Lecture 2 Flashcards

Question

Affinity chromatography

Answer 1

Drug molecule connected to solid support resin by a linker. Receptor added to column containing affinity resin. Gradient if free drug is run through column. Record free drug concentration required to elute receptor. Advantage: no need to label drug. Disadvantage: requires DR complex with long half life i.e. Kd less than or equal to 100nM. Half life= time to reach equilibrium. If your Kd is larger than 100nM, then this method doesn't work well.

Answer 2

Makes use of cross-linked polysaccharide beads that allow small molecules to penetrate but excludes large molecules. Beads of many sizes and degrees of exclusion are available commercially. Usually run in glass columns that filter by gravity, but there are many kinda of spin columns that are run in a centrifuge to speed passage through the columns.

Answer 3

1. Load receptor onto column containing resin-bound ligand. 2. Run gradient of free ligand into column to compete with bound ligand for receptor. 3. Record free ligand concentration at which receptor is eluted from the resin. Since free drug is eluted, it doesn't need to be labeled.

Answer 4

There are pores in the beads. The chosen size allows for small molecules such as the free drug to travel through the pores, as well as around the beads. The drug receptor complex and free receptor are too big so they have to travel around the beads only. The volume outside if the gel beads is small than the volume within, so the DR and R elute first. Use radio labeling.

Answer 5

Choose the right pore size. Can also use FPLC forced pressure chromatography. Basically working to pump the solution through the column rather than waiting for gravity flow.

Answer 6

- bead volume: volume of the column taken up by the beads and surrounding buffer. - void volume: Bed volume - bead volume (aka excluded volume). Can determine void volume by putting something known to be too large to flow through the beads, and is something that you can readily detect. For instance, a dye molecule conjugated to a large protein. Look at what fractions of the dye show up at certain times. Gel filtration can be used to find Kd or to determine if two drugs bind to different sites.

Answer 7

DR will come out first and will come out in excluded volume (10-7)= 3ml. So it will start to come out in tube number 4. First 3 mls that come out will represent excluded volume that was already in the column, and by the fourth tube youll start to see DR complex coming through. Then finaly you should see free drug coming through with the void volume.

Answer 8

- choose a bead size that excludes all R. - volume sample (D+R) should be less than 5% of the column volume (100ml column = less than 5ml of solution loaded). - large length to diameter ratio for the column. - run separation slowly (can run faster if using FPLC system). If you try to speed it up using gravity it can mess up your results. Typically want a tall thin column as opposed to a short wide column.

Answer 9

Pre-equilibrate column with non-radiolabeled drug. Bump in drug concentration is due to more of the label coming off in fractions with the receptor also present. If you have a lower number of drug relative to receptor, you may actually get a dip instead. Height of peak is proportional to amount of DR complex.

Answer 10

So if we have a high Kd and low affintiy, we can do equilibrium gel filtration. Done the same way, but the column is pre-equilibrated with free drug at the concentration that we are measuring. So in addition to having a fixed concentration of drug added to a fixed concentration of receptor in a tube, and then adding it to the top, we also have a buffer in which the gel is suspended pre-equilibrated by running an excess of the drug concentration. This can be time intensive unless you use multiple columns.

Answer 11

2 peaks seen. First is receptor bound to drug and the second is the free drug.

Answer 12

If you have a numerically small Kd, generally a Kd less than micromolar concentration is considered high affinity. Want a high affinity situation for gel filtration because then the drug and receptor would stay together well enough to keep the DR in tact as it goes through the volume. Something with a micromolar or above Kd would probably tend to fall apart once free drug is removed. So you would get a falsely low DR complex measurement at a given drug concentration. So we ant a small Kd, and high affinity.

Answer 13

showed that tubulin was associated with a number of drugs known to depolymerize microtubules. Can use these peaks to analyze if there is competition binding. If a competitor is added, then the first peak will disappear. So this would be like in the presence of Podophyllotoxin. If Vinca Alkaloids were added, then you would get the same peaks as seen above since it doesnt compete with binding of colchicine to microtubules (there are multiple drug binding sites).

Answer 14

These are non-equilibrium methods that are rapid and inexpensive. Examples are the use of activated charcoal, that adsorbs aromatic compounds and can quickly deplete the free drug concentration by centrifugation. Or adsorption of the target (receptor) onto charged surfaces. These approaches often have important limitations due to the possible rapid dissociation of the drug receptor complex, poor adsorption efficacy, dissociation of the complex from the absorptive surface, etc. - wouldn't work well if the drug bound to charcoal can still bind to the receptor, resulting in the receptor being peletted out as well and not in the supernatant.

Answer 15

Can do batch methods. Have an antibody to tubulin that is bound to beads that you pour into a column. As you pour the DR complex over, it gets trapped. Often times when dealing with a crude extract, even though you centrifuge, you may get particulate matter which causes the column to clog. So using a batch method would help with this. Batch method is simply an alternative to the column method.

Answer 16

If a receptor has a net negative charge, you can use a filter paper that has the opposite charge and use it to pull out the receptor: has been used for isolating tubulin-colchicine complexes.

Answer 17

HPLC : high pressure liquid chromatography. Excellent for small molecule separation/ identification (measures free drug). If the drug is too big, it may not separate enough. Fluorescence spectroscopy: increase or decrease in fluorescence in response to drug binding. Most receptors have aromatic amino acids close to the surface, which can undergo fluorescence. Circular Dichroism (CD) spectra: change in absorbance difference between left and right rotated polarized light upon drug binding. Surface Plasmon Resonance

Answer 18

In some cases, after binding you have an aromatic amino acid becoming exposed, thereby increasing fluorescence, in other cases you have the aromatic amino acid being pulled in deeper into the receptor, resulting in fluorescence quenching. The latter is a more accurate way to measure drug binding to receptor. Can also be looking at drug fluorescence. Advantages of fluorescence spectroscopy: 1.) no radioactivity involved, 2.) you dont have to separate the drug from DR complex. So you can work with a fairly low affinity interaction and dont have to worry about dissociation because the drug is always present in solution.

Answer 19

Two peaks. Shift in fluorescence in response to drug binding to receptor. Advantage: can make 2 measurements, both of which are proportional to amount of drug bound. So if something goes wrong with one measurement you can tell based on the other measurement.

Answer 20

In drug quenching, the higher the concentration of drug the lower amount of fluorescence. The opposite is true if dealing with increased fluorescence in response to drug binding.

Answer 21

Receptor is immobilized on a dextran matrix attached to a thin layer of nanogold particles Drug at various concentrations flows over the dextran-gold surface Polarized light is reflected off of the gold layer The angle of reflection is altered by binding of the drug in a manner proportional to the amount of drug bound Real time measurements allows determination of off and on rate constants Kd can be calculated from koff/kon ratio

Answer 22

thin layer of gold provides a good mirror for polarized light. As you bind drug you alter the ability of gold layer to reflect the polarized light (changes the angle), which is proportional to the amount of drug bound. So the more drug bound to the receptor, the greater perturbation of reflected polarized light. So by measuring the angle of reflection you can get the amount of binding and can directly measure the rate constant from which you can derive the Kd.

Answer 23

very expensive mainly due to instruments. Since the free drug is present, this is basically an equilibrium-type method. So you can use this with low-affinity binding situations.

Answer 24

What they are actually showing is binding of an antibody to some tethered antigen. Typically we’re looking for drug receptor binding, with the receptor linked to the dextran surface and the drug is free in solution. Whats shown is somewhat reversing this idea, so you can do it in both ways. The key feature is that whatever is bound, needs to have its surface that is going to interact with its free agent not hindered by the attachment. So lets say we have the receptor attached to the surface, but the attachment blocks the binding site to the drug. Obviously this wouldn’t work well. Its generally easier to tether the receptor without interfering with drug binding, so typically the receptor is covalently attached to the dextran.

Answer 25

- need for a labeled drug. SPR doesn't. - sensitivity (how much receptor do you need?) SPR is the most sensitive. - stability of DR complex. - susceptibility of receptor to denaturation (important for assays that take a long time).