ISOELECTRIC FOCUSING

Overview

Isoenzymes are enzymes with the same catalytic function but which are structurally different, and those belonging to certain enzyme systems are often characteristic of different species and strains of schistosome (Wright et al, 1979 b\ Fletcher et al, 1981; Wright & Ross, 1983). Every protein possesses an isoelectric point (pi): the pH at which its net charge is zero and consequently migration in an electric field ceases. Polyampholines, amphoteric molecules (acidic and basic character) are contained within the gel, and align to form a stable pH gradient when a charge is applied. Isoelectric focusing is an electrophoretic technique in which proteins are separated on a polyacrylamide gel with a pH gradient running between the electrodes - proteins migrate through the gel until they reach the pH corresponding to their pi. The technique usually results in a very good separation of isoenzyme bands, and the pH gradient of the gel can be substantially modified by mixing carrier ampholytes. Isoenzyme separation is visualized by overlaying the gel with agar containing the appropriate enzyme substrate, which produces a strong colour in the presence of the enzyme.

Sample preparation

1. Worms were recovered from liquid nitrogen and thawed 2. Each worm was transferred into a separate 1.5ml tube

3. 12pl of cold ddH2Û was added and the worms homogenised thoroughly using a pestle.

4. The tubes were centrifuged at 4°C, 13 000 rpm for 10 mins

5. The supernatants were transferred to fi*esh tubes and kept on ice until ready for use (NB. The technique depends upon the enzyme remaining active, and therefore samples were kept on ice as much as possible).

Preparation of acrylamide gels (for the LKB Multiphor Horizontal lEF System)

1. A small quantity of ddHiO was applied to a plain glass plate and a sheet of GelBond® Pag film (fi*om Amersham Pharmacia Biotech) rollered onto it

2. Another glass plate with spacers was clipped onto the GelBond® and the cassette stood upright

3. The acrylamide gel solution was made up as below:

Reagent Volume

ddHzO 12.0 ml

Acrylagel (National Diagnostics) 3.15 ml Bis-acrylagel (National Diagnostics) 1.50 ml

Ampholine (pH 4-7.5 for G6PD; 1.45ml

pH 3.5-9.5 for AcP) (Amersham)

10% ammonium persulphate 94pl

4. The solution was drawn into a syringe and a wide bore needle attached

5. The needle was inserted between the gel plates, and the solution poured into the cassette 6. The gel was left for 30 mins to polymerise

7. Once set, a scalpel was inserted between the gel bond and the plain glass plate to remove the latter, and the GelBond® peeled away from the remaining plate

8. Gels were used immediately or stored overnight at 4°C in a sealed polythene bag.

Pre-focusing of gels to establish the pH gradient

1. Two wicks were soaked in anode electrolyte (IM H3PO4) and cathode electrolyte (IM

NaOH), respectively; blotted, and the ends trimmed to the length of the gel

2. 2ml of 0.1% Triton-X was applied to the gel cooling plate of the electrophoresis tank, and the gel lowered on top, ensuring no air bubbles were trapped.

3. Any excess liquid was blotted from around the gel, and the anodic wick placed along the top edge of the gel, and the cathodic vdck along the bottom edge of the gel

4. The electrodes were positioned over the wicks

5. The gel was pre-focused for 20 mins at the following settings:

1 gel 2 gels

Power (W) 15 30

Voltage (V) 2000 2000

Focusing

1. When pre-focusing was complete, 5 pi of each sample was applied to the gel, at the cathodic end for G6PDH and at the anodic end for AcP

2. At the far right-hand end of the gel 2pl of haemaglobin was applied at the anode as a focusing control

3. The gel was focused for 1 hr at the following settings:

For G6PD For AcP

Power (W) 25 25

Voltage (V) 2000 2000

Current (mA) 30 50

4. While the gel was running, the enzyme development mixture was prepared as below 5. When the gel was focused and the enzyme development mixture was ready, the gel was

removed from the electrophoresis tank, placed in a developing tray, and the wicks discarded.

Enzyme development

1. 100ml of buffer (0.2M Tris-HCl, pH 8.0 for G6PDH; O.IM sodium acetate, pH 5 for AcP) was boiled, and, while mixing fast, 1.5g of agar added

2. Heating was continued until the agar had dissolved, being careful not to let it boil over. 3. The agar was kept at approx 60°C until required.

4. 50ml of the enzyme development mixture was prepared (section 2.5.5) and combined with the hot agar

5. The development/agar solution was poured over the gel in the developing tray

6. For G6PDH, the tray was covered with a light-proof lid so that the gel was in darkness (the enzyme reaction is light sensitive)

7. When the agar had set, the tray was placed at 37°C to facilitate the enzyme reaction and speed up colour development.

8. When the enzyme reactions were sufficiently developed, they were stopped with a solution of 5% glacial acetic acid and 4% glycerol

10. The stop solution was then poured off, and the gel washed with a solution of 5% glacial acetic acid and 3% glycerol.

11. The wash solution was changed 2-3 times daily until the gel background had completely de-stained.

12. The agar was then carefully peeled away and discarded, and the gel (on its gel-bond mount) placed on a plate warmer at 40°C for approximately 24 hours until dried down.