration upon Lipid
Binding”, Biochemistry 43(2004) 12258-12264
59. Seelig J. and Ganz P. “Nonclassical Hydrophobic
Effect in Membrane Binding Equilibria”, Biochemistry
30(1991) 9354-9359
60. Lightfoot E. N., Perkins T. W., Mak D. S. and Root T.
W. “Protein retention in hydrophobic interaction
chromatography:modeling variation with buffer ionic
strength and column hydrophobicity”, Journal of
chromatography A 766(1997) 1-14
61. Pavey K. D. and Olliff C. J. “SPR analysis of the
total reduction of protein adsorption to surfaces
coated with mixtures of long- and short-chain
polyethylene oxide block copolymers”, Biomaterials 20
(1999) 885-890
62. Ding H. M., Shao L., Liu R. J., Xiao Q. G. and Chen J.
F. “Silica nanotubes for lysozyme immobilization”,
Journal of Colloid and Interface Science 290(2005) 102-
106
63. Woodle M. C. “Controlling liposome blood clearance by
surface-grafted polymers”, Advanced Drug Delivery
Reviews 32(1998) 139-152
64. Jeon S. I., Lee J. H., Andrade J. D. and de Gennes P.
G. “Protein-surface interactions in the presence of
polyethylene oxide”, J. Colloid Interface Sci. 142
(1991) 149-158
65. Ostuni E., Chapman R. G., Holmlin R. E., Takayama S.
and Whitesides G. M. “A Survey of Structure-Property
Relationships of Surfaces that Resist the Adsorption
of Protein”, Langmuir 17(2001) 5605-5620
66. Vailaya A. and Horváth C. “Solvophobic theory and
normalized free energies of nonpolar substances in
reversed phase chromatography”, J. Phys. Chem. B 101
(1997) 5875-5888
67. Tasaki K. “Poly(oxyethylene)-Water Interactions: A
Molecular Dynamics Study”, J. Am. Chem. Soc. 118
(1996) 8459-8469