Wednesday 16 May 2012

Mixed opinions, disturbed flow


Loading of unequal amounts of protein at different temperatures and different buffer conditions (Tables 1 and 2). 
How to compare 20 mg loaded at 4C in 50 mM PBS (pH 7.4) to 125 mg loaded at 37C in 50 mM PBS (pH 7.4) to determine temperature effects on binding, elution etc?




 But besides the salt in the buffer, there's also more protein loaded at 24 C and 37 C than at 4 C (Tables 1 and 2 above). Wouldn't that also result in more sorbent binding and hence, eluting?



    
Figures 5, 6, 9, 10 and the paragraph above: decreasing the pH to 4 elutes the protein from both ligands.  Lower pH = higher concentrations of hydrogen ions which can protonate ligands AND the proteins in the mixture.
Maybe increasing protonation of IgG and other proteins decreases their negative charge and the protonation of ligands would decrease electrostatic interactions between basic nitrogen of the ligand and negatively charged groups of acidic amino acid residues of IgG (and other proteins). 
This means that pH 4 can elute these proteins better than higher pH.
But the lanes in the gels containing pH 5.5 eluates also contain IgG, much like the lanes in the gels containing pH 4 eluates. So why does "pH 5.5 not favour adsorption of IgG"?




               

 Increasing salt concentration has been used to elute many proteins from (especially) hydrophobic supports.   It disrupts electrostatic interactions between the ligand (support) and the proteins and helps their elution.  So how can it be said that "at higher concentration of salt (0.6 M and 0.8 M NaCl) no protein elutes instead it binds more strong to the column due to hydrophobic interaction (not shown in gel)"? 
It may well be that whatever binds predominantly through electrostatic interactions is eluted with increasing concentrations of salt (and increasing this beyond a certain threshold doesn't help) and beyond this, whatever's stuck on the ligand/support is mostly (or more) through hydrophobic interactions so only very harsh conditions can elute this. 
It may also be that salt molecules attract water molecules towards themselves and create a "hydrophobic" environment in the vicinity of the ligand-protein pairs. 
But does it mean that high concentrations of salt necessarily create that much hydrophobicity to make the proteins stick to the column more?  It would be interesting to see this data. 


 Given that loaded amounts are different for the two buffers and the two ligands at different temperatures, how accurate are these statements about binding capacity?











Ranjini, S.S., Bimal, D., Dhivya, A.P. & Vijayalakshmi, M.A. (2010). Study of the mechanism of interaction of antibody (IgG) on two mixed mode sorbents, Journal of Chromatography B, 878 (15-16) 1037. DOI:

No comments:

Post a Comment