Recrystallization of Catalase

Although the protocol to prepare the crystals is straightforward, the size and quality of the crystals depend on many factors. It is therefore best to perform the protocol in duplicate to increase the chances of getting good crystals.

The crystals are prepared based on the method of Sumners and Dounce (J.B. Sumner and A.L. Dounce, Meth. Enzymol. II (1955) p. 775). All operations should be carried out at 4 degrees C unless otherwise noted.

Materials Needed

• 2X crystallized bovine liver catalase (Sigma: C-100, 500 mg)
• 3 L 1 mM NH₃Cl
• 10% NaCl, 2 mM KH₂PO₄, pH 5.5

Directions

1. Wash 100 mL of bovine liver catalase three times in distilled water to remove the antibacterial agent thymol.
2. Dissolve in 1 mL 10% NaCl, 2 mM KH₂PO₄, pH 5.5 by rocking 1 h at room temperature.
3. Spin at 14,000 rpm for 20 min in an Eppendorf tabletop centrifuge to remove any insoluble protein.
4. Dialyze two nights against several changes of 1 mM ammonium chloride, pH 5.5 to recrystallize the catalase (at least 10 h per dialysis). After the first dialysis few or no crystals may be present; the additional dialysis steps are necessary to get rid of all of the salt.
5. The crystals will be in suspension mixed in with noncrystallized protein (Fig. 1). To remove protein that did not crystallize, allow the crystals to pellet by gravity (the crystals probably need to settle out over a period of a few nights to get crystals of the right size). Remove the supernatant, then gently resuspend the crystals in distilled water by inverting the tube. The crystals can be shattered, so it is imperative to resuspend them gently. After washing, the crystals should ideally look like those in Fig. 2.
6. Apply 4 mL of the crystal suspension per carbon-coated grid and stain with 1% uranyl acetate. It may take a while to find the right size and quality crystals.

Figure 1. Catalase crystals after dialysis and before washing with water. The crystals are mixed with noncrystallized protein. Bar, 5mm.	Figure 2. Good crystals used for the TEM automation. Bar, 5mm.

Store the catalase crystals in the refrigerator. It should last about two weeks.

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