Time Is Tissue

by John Osterhout, PhD

Ok so now you’ve done it. You’ve gone and got yourself snake bit. A good big chomp right on the hand. The good news is that medical science can do something about it. The bad news is, well, the rest of the news…

So here’s what happens: If you are lucky, they get you into an ambulance right away and whoop whoop whoop off you go to the hospital. Also, if you are lucky, someone will phone ahead and tell the emergency room guys that you are coming in so they can scrounge around and make sure they actually have some antivenom in the room when you get there.

An aside: the average number of vials of antivenom to treat a snake bite is 20, 12-40 is common and over 100 is possible. The cost of the antivenom is $2000 – $3000 per bottle (bad news). Each bottle has a gram of protein that goes right into your bloodstream (more bad news) and this can cause shock that can be as deadly as the snake bite (yet more bad news). Furthermore, there are some patients who, after surviving the initial treatment, have an episode of serum sickness where the immune system tries to reject the horse or sheep serum from the antivenom (not the last of the bad news). This happens ten or so days after leaving the hospital (ok, this is the last of the bad news). So don’t get snake bit – it’s really bad news! Now back to the regular programming…

Finally you arrive at the hospital. Someone takes a long look at your hand and determines that, yes, the sorry low- down son of a snake that bit you did, in fact, shoot you up with venom. (Sometimes an older, experienced snake will give you just the fangs to encourage you to leave it alone.) Now they spring into action. Someone pulls down a vial of antivenom and squirts in 10 ml of physiological saline. Then the fun starts. They put the bottle on a very slow shaker and you get to watch it rock slowly back and forth. Back... forth... back... forth... back (well, you get the idea). This rocking goes on for 45 minutes. Yes, forty plus five long minutes. Back... forth... back... forth... Meanwhile every ten minutes a cheerful nurse brings out a magic marker and makes a nice neat black mark on your arm to locate the extent of the swelling. Hint: the swelling marks where the tissue damage will be. In 45 minutes it’s usually up past your elbow and rising fast. At the end of 45 minutes the vial is whipped off the shaker, the contents are shot into a saline bag and they start it dripping into you, drip drip drip.

So, the big question is “Why do they rock it for 45 minutes while you watch your arm self-destruct instead of just banging it into you?” There is a very good answer to this question, but you should be warned, it involves... biochemistry! You see, when you shake a protein in water, it foams (think beer, wait, no, don’t think beer – that might have gotten you in this fix in the first place). Numerous serious studies of this phenomenon have shown that proteins unfold when you foam them. The fear is that vigorous shaking of the antivenom will foam it, that the foaming will unfold the protein and that it will not fold back, essentially ruining the antivenom. So they rock the antivenom back and forth … back …. forth for 45 minutes, which is the amount of time believed to be required to dissolve the antivenom, before they will stick it in you.

But wait! Every good biochemist also knows that proteins can fold back all by themselves. What if the protein goes ahead and refolds after it is foamed. It might be good to go after only five minutes. Forty minutes worth of tissue damage could be avoided.

This was the problem posed to Tony Kanavage, biochemistry undergraduate in need of senior thesis, by Leslie Boyer, M.D. and Medical Director of the Arizona Poison and Drug Information Center at the University of Arizona. Tony needed some biochemical help. Eventually he found his way to the Department of Biochemistry and Molecular Biophysics and John Osterhout, and a collaboration was forged.

	Tony Kanavage running SDS gel electrophoresis on rattlesnake venom
	Tony Kanavage, Dr. Leslie Boyer, and Dr. John Osterhout

The scientific attack on the problem was simple. Samples of antivenom were vigorously foamed and then spun in a desk top centrifuge to settle the foam. If the protein were seriously unfolded it might aggregate, the centrifugation step would remove any aggregated protein and the protein concentration of foamed and unfoamed antivenom would be different. The protein concentrations of foamed samples and unformed samples was measured. The result was that for all three of the available antivenoms the concentration of protein did not change due to foaming.

But wait! What if the protein is messed up but still soluble? Then the concentration would not change but the antivenom would still be ruined! To investigate this question Tony used circular dichroism spectroscopy. Circular dichroism (CD) is an optical technique in which the difference between the absorbance of left and right circular polarized light is measured. Far ultraviolet (UV) CD spectra tell us about the secondary structure in the proteins while near UV CD spectra monitor the formation of tertiary structure. Tony measured both kinds of CD spectra all three antivenoms both foamed and not foamed. In all cases the foamed samples were not significantly different from the unfoamed samples.

These results indicate that all of the snake antivenoms available in the U.S. will refold completely after intentional foaming. This means that the rocking procedure may not be necessary and it may be possible to significantly speed up the treatment procedure. Save time. Save tissue. Save the foam for the beer.