Impact of denaturation with urea on recombinant apolipoprotein A-IMilano ion-exchange adsorption: equilibrium uptake behavior and protein mass transfer kinetics.
We have studied the equilibrium uptake behavior and mass transfer rate of recombinant apolipoprotein A-I(Milano) (apo A-I(M)) on Q Sepharose HP under non-denaturing, partially denaturing, and fully denaturing conditions. The protein of interest in this study is composed of amphipathic alpha helices that serve to solubilize and transport lipids. The dual nature of this molecule leads to the formation of micellar-like structures and self association in solution. Under non-denaturing conditions equilibrium uptake is 134 mg/mL media and the isotherm is essentially rectangular. When fully denatured with 6 M urea, the equilibrium binding capacity decreases to 25 mg/mL media and the isotherm becomes less favorable. The decrease in both binding affinity and media capacity when the protein is completely denatured with 6 M urea can be explained by the loss of all alpha helical structure. The rate of apo A-I(M) mass transfer on Q Sepharose HP was characterized using a macropore diffusion model. Results of modeling studies indicate that effective pore diffusivity increases from 4.5 x 10(-9) cm2/s in the absence of urea to 6.0 x 10(-8) cm2/s when apo A-I(M) is fully denatured with 6 M urea. Based on light-scattering data reported for apo A-I, protein self association appears to be the dominant cause of slow protein mass transfer observed under non-denaturing conditions.
Hunter
Alan K
AK
Pfizer Inc., Global Biologics, Chesterfield, MO, USA. alan.k.hunter@pfizer.com
Suda
Eric J
EJ
Das
Tapan K
TK
Shell
Robert E
RE
Herberg
John T
JT
Ramasubramanyan
Natraj
N
Gustafson
Mark E
ME
Ho
Sa V
SV
D000327:Adsorption; D016632:Apolipoprotein A-I / Q000032:analysis* / Q000737:chemistry*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011489:Protein Denaturation; D014508:Urea / Q000737:chemistry* / Q000378:metabolism*
D016632:Apolipoprotein A-I; D014508:Urea