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Status: chipping away [enzymes]

This head cold started in earnest last Thursday, but I seem to be on the mend already, so I can't really complain. Mostly it has just put a damper on rowing. I had hoped to leap out of bed this morning, but with the way this cold works I've been waking up several times a night with some sort of unproductive choking-cough (i.e. dangerously close to a bronchitis cough). Taking a small sip of water helps, so I am now quite well hydrated, thankyouverymuch, but I'm not well-rested enough to go back to the boathouse yet. Soon.

It's irksome to teeter on that edge where I'm not *quite* sick enough to justify staying home, but am still coughing/sneezing more than I'd like to at work.

Instead, I'm at work, chipping away at the project of sorting out how one should quantify the activities of various enzymes in various cricket tissues. We're planning on following the action of the following five enzymes, involved in glycolysis and energy metabolism: hexokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, citrate synthase, and cytochrome c oxidase.

Things I have learned:

-Know your buffer solutions. Unfortunately, for the combination of enzymes I have selected, I don't know that there's a single good buffer for all of the assays. Yargh. I would have to test each assay individually to see if this is the case. The thing is that different enzymes may have optimal activity at different pH values, and other aspects of the buffers may also be important because enzymes can be finicky molecules. It would be best if I could use the same buffer across multiple enzymes in this case, and I might be able to, but I could also spend an inordinate amount of time trying to figure out how to do so. So, time to consult TZ, the Enzyme Master, hopefully...

-A clever element for enzyme assays is the use of a "coupled reaction." Basically, if the enzyme doesn't catalyze a reaction that directly involves some sort of measurable color change, then instead that reaction is coupled to a second reaction that *does* involve a color change. For example, glucose-6-phosphate dehydrogenase takes a hydrogen atom off of glucose-6-phosphate and slaps it onto NAD+. This converts NAD+ to NADH, which generates a change in color that can be measured at a light wavelength absorbance of 340 nanometers. We'll take advantage of this same reaction for assaying hexokinase, an enzyme which breaks down glucose to make glucose-6-phosphate. There are similar fun coupled reactions for pyruvate kinase and citrate synthase.

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Comments

( 3 remarks — Remark )
randomdreams
Feb. 24th, 2016 02:55 am (UTC)
Oh, man, you're doing exactly the kind of stuff I wanted to do when I was in school. That's awesome.
I hope you get well soon.
rebeccmeister
Feb. 26th, 2016 07:17 pm (UTC)
It's Mad Science, I tell ya!

It makes me a little bit nervous to go in this direction because I am slightly too aware of how many underemployed biochemists are out and about in the world. On the other hand, putting these things to work in an interesting context could send me in a good direction.
randomdreams
Feb. 27th, 2016 02:32 am (UTC)
<- underemployed biochemist, at least as regards my employment in biochemistry.
( 3 remarks — Remark )

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