Wednesday, 28 January 2015

Enzyme rates: the good, the bad and the ugly!

This week we started measuring the activity of Glutamate Dehydrogenase in solution, using the reduction of NAD+ to NADH, which gives an increase in absorbance at 340nm. Having made up the stock solutions of glutamate and NAD+, the enzyme (diluted from a concentrated suspension in saturated ammonium sulphate) in phosphate buffer at pH7.6, was added at a volume determined by you to give good initial rates. So what is a "good" initial rate? 

The Ugly! I am going to use the "Good the Bad and the Ugly" in reverse order, extending the use of a literary concept, to identify the "Goldilocks" initial rate! This is an unacceptable initial rate. Here something has gone wrong. This might present as no observed change in absorbance: you forgot to add an ingredient, say. (Familiarise yourself with the height of a 1ml volume in your cuvette!). The absorbance starts shooting up in the absence of enzyme. This can occur if your solutions (which may be on ice) create condensation on the optical face of the cuvette. And then, there is the passing increase, as a hair or piece of chocolate floats past the light beam! These represent examples of the ugly side of initial rate measurement! They are all a result of a lack of care!

The Bad! A bad rate is a rate that is either too fast or too slow to capture. I am referring here to rates measured on general lab spectrophotometers, and in our case we are using good quality specs but in a simple manual mode. Therefore you will take absorbance readings against a blank sample every 15/30s. If the rate is too sow, ie the absorbance changes by 0.001 at each reading, this is bad for productivity (but it may be good for the determination of the tangent). However, on balance we try to obtain good slopes, in a reasonable time scale ( maybe 10 minutes). If the rate is too fast, this is worse. The slope is hard to determine and the reaction is over before you have had time to write down the first absorbance reading!

Good! When the data are plotted (absorbance versus time) and the first 50% of the plot lies on an approximate 45 degree straight line, you have obtained the Goldilocks conditions! This is a balance of the substrate concentrations and the amount of enzyme added to initiate the reaction. The rate shown below was obtained by someone and is in my view satisfactory. I want to replace this with a post of your rate measurements tomorrow, to highlight the best result in the class and to improve on the one below! If you could show me your initial rates, I'll choose the best!

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