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!

Saturday, 24 January 2015

We have a lovely bunch of coconuts in the lab!

I was delighted to see the natural product experimental planning taking shape this week. Last year, lemons proved the most popular source of potential ant-bacterials, but this year coconuts are topping the bill! It has prompted me to use coconuts as the backdrop to help with your experimental planning. The first thing I noticed was an overwhelming rush of enthusiasm to make the plant extracts. However, how many of you weighed the item? Did you measure the volume of the coconut (or kiwi fruit etc)? If you did, how did you do it? How did you extract the "milk"? What was the volume? How did you store it?

These were the questions that I asked several groups and the reason for asking is simple. Whenever you embark on an experiment, you must plan to record all of your observations. Before you extract the coconut milk, weigh the coconut. You should then compare (for example) 3 coconuts. Are they the same weight? What is the average weight? The same applies to the volume (think ancient Greeks taking baths!). Ask yourself why these measurements are important.

Now we come to the fluid inside the coconut: the so called coconut milk. Milk is usually defined as the fluid from a mother's breast or from the udder of a cow. It is a mixture rich in proteins and fats. So what is in coconut milk? You should consider the coconut carefully. Define its structure, consider the biological origin of its distinctive structures and then think about the source and composition of the "milk". You should then plan how you make various extracts, with reference to the chemical composition of the various parts of the "nut".

The search for lauric acid (above) was a topic of discussion with two groups. This is one of the therapeutic molecules found in coconut milk. If I tell you that chemistry is full of alternative names for the same thing (trivial and systematic), you may be surprised to find that lauric acid, which is claimed to be of therapeutic value, is very similar to the detergent we use to denature proteins, sodium dodecyl sulphate. So how can two very similar molecules have such different properties! 

What I want you all to do is to measure everything you possibly can before you commit to any destruction of the plant or fruit. Take photos and annotate the structures. Research what is known about your choice of plant and then consider whether you are likely to find water soluble or lipid soluble antibacterials....and how you might plan for both! But don't lose that enthusiasm!!!