Friday, 4 May 2012

Protein Chromatography: Lectures 2 and 3

- Kd affects a
---> sub milimolar to mM range
- if mM is high
 ---> may not interact
 ---> a may be low
- If Kd is decreased, a is smaller than 0.5
- Kd is the key to altering a.

- Assume binding site is equal to strength
----> not true
- Range of Kd's
---If Kd is said to be 50 mM, it's + or -
- [Protein] is not constant
- Pump to top of column ---> flows down ---> diffusion ---> [Proteins] decreases as it goes down
- If from 10 ml of protein goes to 1 ml,
---> protein is eluted at bottom
--->as it flows, molecules diffuse out.
- Avidity effect affects protein binding.
- Manmade surface interacts with protein
--- heterogeneous
--- solid-phase.


Dynamic effects
- Thermodynamics take time
- must equilibrate
- Flow rate is important
- If elution flow rate too slow, diffusion/dispersion overtakes dissociation/unbinding.
- With fast elution, 2 bands/fractions can overlap, once fractions of very close properties is immobilised.
- If too slow, bands get wider and overlap.
- High concentrated binding at start
---> when eluted, not all dissociates
--->some remnant binds at lower columns.
- If too fast or slow ---> inferior quality separation.
- Equilibrium must establish.
- With a wider band of protein
---> multiple bands ---> overlap ---> mix
---> bind to column ---> elute
---> proteins flow down at different rates due to design of the column
---> if it takes too long, the proteins will mix
- spreading is called dispersion
---> dictates how well we separate 2 proteins
- When protein hits column it sticks to the top of the column
--- form region/band of protein
---buffer does not stick to column
--- continually pump in buffer
--- immobilise protein ---> flow with buffer
---> gets bigger as it flows down
---> due to diffusion

- Protein bands move down column when mobilised
---come off column at different times
--- may overlap
---> get mixed proteins at bottom
--- when mobile ----> diffuse and spread


-Diffusion
---molecules diffuse from area of high concentration to area of low concentration


- Liquid flows through very porous solid phase
--- like stream going on pebbles
--- should be minimised
--- diffusion makes bands bigger
--- dilute protein


Diffusion
- Comes out at bottom
--- collect in fractions
--- measure [protein] as it exits column
--- use UV light


- Protein has tryptophan ---> use UV light
--- fluorescent
--- emission alpha to protein


- Resolution is denoted by position of 2 peaks
- Separate further ---> narrower bands
- Narrower bands ---> higher resolving power
- Less diffusion ---> better selectivity
--- as you go down column


Turbulent flow

  • Solid phase forces liquid past
  • Eddy currents
  • Fast flow decreases diffusional broadening ---> increase eddy currents
  • Slow flow hs opposite effet
  • When making proteins by kilos, must be efficient
  • Characterise column with standards
  • Predict resolution

Turbulent flow
  • don't remember equation
  • L = length of column
  • D = how fast protein flows
  • dp = how finely mil solid phase
  • td= off rate, Kd
  • lambda= friction, how rough solid phase is
Resolution/ band spreading (correct equation)
  • High diffusion decreases resolution, increases band spreading
  • Slower flow ---> equilibrium has time to establish
  • Turbulent flow
---> larger diameter, more voids
---> more diffusion
---> more rough
---> all this must be minised.
  • Porous ---> can't flow liquid through
Turbulent flow
  • Big proteins diffuse slowly
  • Larger column, more time to separate bands out
Example 2 "Self-sharpening"
  • What works for one works for another
  • If flow is 1/2 mil/min ---> slanted tail (see diagram) ---> can affect resolution
  • may get undesirable proteins for another curve. 
  • How does PT affect Langmuir?
  • Protein regions at start move down column
  • regions of diffusion
  • PT is lower in one region than other
---- as it diffuses out
--- low PT ---> potential to change equilibrium
  • Series of binding sites on wall
  • Can find one and bind
  • decrease number,
  • increase chances of binding site.
  • Proteins slow down column
  • binds more than if in higher concentration
  • Lower protein concentration, more likely to find binding site, as with high concentrated sites may be saturated ---> self-sharpening
  • Plug PT into Langmuir isoferm
  • As protein flows down the column
---> centre of protein region moves at particular rate
---> low [protein] moves at slow rate
---> Protein at back tails away
----> Protein in front catches up
-----> tailing effect

Adsorption/desorption types

IEC
  • Protein-friendly
  • Based on charge
  • positive, negative, how much charge
  • most common method
  • Charged solute and protein interact
  • and opposite charge on solid phase interacts
  • Ionisable side chains
  • Point which positively charged side chains balance negatively-charged sidechains has no net charge
---> PI, isoelectric point
---> go lower PI, protonate negative charge
---> below PI, net positive charge
---> above PI, net negative charge
  • Must know pI to do it
  • Know pH of charge
  • Know how many charges
  • Know protein sequence
  • Nature of solid phase
----Hydrophilic
--- don't want proteins to stick to it
--- Porosity
--- get molecules into and close to surface
--- need big surface area
---rigid
--- milimetre particles
----polysaccharides solved problem
--- carboyhydrates are base substrate
---- derivitise -OH groups

IEC
  • Derivatise with what you want
  • Get IE solid phase
  • DEAE
--- positively charged
--- has one proton
--- increase pH, remove H+ ---> neutral
  • Choose right pH for media.
  • QAE
--- strong base
--- more usual
--- positive charge
---- no proton
--- Nitrogen is valent
--- harder to remove charge
--- stabilise positive charge
  • Chromatofocussing
--- weak acid eg carboxylic acid
--- protonate ---> remove negative charge
  • SP
--- strong acid
--- more usual


Ion exchange chromatography

  • Solid-phase
  • positively charged solit-phase
  • Add salt when handling proteins
  • Proteins prefer a little salt
  • NaCl2
  • Buffer solution is NaCl2
  • positively charged surface
  • positive charges stabilised by negative charges in buffer
  • starting charge is 100mM
  • Protein in same buffer
--- choose pH ---> negative
--- counterbound bysalt ions
  • high charge density on protein
  • solid phase is very positively-charged
  • protein will interact with positive ahrge
  • solvating salt ions dissociate
  • If 100mM salt, Kd=50 mM ---> 100 mM salt will not dissociate it
  • Strong interaction between protein and solid phase
  • Influence Kd ---> change pH
  • Decrease pH to pI ---> neutral charge
  • Make protein positive ---> go below its pI
  • very charged surface, needs buffer
          ---> prevent protein from denaturing
          ---> can't simply add acid
  • H+ and oH- are preferentially displaced or attracted
  • Increase [salt] ---> salt acts as inhibitor ---> displace protein
  • Increase [NaCl] ---> small counterions compete with protein
          ---> insulate, interact
          ---> higher [salt]
          ---> Increase Kd
  • Need molar NaCl to dissociate
  • No issues with pH gradient
  • Hold pH buffer
  • Single pH
  • Increase [salt] flowing down column.
IEC
  • positive protein, negative surface ---> 1.2 kJ of free Energy
  • dG = -RT ln K
  • K is dissociation constant
  • Factor of to ---> 6 kJ mol-1    
          ---> 4 switches in charges
          ---> 4 ionisations ---> switch from binding to unbinding
IEC
  • 8,9, 10 pH ---> don't want protein samples to be there
  • pH 5-9 at all times
          ---> protein comfortable in that range
IEC
  • Choose media and identiy [protein]
  • binding capacity must be greater than [protein]
  • Specification will say for example 25 mg/ml on MW
  • must have charge on protein
  • pH of surface exchanger
          ---> don't remove charge on surface
          ---> if removed, all proteins come off
  • Minimise pH gradients near solid surface
  • Protons are excluded near the charged surface
  • Use high buffering capacity
  • Stay away from charge buffers
          ---> buffers may bind column
          ---> avoid this
  • Work out pI, choose column, pH 1 or 2 away from pI, elute with increasing salt gradient
  • Start with 50mM [salt] ---> 500 mM [salt]
Modern protein chromatographic system
  • Take solution and flow it onto column
  • Take 2 buffers: 1 low salt, 1 high salt
  • Go into green square (mixer)
  • Inject protein in injection valve
  • Stop chromatographic pumping and start again
  • Pump onto column in pink
  • X exits column enters UV conductance
          ---> UV measures protein
          ---> conductance measures [salt]
  • Go in fraction collector
  • Elute with smooth salt gradient
          --- gradual gradient fr 50 to 100 for example
  • Better salt gradient, better separation and resolution
Example 4: Band sharpening
  • Reverse process
  • Salt gradient reverses process
  • Protein bound column
          ---> elute
          --- change pH
          --- get  tailing band
  • Linear gradient across protein region
  • Front of band has lower [salt] than back of band
  • Salt increases Kd
           --- back of region has high Kd
  • Less time on column ---> more mobile when flowing down
  • Back of band [salt] higher ---> speeds up















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