I finished prepping the arteries, cleaned up the lab a bit, and we went to lunch. Deli sandwiches in Michigan are not as good as they are in New York, but it was a good lunch. I went back to the lab after lunch very happy and ready to get on with the experiments to produce lots of data. Following on my theme of working hard to not work hard, I had planned to set up a long-term function study and start a NMR study at the same time. The trick with this strategy was that each needed fresh living arteries and the experiments were to be performed in different buildings. But the data produced by such planning would be valuable and would keep me from having to take twice as long to get the same amount of data. So I set up the function experiments in the physiology lab right away and gave the arteries time to stabilize. They needed a couple of hours to become stable after being collected from the pigs and put into the function machine. Next, I went to the NMR lab and took a couple of hours to get that experiment set up. I used the program I wrote to do the whole experimental protocol and went back to the physiology lab and did the rest of the function experiments.

Function experiments for a pig artery are a lot like making a fist—an artery can clamp around in a grip as we do when we grasp something. So I would make an artery contract its muscle like this and measure the force it could generate. The more force, the better the function.

So, now I was collecting data in both the NMR lab and the physiology lab. I could maximize the data being collected by doing these simultaneous experiments on carotid arteries from the same pig. I just needed to make sure the programs were working and get them started such that they could go for hours or even a day or two. I would be working collecting data while at home asleep in bed.

After three months of research and protein purification, I was getting a solution that I knew contained the protein creatine kinase. The solution also contained some salts that I did not want with the protein. I needed a solid protein without solution or salts. This is called crystallization. This meant getting protein crystals to form in the solution so I could collect pure protein. Dr. Dillon suggested that I add some pure creatine kinase purchased from a chemical company to seed the crystals. At first I thought what he was suggesting sounded too much like cheating. Was he telling me to add someone else’s protein to mine and claim it to be mine? While it may sound a little suspicious, this is actually more like using a starter when baking bread. You start with a little dough from an earlier batch and add that to the ingredients of a new batch of bread. The new dough rises and acts like bread because of the yeast and other ingredients in the starter. Before you bake the new bread you save a bit of the dough for a new starter. The newly baked bread is your bread because you made it and now the new starter contains mostly your own bread dough. Using a few “seed” crystals in this case was very similar. I added a small amount of purchased creatine kinase protein and ended up with a lot more protein than I had added. I used some of that new protein for experiments and saved some to seed the next purification. As an example, if I added 1 ounce of protein as seed and got back 9 ounces, my first batch was 90% my protein. If I did it again, with seed crystals from my first batch, then my second batch would be 99% my protein, and so on. So I used someone else’s starter to get my purification going and quickly had protein that was pure and without cheating on my research results.

The data that resulted were showing that the enzyme creatine kinase and creatine were very important to normal artery function and metabolism. Creatine and creatine kinase gave the artery the energy it needed to work and function normally. This was a neat little discovery. I knew from the research of others that when hearts and arteries were diseased they always lost creatine and even creatine kinase into the bloodstream. When a heart attack was suspected physicians would do a blood test to see if creatine kinase was in the blood. The heart cells and sometimes the artery cells lose creatine kinase when sick or injured. Thus my data might be useful in understanding cardiovascular disease and perhaps someday devising a treatment for patients who were losing creatine and creatine kinase from their muscle. My research might be able to someday “help people”— with luck lots and lots of them.