本研究藉恆溫滴定微卡計(isothermal titration calorimetry)之量測，探
討蛋白質與吸附基材表面作用之機制。研究之系統包括疏水作用層析
(hydrophobic interaction chromatography, HIC)、固定化金屬親和性層析
(immobilized metal ion affinity chromatography, IMAC)、及離子交換層析
(ion-exchange chromatography, IEC)等。另外，也利用van’t Hoff plot 及相
關之熱力學關係式來分析以疏水作用力為主之程序(如疏水性氣體溶於水
和疏水性氨基酸滯留於疏水作用層析管柱之行為)。由此所獲致之結果與
數據將有助於更進一步瞭解生化反應程序中蛋白質於界面之行為，並可
應用於蛋白質之純化。
在HIC 系統的研究方面，ITC 的結果顯示鹽類對蛋白質作用機制主
要的影響在於降低去水合所需之熱量。固定不同疏水基(n-butyl 和n-phenyl)
之吸附基材之差別則在於後者與蛋白質間有p-p作用力，而導致兩吸附基
材間有明顯不同的焓和火商值。另外，蛋白質與固定n-butyl 之吸附基材之
吸附焓為負值，但與固定n-octyl 的吸附基材則為正值，顯示蛋白質與nbutyl
膠體之作用機制主要為吸附作用，而與n-octyl 膠體則為分布行為。
而且HIC 系統之焓值也隨吸附基材疏水基之密度或蛋白質之疏水表面積
之增加而增加。換言之，當HIC 系統之疏水作用增加，焓值也會隨著增
加，表示蛋白質之作用機制可能由吸附為主的作用轉為以分布為主的行
為。另外，本研究也利用ITC 的量測，探討蛋白質與蛋白質間之作用行
為。例如，蛋白質a-chymotrypsinogen A 和trypsinogen 與疏水性吸附基材
作用之焓值，皆隨蛋白質吸附量之增加而增加，而且trypsinogen 之焓值
隨吸附量增加的幅度較a-chymotrypsinogen A 的大，表示trypsinogen 分子
間之斥力可能較a-chymotrypsinogen A 間的大。
v
在IMAC 系統的研究方面，由ITC 所量得的數據得知，lysozyme 與
固定化Fe(III)作用之焓值隨pH 值或NaCl 濃度增加而降低，而且lysozyme
與固定化Cu(II)作用之焓值比Fe(III)小，表示lysozyme 與固定化Cu(II)間
產生之配位鍵所放出之熱量較Fe(III)多。另外，本研究也將一基因重組蛋
白穀胱甘月太轉移酵素(Schistosoma japonicum glutathion Stransferase,
SjGST)融合一含六個histidine 的標籤，而成為SjGST/His
來探討IMAC 系統之作用機制。ITC 之結果顯示，SjGST/His 與IMA 膠
體(NTA-Ni)作用之吸附焓較SjGST 的小，這是因為SjGST/His 與NTA-Ni
間產生較多的配位鍵。另外，SjGST/His 在正常(nature)和失活(denaturing)
狀態下與IMA 膠體(TALON)作用之吸附焓相差不大，顯示蛋白質之作用
機制在此兩種狀態下差別不大。
在IEC 系統的研究方面，是利用ITC 的量測探討蛋白質b-lactoglobulin
A & B 在蛋白質等電點(isoelectric point, pI)時與陰離子交換基材(QSepharose)
之作用機制。結果顯示在pH 值接近等電點時，蛋白質與QSepharose
間之作用主要受靜電力與疏水力之影響，而在高NaCl濃度(0.3 M)
下，則以疏水作用為主。另外，在不同溫度下所量得之吸附焓值顯示，
溫度對蛋白質與Q-Sepharose 作用機制之影響與鹽濃度有關。例如在高
NaCl 濃度(0.3 M)下，以疏水作用為主，因此對溫度的效應較明顯。

This study presents a thermodynamic framework employed by isothermal
titration microcalorimetric (ITC) measurements to explore the interaction mechanisms
between proteins and solid surfaces in various modes of liquid chromatography such
as hydrophobic interaction chromatography (HIC), immobilized metal ion affinity
chromatography (IMAC), and ion-exchange chromatography (IEC). The approach for
evaluating thermodynamic parameters based on van’t Hoff analysis associated with
hydrophobic interactions involving processes such as dissolution of non-polar gases in
water and HIC retention of dansyl amino acids was also reported. The data obtained
and conclusion reached in this manner provide valuable information about the
behaviours of biomaterials at interfaces at the molecular level and facilitate the
purification of proteins by liquid chromatography in the industrial application.
Specifically, in the study of HIC, the adsorption enthalpies of the proteins with
the hydrophobic sorbents were measured by ITC under various experimental
conditions. The results show the dependence of the free energy change for protein
adsorption to HIC sorbents on the salt composition can be mainly attributed to the
enthalpy changes associated with the protein and sorbent dehydration and
hydrophobic interactions. Differences in binding mechanisms between the n-butyland
phenyl-HIC sorbents were evident. In the latter case, the participation of p-p
hydrophobic interactions leads to significant differences in the associated enthalpy
and entropy changes. Also, the adsorption of the proteins on butyl containing
adsorbents was exothermic, while their adsorption on octyl ones was endothermic,
indicating the binding of both the proteins with butyl ligand is basically an adsorption
process, whilst the binding with octyl ligand is adsorption and partition processes.
Moreover, the enthalpy and entropy changes became increasingly positive as the
ligand density of the sorbents or exposed hydrophobic surface area of the proteins
vii
increased. As a consequence, an increased contribution from the entropy change to the
respective change in free energy occurs when HIC sorbents or proteins of higher
hydrophobicity are employed, with these larger entropy changes consistent with a
change in the interaction mechanism from a binding event dominated by adsorption to
a partitioning-like process.
Moreover, as temperature is increased from 298 to 310 K, the enthalpy change
of a-chymotrypsinogen A with butyl-Sepharose increases, while the value of
trypsinogen is deduced. This is likely owing to a-chymotrypsinogen A has a higher
area of exposed hydrophobic segments than trypsinogen does. This observation also
implies that as temperature increases, the interaction mechanism of a-
chymotrypsinogen A with butyl-Sepharose transfers from an adsorption dominated to
a partitioning process.
In addition, ITC measurements can be used to probe protein-protein interaction.
For example, the enthalpy changes of the proteins increased as the amount of bound
protein increased, and the enthalpy increase of trypsinogen appeared to be higher than
that of a-chymotrypsinogen A. This observation indicates the protein-protein
repulsion was stronger among trypsinogen molecules in this experiment.
In IMAC, various effects on the interaction mechanisms between the proteins
and the immobilized metal ions were investigated by ITC measurements. The results
reveal that the enthalpy changes decreased with the pH values and NaCl
concentrations. Furthermore, the enthalpy of lysozyme with Fe(III) is higher than that
with Cu(II) implying the heat generated from the formation of the coordination bonds
with Cu(II) is higher than with Fe(III). In addition, a recombinant protein,
Schistosoma japonicum glutathione-S-transferase (SjGST) was fused with a Cterminal
hexa-histidine tag to obtain SjGST/His. Both the proteins were also used to
probe the interaction mechanisms with two commercial affinity resin, Ni-NTA and
TALON (Co2+), under the conditions of with and without the presence of denaturant.
The result demonstrates that SjGST/His had a lower enthalpy change with Ni-NTA
than did SjGST, mainly attributed to the formation of more coordination bonds with
viii
or a stronger binding with Ni-NTA. Moreover, the difference between the enthalpy
change of SjGST/His onto TALON under the nature and denaturing condition were
insignificant, implying the binding topography of the hexa-histidine tail with
immobilized Co2+ was not significantly changed with the presence of denaturant. As a
consequence, the proposed binding models and the directly measured adsorption heat,
can be combined to elucidate the difference in the interaction mechanisms of the
proteins adsorption onto the sorbents containing transition metal ions in
thermodynamic perspectives.
In IEC study, the interaction mechanisms between b-lactoglobulin A and B
(Lg A, Lg B) with an anion exchanger, Q-Sepharose at pH near isoelectric point (pI)
were examined under various NaCl concentrations and temperature by the equilibrium
binding analysis and the adsorption enthalpy directly measured by ITC. The results
demonstrate that the involvement of electrostatic and hydrophobic forces collectively
affect the interaction behaviors of Lg A or Lg B with Q-Sepharose at pH near pI,
leading to the changes in the binding affinities, capacities and adsorption enthalpies.
Specifically, at a higher NaCl concentration (0.3M), the hydrophobic interactions
becoming a more important contributor to the adsorption, while at 0.03 M NaCl, the
electrostatic attraction was dominated. Furthermore, the enthalpy change values
measured at pH near pI under various NaCl concentrations and temperature have
confirmed that the effects of temperatures on equilibrium binding behaviors of Lg A
or Lg B with Q-Sepharose were salt concentration-dependent, due to their different
interaction mechanisms at 0.03 M and 0.3 M NaCl.

1. Yamamoto, S., and Ishihara, T., “ion-exchange chromatography of proteins near
the isoelectric points”, J. Chromatogr. A 852 (1999) 31.
2. Lin, F.-Y., Chen, C.-S., Chen, W.-Y., and Yamamoto, S., “microcalorimetric
studies of the interaction mechanisms between proteins and Q-Sepharose at
pH near pI: effects of NaCl concentrations, pH values, and temperature”, J.
Chromatogr. A 912 (2001) 281.
3. Ruckenstein, E., Marmur, A., and Gill, W. N., “feasibility of colloid particles
separation by potential barrier chromatography”, J. Colloid. Interface Sci. 61
(1977) 183.
4. Ruckenstein, E., and Srinivasan, R., “potential barrier chromatography: an HPLC
method for protein separation”, Sep. Sci. Technol. 17 (1982) 763.
5. Lesins, V., and Ruckenstein, E., “control of the interaction potential for improved
resolution in potential barrier chromatography”, Sep. Sci. Technol. 19 (1984) 219.
6. Jun, S.-H., and Ruckenstein, E., “separation of immunoglobulins by potential
barrier chromatography”, Sep. Sci. Technol. 19 (1984) 531.
7. Ruckenstein, E., and Lesins, V., “protein separation by potential barrier
chromatography”, Biotechnol. Bioeng. 28 (1986) 432.
8. Jun, S.-H., and Ruckenstein, E., “separation of a multicomponent mixture of
proteins by potential barrier chromatography”, Sep. Sci. Technol. 21 (1986) 111.
9. Ruckenstein, E., Chillakuru, R., “retention studies and protein separation by
potential barrier chromatography,” Sep. Sci. Technol. 25 (1990) 207.
10. Norde, W., J., “energy and entropy of protein adsorption”, Disper. Sci. Technol.
13 (1992) 363.
11. Norde, W., “protein adsorption at solid surfaces: a thermodynamic approach”,
Pure Appl. Chem. 66 (1994) 491.
12. Norde. W, “adsorption of proteins from solution at the solid interface”, Adv.
Colloid Interface Sci, 25 (1986) 267.
13. Lin, F.-Y., Chen,W.-Y., Ruaan, R.-C., and Huang, H.-M., “microcalorimetric
studies of interactions between proteins and hydrophobic ligands in
hydrophobic interaction chromatography: effects of ligand chain length,
94
density and the amount of bound protein”, J. Chromatogr. A 872 (2000) 37.
14. Vailaya, A., and Horvath, Cs., “enthalpy-entropy compensation in hydrophobic
interaction chromatography”, J. Phys. Chem. 100 (1996) 2447.
15. Vailaya, A., and Horvath, Cs., “retention thermodynamics in hydrophobic
interaction chromatography”, Ind. Eng. Chem. Res. 35 (1996) 2964.
16. Vailaya, A., and Horvath, Cs., “retention in hydrophobic interaction
chromatography and dissolution of non-polar gases in water”, Biophys. Chem. 62
(1996) 81.
17. Dec, S.F. and Gill, S.J., “heats of solution of gaseous hydrocarbons in water at 25
℃”, J. Sol. Chem. 13 (1984) 27.
18. Cabani, S., Gianni, P, Lepori, L. and Mollica, V., “group contributions to the
thermodynamic properties of non-ionic organic solutes in dilute aqueoussolution”,
J. Sol. Chem. 10 (1981) 563.
19. Makhatadze, G. I., and Privalove, P.I., “contribution of hydration to protein
folding thermodynamics. I. the enthalpy of hydration., J. Mol. Biol. 232 (1993)
639.
20. Haidacher, D., Vailaya, A., and Horvath, Cs, “temperature effects in hydrophobic
interaction chromatography”, Proc. Natl. Acad. USA 93 (1996) 2290.
21. Hearn, M.T.W. and Zhao, G., “investigations into the thermodynamics of
polypeptides interaction with nonpolar ligands”, Anal. Chem. 71 (1999) 4874.
22. Boysen, R.I., Wang, Y., Keah, H.H., and Hearn, M.T.W., “observation on the
origin of the non-linear van’t Hoff behaviour of peptides in hydrophobic
environments”, Biophys. Chem. 77 (1999) 79.
23. 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”, J. Colloid Interface Sci. 229
(2000) 600.
24. Diogo, M. M., Cabral, J. M. S., and Queiroz, J. A., “preliminary study on
hydrophobic interaction chromatography of chromobacterium viscosum lipase on
polypropylene glycol immobilized on sepharose”, J. Chromatogr. A 796 (1998)
171.
25. Melander, W., and Horvath, Cs., “salt effects on hydrophobic interactions in
precipitation and chromatography of proteins: an interpretation of the lytropic
95
series”, Arch. Biochem. Biophys. 183 (1977) 200.
26. Melander, W., Corradoni, D., and Horvath, Cs., “salt-mediated retention of
proteins in hydrophobic-interaction chromatography application of solvophobic
theory”, J. Chromatogr. 317 (1984) 67.
27. Szepesy, L., and Horvath, Cs., “specific salt effects in hydrophobic interaction
chromatography of proteins”, Chromatographia 26 (1998) 13.
28. Schmuch, M.N., Nowlan, M.P., and Gooding, K.M., “effects of mobile phase and
ligand arm on protein retention in hydrophobic interaction chromatography”, J.
Chromatogr. 371 (1986) 55.
29. Perkins, T.W., Mak, D.S., Root, T.W., and Lightfoot, E.N., “protein retention in
hydrophobic interaction chromatography: modeling variation with buffer ionic
strength and column hydrophobicity”, J. Chromatogr. A 766 (1997) 1.
30. Gill, D.S. Roush, D.J., Shick, K.A., and Willson, R.C., “microcalorimetric
characterization of the anion-exchange adsorption of recombinant cytochrome b5
and its surface charge mutants”, J. Chromatogr. A 715 (1995) 81.
31. Norde, W., and Anusiem, A. C. I., “adsorption, desorption and re-adsorption of
proteins on solid surfaces”, Colloid. Surface. 66 (1994) 73.
32. Norde, W., and Favier, J. P., “structure of adsorbed and desorbed proteins”,
Colloid. Surface. 64 (1992) 87.
33. Arai, T., and Norde, W., “the behavior of some model proteins at solid-liquid
interfaces. 1. adsorption from single protein solution”, Colloid. Surface. 51 (1990)
1.
34. Sun, L., and Carr, P. W., “mixed-mode retention of peptides on phosphatemodified
polybutadiene coated zirconia”, Anal. Chem. 67 (1995) 2517.
35. Sun, L., and Carr, P. W., “chromatography of proteins using polybutadiene-coated
zirconia”, Anal. Chem. 67 (1995) 3717.
36. Ruaan, R.-C., Yang, A., and Hsu, D., “bifunctional adsorbents for hydrophobic
displacement chromatography of proteins”, Biotechnol. Prog. 16 (2000) 1132.
37. Baker, B. M. and Murphy, K. P., “evaluation of linked protonation effects in
protein binding reactions using isothermal titration calorimetry”, Biophys. J. 71
(1996) 2049.
38. Garciafuentes, L., Camaraartigas, A., Lopezmayorga, O., and Baron, C., “a
calorimetric study of the binding of AMP to liver-glycogen phosphorylase-b”,
96
Biochim. Biophys. Acta Protein Struc. Mol. Enzymol. 1294 (1996) 83.
39. Gorshkova, I., Moore, J. L., McKenney, K. L., and Schwarz, F. P.,
“thermodynamics of cyclic nucleotides binding to the cAMP receptor protein and
its T127L mutant”, J. Biol. Chem. 270 (1995) 21679.
40. Porath, J. Carlsson, J. Olsson, I., and Belfrage, G., “metal chelate affinity
chromatography - a new approach to protein fractionation”, Nature 258 (1975)
598.
41. Wu, C.-F., Chen., W.-Y. and Lee, J.-F., “microcalorimetric studies of the
interactions of imidazole with immobilized Cu(II): effects of pH value and salt
concentration”, J. Colloid Interface Sci. 183 (1996) 236.
42. Chen,W.-Y., Wu, C.-F., and Tsao, H.-K., “microcalorimetric studies of the
interactions of lysozyme with immobilized Cu(II) - effects of pH value and salt
concentration”, J. Colloid Interface Sci. 190 (1997) 49.
43. Lin, F.-Y., Chen, W.-Y., and Sang, L.-C., “microcalorimetric studies of the
interactions of lysozyme with immobilized metal ions: effects of ion, pH value,
and salt concentration”, J. Colloid Interface Sci. 214 (1999) 373.
44. Chen, W.-Y., Lin, F.-Y., and Wu, C.-F., “Microcalorimetric Studies of
Biomaterials at Interface” in: Kallay, N., (Eds.), Interfacial Dynamics, Marcel
Dekker, New York, 1999, p. 419.
45. Lin, F.-Y., Chen, W.-Y. and Chen, H.-M., “microcalorimetric study of the effect
of hexa-histidine tag and denaturant on the interaction mechanism between
protein and metal-chelating gel”, J. Colloid Interface Sci. 238 (2001) 333.
46. Chattopadhyay, D., Stewart, J. E., and Delucas, L. J., “large-scale preparation of
the D10 form of staphylokinase by in vitro processing of recombinant
staphylokinase with purified human plasminagen”, Appl. Biochem. Biotechnol. 69
(1998) 147.
47. Armisen, P., Mateo, C., Cortes, E., Barredo, J. L., Salto, F., Diez, B., Rodes, L.,
Garcia, J. L., Fernandez-Lafuente, R., and Guisan, J. M., “selective adsorption of
poly-His tagged glutaryl acylase on tailor-made metal chelate supports”, J.
Chromatogr. A 848 (1999) 61.
48. Cha, H.J., Dalal, N.G., Pham, M.-Q., and Bentley, W.E., “purification of human
interleukin-2 fusion protein produced in insect larvae is facilitated by fusion with
green fluorescent protein and metal affinity ligand”, Biotechnol. Prog. 15 (1999)
97
283.
49. Chen, W.-Y., Wu, C.-F., and Liu, C.-C., “interactions of imidazole and proteins
with immobilized Cu(II) ions: effects of structure, salt concentration, pH in
affinity and binding capacity”, J. Colloid Interface Sci. 180 (1996) 135.
50. Lin. F.-Y., Chen, W.-Y., and Huang, S.-Y., “selective separation of caseinophosphopeptides
through immobilized metal ion affinity chromatography: effects
of pH values, salt concentrations and degree of phosphorylation”, Bioproc. Eng.
23 (2000) 467.
51. Vailaya, A., and Horvath, Cs., “exothermodynamic relationships in liquid
chromatography”, J. Phys. Chem. B 102 (1998) 701.
52. Finette, G. M. S., Mao, Q. M. and Hearn, M. T. W., “Comparative studies on the
isothermal characteristic of proteins adsorbed under batch equilibrium conditions
to ion-exchange, immobilized metal ion affinity and dye affinity matrices with
different ionic strength and temperature conditions”, J. Chromatogr. A 763 (1997)
71.
53. Fang, F., Aguilar, M. I., and Hearn, M. T. W., “influence of temperature on the
retention behavior of proteins in cation-exchange chromatography”, J.
Chromatogr. A 729 (1996) 49.
54. Fang, F., Aguilar, M. I., and Hearn, M. T. W., “temperature-induced changes in
the bandwidth behaviour of proteins separated with cation-exchange adsorbents”,
J. Chromatogr. A 729 (1996) 67.
55. Milton, M. T. W., “physicochemical factors in polypeptide and protein
purification and analysis by high-performance liquid chromatographic techniques:
current status and challenges for the future”, Sep. Sci. Technol. 2 (2000) 71.
56. Haynes, C. A., Sliwinsky, E., and Norde, W., “structural and electrostatic
properties of globular-proteins at a polystyrene water interface”, J. Colloid
Interface Sci. 164 (1994) 394.
57. Koutsoukos, P. G., Norde, W., and Lyklema, J., “protein adsorption on hematite
(a-Fe2O3) surfaces”, J. Colloid Interface Sci. 95 (1983) 385.
58. Lin, F.-Y., Chen,W.-Y., and Hearn, M. T. W., “microcalorimetric studies of
the interaction mechanisms between proteins and hydrophobic solid surfaces
in hydrophobic interaction chromatography: effects of salts, hydrophobicity of
the sorbent and the structure of proteins”, Anal. Chem., (2001) in press.
98
59. Lin, F.-Y., and Chen, W.-Y., “Microcalorimetry of Solid Surfaces” in Hubbard, A.
T. (Eds.), Encyclopedia of Surface and Colloid Science, Marcel Dekker, New
York, 2001, in press.
60. Horvath, Cs., Melander, W., and Molnar, I., “solvophobic interaction in liquid
chromatography with nonpolar stationary phases”, J. Chromatogr. 125 (1976)
129.
61. Leffler, J., and Grunwald, E., Rates and Equilibrium of Organic Reactions,
Wiley- InterScience, New York, 1963.
62. Melander, W. R., Horvath, Cs., and Mannan, C. A., “mobile phase effects in
reversed-phase chromatography .4. retention by normal-alkylbenzenes as a
function of column temperature and the nature and concentration of organic
cosolvent”, Chromatographia 15 (1982) 611.
63. Krstulovic, A. M., Colin, H. and Guiochon, G., “comparison of methods used for
the determination of void volume in reversed-phase liquid-chromatography”,
Anal. Chem. 54 (1982) 2438.
64. Tchapla, A. M., Colin, H. and Guiochon, G., “Linearity of Homologous Series
Retention Plots in Reversed-Phase Liquid-Chromatography”, Anal. Chem. 56
(1984) 621.
65. Lesellier, E., Tchapla, A. M., and Krstulovic, A. M., “use of carotenoids in the
characterization of octadecylsilane bonded columns and mechanism of retention
of carotenoids on monomeric and polymeric stationary phases”, J. Chromatogr.
645 (1993) 29.
66. Lee, B., “isoenthalpic and isoentropic temperatures and the thermodynamics of
protein denaturation “, Proc. Natl. Acad. Sci. USA 88 (1991) 5154.
67. Lee, B., “enthalpy-entropy compensation in the thermodynamics of
hydrophobicity”, Biophys. Chem. 51 (1994) 271.
68. Hermann, R. B., “theory of hydrophobic bonding II: the correlation of
hydrophobic solubility in water with solvent cavity surface area”, J. Phys. Chem.
76 (1972) 2754.
69. Raffa, R. B., “extrathermodynamics of the drug-receptor interaction”, Life
Science 65 (1999) 967.
70. Sandi, A., and Szepesy, L., “characterization of various reversed-phase columns
using the linear free energy relationship: I. evaluation based on retention factors”,
99
J. Chromatogr. A 818 (1998) 1.
71. Sandi, A., and Szepesy, L., “characterization of various reversed-phase columns
using the linear free energy relationships: II. evaluation of selectivity”, J.
Chromatogr. A 818 (1998) 19.
72. Sandi, A., Nagy, M., and Szepesy, L., “characterization of reversed-phase
columns using the linear free energy relationship: III. effect of the organic
modifier and the mobile phase composition”, J. Chromatogr. A 893 (2000) 215.
73. Li, J., and Carr, P. W., “characterization of polybutadiene-coated zirconia and
comparison to conventional bonded phases by use of linear solvation energy
relationships”, Anal. Chim. Acta 334 (1996) 239.
74. Abraham, M. H., Poole, C. F., and Poole, S. K., “solute effects on reversed-phase
thin-layer chromatography: a linear free energy relationship analysis”, J.
Chromatogr. A 749 (1996) 201.
75. Richmond, T. J., “solvent accessible surface-area and excluded volume in
proteins - analytical equations for overlapping spheres and implications for the
hydrophobic effect”, J. Mol. Biol. 178 (1984) 63.
76. Privalove, P. L., and Makhatadze, G. I., “contribution of hydration to protein
folding thermodynamics. II. the entropy and Gibbs energy of hydration”, J. Mol.
Biol. 232 (1993) 660.
77. Murphy, K. P., and Freire, E., “thermodynamics of structural stability and
cooperative folding behavior in proteins”, Adv. Protein Chem. 43 (1992) 313.
78. Sharp, K. M., Nicholls, A., Friedman, R., and Honing, B., “extracting
hydrophobic free-energies from experimental-data - relationship to protein
folding and theoretical-models”, Biochem. 30 (1991) 9686.
79. Lumry, R., and Rajender, S., “enthalpy-entropy compensation phenomena in
water solutions of proteins and small molecules: a ubiquitous property of water”,
Biopolymers 9 (1970) 1125.
80. Morin, N., Guillaume, Y. C., Peyrin, E., and Rouland, J.-C., “peculiarities of an
imidazole derivative retention mechanism in reversed-phase liquid
chromatography: b-cyclodextrin concentration and temperature consideration”, J.
Chromatogr. A 808 (1998) 51.
81. Liu, L., Yang, C., and Guo, Q.-X., “a study on the enthalpy-entropy
compensation in protein unfolding”, Biophys. Chem. 84 (2000) 239.
100
82. Basiuk, V. A., and Gromovoy, T. Y., “comparative study of amino acid adsorption
on the bare and octadecyl silica from water using high-performance liquid
chromatography”, Colloid. Surface. A 118 (1996) 127.
83. Jensen, W. A., Armtrong, J. M., De Giorgio, J., and Hearn, M. T. W.,
“thermodynamic analysis of the stabilisation of pig heart mitochondrial malate
dehydrogenase and maize leaf phosphoenolpyruvate carboxylase by different
salts, amino acids and polyols”, Biochim. Biophys. Acta 1338 (1997) 186.
84. Ecaterina, T., and Tatiana, O., “influence of temperature and n-alkane pair on the
methylene increments to the thermodynamic functions of solution on SE-30 and
Carbowax capillary columns”, J. Chromatogr. A 844 (1999) 201.
85. Murphy, K. P., and Gill, S. J., “group additivity thermodynamics for dissolution
of solid cyclic dipeptides into water”, Thermochim. Acta 172 (1990) 11.
86. Murphy, K. P., Privalov, P. L., and Gill, S. J., “common features of protein
unfolding and dissolution of hydrophobic compounds”, Science 247 (1990) 559.
87. Naghibi, H., Dec, S. F., and Gill, S. J., “heat of solution of methane in water from
0 to 50 ℃”, J. Phys. Chem. 90 (1986) 4621.
88. Naghibi, H., Dec, S. F., and Gill, S. J., “heats of solution of ethane and propane in
water from 0 to 50 ℃”, J. Phys. Chem. 91 (1987) 245.
89. Naghibi, H., Ownby, D. W., and Gill, S. J., “enthalpies of solution of butanes in
water from 5 to 45 ℃“, J. Chem. Eng. Data 32 (1987) 422.
90. Uhlig, H. H., J. Phys. Chem. 42 (1937) 1215.
91. Sinanoglu, O., in Pullman, B. (Eds.), Molecular Associations in Biology,
Academic Press, New York, 1968, p.427.
92. Tanford, C., in Physical Chemistry of Macromolecules, Wiley, New York, 1961.
93. Kirkwood, J. G., in Proteins, Amino Acids and Peptides, Reinhold, New York,
1943.
94. Snyder, L. R., in High Performance Liquid Chromatography: Advances and
Perspectives, Academic Press, New York, 1980.
95. Signanoglu, O., in Ratajczak, H., and Orville-Thomas, W. J., (Eds.) Molecular
Interactions, J. Wiley, New York, 1982, p.283.
96. Melander, W. R., and Horvath, Cs., in McGuire, M. J., and Suffett, I. H., (Eds.)
Activated Carbon Adsorption of Organic from the Aqueous Phase, ann Arbor
Science, Ann Arbor, MI. 1980, p.65.
101
97. Signanoglu, O., J. Chem. Phys. 755 (1981) 463.
98. Girifalco, L. A., and Good, R. J., J. Phys. Chem. 61 (1957) 904.
99. Chothia, C., “the nature of the accessible and buried surfaces in proteins”, J. Mol.
Biol. 105 (1976) 1.
100. Naghibi, H., Tamura, A., and Sturtevant, J. M., “significant discrepancies
between van’t Hoff and calorimatric enthalpies”, Proc. Natl. Acad. Sci. USA 92
(1995) 5597.
101. Liu, Y., and Sturtevant, J. M., “significant discrepancies between van’t Hoff and
calorimatric enthalpies. II”, Protein Sci. 4 (1995) 2559
102. Liu, Y., and Sturtevant, J. M., “significant discrepancies between van’t Hoff and
calorimatric enthalpies. III”, Biophys. Chem. 64 (1997) 121.
103. Katti, A., Maa, Y. F., and Horvath, Cs., “protein surface area and retention in
hydrophobic interaction chromatography”, Chromatographia 24 (1987) 646.
104. Makhatadze, G. I., and Privalov, P. L., “energetics of interactions of aromatic
hydrocarbons with water”, Biophys. Chem. 50 (1994) 285.
105. Lenk, T. J., Ratner, B. D., Gendreau, R. M., and Chittur, K. K., “IR spectral
changes of bovine serum-albumin upon surface-adsorption”, J. Biomed. Mater.
Res. 23 (1989) 549.
106. Lu, D. R., and Park, K., “effect of surface hydrophobicity on the conformationalchanges
of adsorbed fibrinogen”, J. Colloid Interface Sci. 144 (1991) 271.
107. van Oss, C. J., Absolom, D. R., and Neumann, A. W., in: Gribnau, C. J., Visser,
J., Nivard, R. J. K., (Eds.), Affinity Chromatography and Related Techniques,
Elsevier, Amsterdam, 1982, p. 29.
108. Hofmeister, F., Arch. Exp. Pathol. Pharmakol. 24 (1888) 247.
109. Hofstee, B. H. J., “immobilization of enzymes through non-covalent binding to
substituted agarose”, Biochem. Biophys. Res. Commun. 53 (1973) 1137.
110. de Araujo, A. F. P., Pochapsky, T. C., and Joughin, B., “thermodynamics of
interactions between amino acid side chains: experimental differentiation of
aromatic-aromatic, aromatic-aliphatic, and aliphatic-aliphatic side-chains
interactions in water”, Biophys. J. 76 (1999) 2319.
111. Reubsaet, J. L. E., and Vieskar, R., “characterisation of π–π interactions
which determine retention of aromatic compounds in reversed-phase liquid
chromatography”, J. Chromatogr. A 841 (1999) 147.
102
112. Staros, J. V., Wright, R. W., and Swingle, D. M., “enhancement by nhydroxysulfosuccinimide
of water-soluble carbodiimide-mediated coupling
reactions”, Anal. Biochem. 156 (1986) 220.
113. Smith, M. C., Furman, T. C., and Pidgeon, C., “immobilized iminodiacetic acid
metal peptide complexes. identification of chelating peptide purification
handles for recombinant proteins”, Inorg. Chem. 26 (1987) 1965.
114. Ericson, M. L., Mooney, N. A., and Charron, D. J., “Prokaryotic production of
the human T cell receptor Vb2 chain and binding to toxic-shock syndrome toxin-
1”, Gene 168 (1996) 257.
115. Min, C., and Verdine, G. L., “immobilized metal affinity chromatography of
DNA”, Nucl. Acid. Res. 24 (1996) 3806.
116. Chen, H.-M., and Chen, K.-T., “production and characterization of glutathione-
S-transferase fused with a poly-histidine tag”, Enzyme Microb. Technol. 27 (2000)
219.
117. Chen, H.-M., Luo, S.-L., Chen, K.-T., and Lii, C.-K., “affinity purification of
Schistosoma japonicum glutathione-S-transferase and its site-directed mutants
with glutathione affinity chromatography and immobilized metal affinity
chromatography”, J. Chromatogr. A 852 (1999) 151.
118. Habig, W.H., Pabst, M. J., and Jakoby, W. B., “glutathione-S-transferase”, J. Biol.
Chem. 249 (1974) 719.
119. Bradford, M. M., “a rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding”,
Anal. Biochem. 72 (1976) 248.
120. George, P., and McClure, D. S., Prog. Inorg. Chem. 1, 418 (1959).
121. Pearson, R. G., “hard and soft acids and bases-the evolution of a chemical
concept”, Coor. Chem. Rev. 100, 403 (1990).
122. Eigel, W. N., Butler, J. E., Ernstrom, C. A., Farrell, H. M., Harwalkar, V. R.,
Jenness, R., and McL. Whitney, R., “nomenclature of proteins of cows milk”, J.
Dairy Sci. 67 (1984) 1599.
123. Chen, W.-Y., Dai, K.-C., Ho, K.-C., Lin, F.-Y., Huang, H.-M., Ruaan, R.-C.,
application of steric-mass action model to triblock copolymer as a displacer in
ion-exchange displacement chromatography”, J. Chin. Inst. Chem. Engrs. 30
(1999) 375.