Enzyme Immobilization

The following procedure is described in detail in the publication by Sturgeon et al. and in the BioRad documentation found here.

Edition 2

Immobilizing enzymes depends heavily on an enzyme’s isoelectric point (pI). If an enzyme’s pI is 7.4, than when it is dissolved in a buffer with a pH above 7.4 the enzyme will develop an overall negative charge. The opposite is true when the enzyme is dissolved into a buffer at a pH lower than its pI, the enzyme will develop an overall positive charge. When this enzyme with a pI of 7.4 is placed into a pH 7.4 buffer, then the overall charge on the enzyme will be zero, or neutral.

This can be explained using glycine (H₂NCH₂CO₂H). Glycine has a carboxyl group and an amine group; the carboxyl group can deprotonate forming a negative charge (a carboxylate) and the amine group can be protonated to form a positive charge (an ammonium).

The pka of the carboxyl group is 2.72, so at a pH above 2.72, the carboxyl group will be deprotonated (ie. negative):

The pka of the amine group is 9.60, so at a pH below 9.60, the amine group will be protonate (ie. positive):

So gylcine can exsit in 3 forms:

cation at pH <2.72	neutral at pH >2.72 but <9.60	anion at pH >9.60
H3N+CH2CO2H	H3N+CH2CO2-	H2NCH2CO2-

Never exists as: H₂NCH₂CO₂H

H₂NCH₂CO₂H ⇌ H₃N⁺CH₂CO₂^-

H2NCH2CO2H ⇌ H3N+CH2CO2−

This is important to know because Affi-Gel 10 has a slightly negative charge whereas Affi-Gel 15 has a slightly positive charge. The objective to immobilizing an enzyme is to use a pH to make your enzyme charged and then use the opposite charged Affi-Gel to allow for optimal coupling. For example, your enzyme has pI of 6.8 and you want to immobilize it using Affi Gel 10 (-) then you should dissolve your enzyme in pH lower than 6.8 like pH 5. By dissolving you enzyme in pH lower then you pI you get a positive charge which will pair nicely with the negatively charged Affi-Gel. Below is the procedure for immobilizing your enzyme after an appropriate pH is pick according to the Affi-Gel you which to use. The procedure below has been modified from the BioRad suggested procedure due to the semester of research spent analysising this procedure. In the orginal proceudre there was a buffer wash after the cold DI water wash, but that was removed when it was realized that it didn't increase the coupling of the enzyme to the Affi-Gel bead. The other portion that was removed was it was suggested to rock for 4 hours, it was determined that there was a small change that wasnt significant to wait for 4hours. Remember throughout this entire process don’t let the Affi-Gel bead bed go dry, if needed you may add a little buffer.

Edition 1

Immobilizing enzymes depends heavily on an enzyme’s isoelectric point (pI). If an enzyme’s pI is at 7.4, than when it is placed in a pH above 7.4 the enzyme will develope negative charge from its carbonyl sites becoming negative as the enzymes amine groups become natural. The opposite is true when you add the enzyme into a pH lower than its pI, the carbonyl groups will become neutral as the amine groups become positive. This is important to know because Affi-Gel 10 has a slightly negative charge whereas Affi-Gel 15 has a slightly positive charge. The objective to immobilizing an enzyme is to use a pH to make your enzyme charged and then use the opposite charged Affi-Gel to allow for optimal coupling. For example, your enzyme has pI of 6.8 and you want to immobilize it using Affi Gel 10 (-) then you should dissolve your enzyme in pH lower than 6.8 like pH 5. By dissolving you enzyme in pH lower then you pI you get a positive charge which will pair nicely with the negatively charged Affi-Gel. Below is the procedure for immobilizing your enzyme after an appropriate pH is pick according to the Affi-Gel you which to use. The procedure below has been modified from the BioRad suggested procedure due to the semester of research spent analysising this procedure. In the orginal proceudre there was a buffer wash after the cold DI water wash, but that was removed when it was realized that it didn't increase the coupling of the enzyme to the Affi-Gel bead. The other portion that was removed was it was suggested to rock for 4 hours, it was determined that there was a small change that wasnt significant to wait for 4hours. Remember throughout this entire process don’t let the Affi-Gel bead bed go dry, if needed you may add a little buffer.

1. Set up equipment

a. Syringe (5mL)

b. Column (731-1550)

c. Couple pieces of para-film

d. Transfer pipets

e. Three way stopper (732-8103)

f. Ring stand

2. Gather Chemicals

a. Enzyme solution

i. Buffer/pH

b. Affi-Gel Beads

c. Cold DI water

3. Assemble

a. Place the column and syringe in the top(para-flim connection and side(that is 90degrees to top, and the syringe will "lock" into the stopper), respectively

4. Re-suspend Beads(shake and stir)

a. Add beads(about 1ml) to column using dropper

5. Wash with 10ml ice cold DI water(dropper)

6. Add Enzyme

a. Take about 20µL of enzyme solution and had to an extra eppendorf (label Initial)

7. Re-suspend in column and rock for 1-4 hours

8. After rocking, take a couple drops into another eppendorf (label final)

a. Remember don’t let the bead bed go dry if needed you may add a little buffer

9. Add 100µL of 1M glycine ethyl ester for blocking

10. Then re-rock for 1 hour