Microcalorimetric studies of interactions between proteins and hydrophobicligands in hydrophobic interaction chromatography: effects of ligand chainlength, density and the amount of bound protein
Fy. Lin et al., Microcalorimetric studies of interactions between proteins and hydrophobicligands in hydrophobic interaction chromatography: effects of ligand chainlength, density and the amount of bound protein, J CHROMAT A, 872(1-2), 2000, pp. 37-47
Using isothermal titration calorimetry (ITC), this investigation directly m
easured the adsorption enthalpies of proteins on various hydrophobic adsorb
ents. Various amounts of butyl and octyl groups were attached onto CM-Sepha
rose to form C-4 and C-8, two types of hydrophobic adsorbents. The adsorpti
on enthalpies of both trypsinogen and alpha-chymotrypsinogen A were measure
d at 4.0 M NaCl and pH 10.0, in which most ionic interaction was suppressed
. The adsorption isotherms of both proteins on various adsorbents were also
measured, thus allowing us to calculate the Gibbs free energy and entropy
of adsorption. Experimental results indicated that the adsorption of both p
roteins on butyl-containing adsorbents was exothermic, while their adsorpti
on on octyl ones was endothermic. In addition, binding of both proteins wit
h the butyl ligand is basically an adsorption process, while binding with t
he octyl Ligand is adsorption and partition processes. Moreover, on both bu
tyl or octyl, the adsorption enthalpy became increasingly positive as the l
igand density increased, while the adsorption entropy became more positive
as the alkyl chain length or density of the adsorbent increased. In additio
n, ITC was used to measure protein-protein interaction. The adsorption enth
alpy of both proteins increased as the amount of bound protein increased, a
nd the enthalpy increase of trypsinogen appeared to be higher than that of
alpha-chymotrypsinogen A. This observation implies that protein-protein rep
ulsion was stronger among trypsinogen molecules in the experiments. (C) 200
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