Question

1. If you were to run a SP-sepharose column equilibrated with a buffer at pH 7, what would be the range of pIs of the proteins that you expect to bind to the column fully?

2. In hemoglobin Rainer, Tyr 145 in the β subunit is replaced by Cys, which forms a disulfide bond with another Cys residues in the same subunit. This prevents the format of ion pairs that normally stabilize the T state. How does hemoglobin Rainer differ from normal hemoglobin with respect to:

a. Oxygen affinity_______ b. The Hill Constant______

3. Enzymes bind to the _____________ and lower the _________ energy.

Answer 1

Answer:

1,  a guanidine-based refold, and the use of anion exchangers then there is a slight chance that a Q strong anion exchange fast flow resin may work at high pH near 10 -11 with significant feedstock dilution to make the binding as strong as possible in the presence of that much chloride conductivity from the refold. Unfortunately your protein is probably not stable to this pH. I believe Pall or Tosoh have an high salt tolerant anion exchanger. Glycerol and lower temperatures may help lower ionic activity but there may be loss of binding capacity. There may also be left over redox couples from the refold and misfolds that can adversely affect the concentrated product at the head of the column. Keep in mind that switching the pH to 4.5 from an alkaline pH may cause on-column precipitation of misfolded product molecules as the column transitions to acidity, especially at the protein concentration at the column head. A multi cycle batch-wise mode with diluted feed stock may be an approach to avoid column issues. This anion exchange strategy is unlikely to work with cation exchange approaches although there may be a high salt tolerant cation exchanger as well. HIC followed by anion exchange may be another strategy where greater purity may be achieved due to greater aggregate removal and the need for high salt for HIC binding.

• TEV protease may still slightly bind at pH 8 due to smaller % of neg. molecules in equilibrium with neutral and pos. species but the conductivity will likely be sufficient to prevent binding.
• Dimethyl urea or dimethyl sulfone (not sulfoxide) can be used if you're concerned about carbamylation
• Imidazole at pH 8 will have tau nitrogen(s) with free pair of electrons in p orbitals providing basic character but not donating to the quaternary amine of Q
• Arginine could be helpful in controlling aggregation
• No guanidine helps so all systems "go" for binding.
• Citric has carboxyl functions so only neutral or negative with pKa's ~3, ~4-5, and ~6-7 as I recall; also carboxyl is a weak acid but phosphate a strong acid. Both will help in the step elution with the NaCl.
• Arginine from refold will be gone once the load stage is complered. But may want to have some there along with some chaotrope to inhibit precipitation/aggregation.
• pH gradients are not straight forward to control, especially in this situation, and slower residence times are needed to develop the pH and allow equilibration of the bound solutes with the pH. This will also allow plenty of time for precipitation/aggregation to occur. You may need chaotropes.
• Probably should elute in batch mode to avoid an unfavorable or precipitating pH value forming at the interface/front between the two buffers in the column mode. May need chaotrope.
• If running in column mode then use limit of linear flow rate from the pressure-flow curve provided by manufacturer to create rapid pH change, but may need chaotrope.
• After loading, might want to rinse the resin with a very low concentration (few millimolar) of the pH 8 load buffer so that your step elution with pH 4.5 buffer will be more easily/quickly changed to pH 4.5. May need chaotrope.
• Don't need FPLC: just a good peristaltic pump (adjustable occlusion) couple of beakers connected by glass U-tube or two-chamber plastic gradient former (BRL) if needing gradient, silicon Pt cured tubing or Sanipure (20-30 psi), mag stirrer for one beaker, feed tubing from buffer A to column, detector and strip chart recorder. With batch loading there will not be much separation so fast flow and low bed height will be consistent in producing low resolution with fast buffer exchange with minimal pressure issues on Fast Flow resins. Manually collect fractions according to recorder trace.

2,

(a) Hemoglobin Rainer differ from normal hemoglobin with respect to oxygen affinity Because the mutation destabilizes the T conformation of hemoglobin Rainier, the R (oxygen)conformation is more stable. Therefore, the oxygen affinity of hemoglobin Rainier is greater than normal.

(b) Hemoglobin Rainer differ from normal hemoglobin with respect to Because the R conformation of hemoglobin Rainier is more stable than the T conformation, even when the molecule is not oxygenated, O₂-binding cooperativity is reduced. The Hill coefficient of hemoglobin Rainier is therefore less than that for normal hemoglobin.

3, Enzymes are large proteins that bind small molecules. When bound to an enzyme, the bonds in the reactants can be strained (that is stretched) thereby making it easier for them to achieve the transition state. This is one way for which enzymes lower the activation energy of a reaction.