The University of Chicago Magazine October 1995
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Building a Better



Take a pound of sheep seminal vesicles, grind well...

Biochemists have been isolating and purifying individual enzymes since the turn of the century, when Eduard Buchner (for whom the laboratory filter-funnel is named) showed that "ferments" could be separted from the cells that contain them.

At Chicago, Michael Garavito (right), Daniel Picot, and Patrick Loll devised the purification scheme that allowed crystallization of PGHS-1 only after exploring an exhaustive maze of long, blind alleys. Often, a technique useful for purification would turn out to prevent crystallization. Even with a procedure established, preparation was slow work: After each step, many small samples had to be assayed for the amount and purity of the enzyme.

In every case, the process begins with purification: isolating a single pure enzyme from the thousands of others in the crushed guts of a cell. Gentle conditions must be maintained so as not to "denature" or unfold the enzyme, which is a protein, from its biochemically active shape. The acidity and salt concentration must be carefully controlled, and everything must be done at ice-cold temperatures.

If purification is like looking for a needle in a haystack, crystallization-the second stage of the process-is like trying to build a card house out of wet tissue paper. Crystal growth demands almost absolute purity and perfect integrity of the protein molecules, and proteins bound to cell membranes-like aspirin's target, the enzyme prostaglandin H2 synthase-are notoriously hard to purify because the detergents needed to free the protein from the greasy membrane inhibit crystallization.

Garavito's long-sought recipe starts with a pound of frozen sheep seminal vesicles, run through a meat grinder and then popped, along with a special solution, into a kitchen blender. Solid cellular debris is spun out using a low-speed centrifuge, and the liquid is filtered through cheesecloth. The cellular membranes are spun from liquid by centrifuging the mixture at 200,000 times the force of gravity. Then the clump of membranes is washed in another solution and centrifuged again.

Next, the membranes are dissolved in a detergent solution and passed over a long filter column, containing a material carrying an ionic charge that holds onto proteins as fats wash through. The proteins are then rinsed free of the column, and this solution is concentrated by forcing out excess liquid through a synthetic filter membrane. The sample gets passed over another column (this one filled with a material that sorts molecules by size, slowing smaller molecules within its pores), reconcentrated, passed over another molecular sizing column, reconcentrated again, passed over another charged-ion column, reconcentrated, and finally applied to a final, high-resolution, molecular sizing column and reconcentrated.

The last column also exchanges the detergent used for purification for one that will allow crystal formation. The researchers end up with a small test tube of solution containing a few hundredths of a gram of very pure PGHS-1.

To grow crystals from this solution, the researchers add a drug for the protein to latch onto. Suspending drops of the solution from glass slides, over a period of weeks they slowly change the composition of the solution and the surrounding vapor. Brown, rod-shaped crystals, less than one sixteenth of an inch long, grow in the hanging drops. These, finally, are the crystals the researchers can probe with X-rays.--William Burton

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