[RASMB] Protein charge

[Tom Laue]

Hi all-
There is increasing interest in the accurate measurement of protein 
charge since charge-charge and charge-dipole interactions contribute 
significantly to solution behavior, particularly at high concentrations. 
Some of you asked me for a quick review on measuring protein charge.

Three methods are available for measuring charge: capillary 
electrophoresis, electrophoretic light scattering and membrane confined 
electrophoresis. Each has its advantages and each has its limitations.

 From my perspective, here are the pluses and minuses for each method.

_Capillary electrophoresis (CE)_
   1. CE, pluses
         1. Very small quantities required
         2. True physical separation of charge isomers
              1. Best method for detecting and quantifying charge isomers
         3. Many (unused) instrument already are in labs
   2. CE, minuses
         1. Capillary wall interactions (e.g. electroendosmosis) 
complicate data analysis
         2. Solvent conditions often dictated by capillary wall 
interactions
         3. Requires hours to complete
         4. Requires separate determination of the Stokes radius (by 
Sed. velocity or dynamic light scattering)

_Membrane confined electrophoresis (MCE)_
   1. MCE, pluses
         1. First principle method, no standards required
               1. Both mobility and steady state measurements are first 
principle
         2. Gold standard charge accuracy from steady state
         3. Only method that can provide direct determination of 
effective charge (steady state)
                 1. No frictional coefficient determination needed
         4. Small volume (20 uL), at  ~0.5 to 1.0 OD at 280
   2. MCE, minuses
         1. Steady state method is SLOW... complete set of measurements 
require days to complete
         2. Solvent interactions with membrane (di- and tri-carboxylic 
acids) limit solvent choices
         3. Steady-state method requires highly purified sample

_Electrophoretic light scattering (ELS)_
  1. ELS, pluses
       1. Fast, fast, fast! Requires ~5 minutes to measure the mobility
       2. Wide range of solvent conditions may be used
       3. Small volume (~200 - 500 uL) and low concentrations (< 1 
mg/ml) required
       4. Only method suitable for rapid survey work
       5. Same instruments can provide Stokes radius via dynamic light 
scattering
  2. ELS, minuses
       1. Scattering method, sensitive to aggregates
       2. Accuracy and precision lower than MCE
             1. Accuracy and limitations still being tested
       3. Some early instruments were inaccurate due to technical problems
             1. Manufacturers are working on fixes and testing

Commercial instruments are available for each of these methods:

CE-     Beckman, Agilent, Applied Biosystems
MCE-  Spin Analytical
ELS-   Malvern, Wyatt, Brookhaven

I have had experience with some of these, but not all.

I'll be glad to answer any questions.
Best wishes,
Tom


-- 
Department of Biochemistry and Molecular Biology
University of New Hampshire
Durham, NH 03824-3544
Phone: 603-862-2459
FAX:   603-862-0031
E-mail: Tom.Laue at unh.edu
www.bitc.unh.edu
www.camis.unh.edu