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At the moment, I'm designing an experiment for a high school lab with no financial resources. I need to assess the impact of a variety of factors on a specific enzyme's reaction with its respective substrate and determine the rate of reaction. Normally this would require some combination of an assay kit and a plate reader. Though cheaper versions of the former are within my budget, the latter is not.

What are the cheapest ways you can think of to:

1) Get access to enzymes and the substrate;

2) Make them react in a controlled manner;

and 3) Assess the rate of reaction from the resultant substance?

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    $\begingroup$ Do you have a specific enzyme and substrate in mind? $\endgroup$ – canadianer Sep 24 at 6:00
  • $\begingroup$ No specific enzyme or substrate in mind. The only condition is that it can't be amylase. $\endgroup$ – Lysander Cox Sep 24 at 10:21
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There is the classic potato starch/iodine/amylase experiment: http://dailynexus.com/2016-08-31/chemistry-101-the-classic-potato-iodine-blueblack-experiment-finally-has-an-explanation/

From memory, when you add iodine to a small piece of potato, the potato starch reacts to produce a dark blue/black colour. The amylase enzyme in human saliva can break down the starch and remove the colour, which is effected by spitting into the test tube.

You might need to try titrations to work out how to do number 3.

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    $\begingroup$ How specifically would I use titrations? $\endgroup$ – Lysander Cox Sep 25 at 3:55
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    $\begingroup$ That's a good question. Now I think about it, I don't know how you would be able to assess the rate of reaction. My thought was to try increasing amounts of saliva until some kind of threshold was reached, but I don't know if that helps at all. $\endgroup$ – Jonathan Moore Sep 25 at 8:44
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Someone has already mentioned catalase and potatoes, but another way to do it would be to use hydrogen peroxide. The students could cut the potato up smaller to investigate the impact of surface area, heat/cool the mixture using a beaker with water in and a thermometer, and change the amount of potato used. So a few variations of conditions for a fairly low cost!

To measure rates, you can pop a bung attached to a tube and gas syringe onto the test tube when the potato is added, and record the volume of oxygen evolved within a given period of time, or the amount present at different time points (say, every thirty seconds). Lots of fun graph drawing for the students, and they can work out the initial rate of reaction etc! :)

If you're looking for a qualitative result, this experiment produces bubbles and foams up so you can go for which reaction produces the greatest height of bubbles in the tube. Perhaps the students could measure this height with a ruler to get quantative data?

I hope this is helpful!

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In order to set up an enzyme assay that allows quantitative kinetic analysis three key requirements come to mind:

  • The assay should be sensitive

  • A plot of velocity versus enzyme concentration should be linear and through the origin at both high and low substrate concentrations.

  • The initial rate should be measured.

Spectrophotometric Assays

Spectrophotometry is a very versatile method for measuring changes in absorbance with time, and if an enzyme produces a product that differs in absorbance (at a given wavelength) from that of the substrate, this can be the basis of a very convenient assay: NAD(P)H absorbs at 340nm whereas NAD$^+$ does not, for example

Furthermore, if the extinction coefficient is known (6220 M$^{-1}$ cm$^{-1}$ for NADH), initial rates may be quantitatively expressed as micromoles of product formed/minute (rather than change in absorbance per min).

Things are even easier if the chromogen absorbs in the visible region.

The p-nitro-phenolate anion is yellow, for instance, and any enzyme that produces p-nitro-phenol from a colorless (or near colorless) substrate at relatively high pH may be assayed by monitoring the increase in absorbance at about 400nm.

A further advantage is that the extinction coefficient is high (Dawson quotes a figure of 18000 M$^{-1}$ cm$^{-1}$ for p-nitro-phenol at pH 8-9 and 400nm, making this assay method roughly three times more sensitive than NAD/NADH).

  • If spectrophotometry is the chosen method, a valid assay requires that The Beer-Lambert law be obeyed within the absorbance range of the experiment.

Possible Examples

Alkaline phosphatase may be assayed with p-nitrophenylphosphate as substrate, producing yellow p-nitro-phenol as product. Thus recording the increase in absorbance at about 400nm with time is the basis of a valid, sensitive assay.

In addition, many proteases and esterases may be assayed with p-nitro-phenylacetate, where the action of the enzyme also produces p-nitro-phenol as product. This includes chymotrypsin.

Both p-nitro-phenylphosphate and p-nitro-phenylacetate these are relatively cheap.

Another possibility is mushroom tyrosinase, which may be assayed spectrophotometically by measuring the decrease in absorbance at 475nm due to the formation of DOPA-quinone from L-DOPA, and there is a very concise and detailed description available.

There are many other possibilities.

Smartphone 'Spectrophometer'

The problem with spectrophotometry is that equipment is expensive. However, a number of authors, have shown that smartphones may be used as 'spectrophotometers' in the visible region.

A very simple and effective method has been described by Kuntzleman & Jacobson (2016), and Kuntzlman has posted an outstanding blog which includes a YouTube video that describes the method in detail.

To understand the method, we need to be aware that a colored solution absorbs light in the region of its complementary color. A solution appears red (to us humans) because green light is absorbed. A yellow-orange solution absorbs (mainly) blue light. (Complementary color may be obtained by consulting a colorwheel)

The method uses a smartphone camera, together with an App to give average red, green and blue (RGB) values for pixels found in a 'detection circle'.

Light reflected from a background complementary to the color of the solution of interest is passed through the solution and is detected by the camera by noting the decrease in either the R, G or B value.

Other than a smartphone, all that is needed is a cardboard box with holes cut to accommodate a cuvette or other vessel and a sheet of paper or cardboard colored in the complementary color, which is placed behind the cuvette (to reflect light of the appropriate wavelength through the solution).

In the video example, red light is detected by using a green background and recording the decrease in G value. The Beer-Lambert law was then be used to convert G values to absorbance. (The video even includes a spreadsheet example showing how to do this).

It should be easy to adapt this method for use in enzyme assay. Use of a timer is all that is required to record changes in absorbance with time, thus allowing the initial rate to be estimated.

There is one caveat. The App by Kuntzleman is colorometer and this does not seem to be available for iPhone 7 and higher. However, it appears that the App is still functional on Android devices.

References

  • Use Your Smartphone as an "Absorption Spectrophotometer" [Tom Kuntzleman blog]
  • Kuntzleman,TS & Jacobson, EC (2016) Teaching Beer’s Law and Absorption Spectrophotometry with a Smart Phone: A Substantially Simplified Protocol J. Chem. Educ. 93, 1249-1252 [ACS Site]

  • Smartphone "Spectrophotometer" [YouTube]

  • Rodriquez, MO & Flurkey, WH. (1992) Biochemistry Project To Study Mushroom Tyrosinase. Enzyme Localization, Isoenzymes, and Detergent Activation J. Chem. Educ. 69 767 [ACS Site]

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  • $\begingroup$ So, using a the colorometer, how would I ascertain the hydrogen peroxide content of a given solution? $\endgroup$ – Lysander Cox Oct 10 at 8:20
  • $\begingroup$ @LysanderCox I think a key difference from what described above is the need to allow the reaction (in the presence of excess enz) to run to completion, rather than measure initial rates, and then use a standard curve to estimate the total amount of hydrogen peroxide in a sample. One widely used enzyme is horse radish peroxidase (especially in ELISAs) and there are many colorimetric substrates. The paper quoted below, for example describes a sensitive assay for H2O2 based on the oxidation of phenol red that is linear up to 60 micromolar H2O2 (detection by measurement of A at 660nm). $\endgroup$ – user1136 Oct 10 at 9:42
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Catalase from blood. Prick your finger, collect a small amount of blood. Add some hydrogen peroxide to the blood, it will foam as the catalase decomposes the hydrogen peroxide. Obviously there are biohazard considerations when dealing with human blood. Some plants, notable horseradish also contain peroxidase if you wish to avoid pinpricks.

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  • $\begingroup$ NO. You can’t do this sort of thing anymore! Not in Britain at least. We used to have medical students do blood glucose on themselves after fasting. That was pre-HIV. Not now. Don’t go there. $\endgroup$ – David Sep 24 at 19:41
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    $\begingroup$ So, what substrate would you recommend I use? Additionally, how would I assess the rate of reaction? $\endgroup$ – Lysander Cox Sep 24 at 22:53
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    $\begingroup$ The substrate is the hydrogen peroxide, the enzyme is contained on blood, horseradish, and a number of microorganisms. You can assess the rate of reaction by observing the bubbles produced, or collecting the oxygen in an inverted jar filled with water. David: calm down. What happens when someone gets a bloody nose or scrapes their knee? Do we call a hazmat team and incinerate the waste? No, we throw them in the trash and move on. Certainly the teacher could prick their own finger to obtain less than 20uL of blood for the sake of an experiment. $\endgroup$ – Sam Sep 25 at 13:39
  • $\begingroup$ You don't need a co-substrate. Peroxidase may act as a catalase where H202 is both the electron donor and electron acceptor (as @sam has pointed out), leading to the evolution of oxygen $\endgroup$ – user1136 Sep 25 at 14:43
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    $\begingroup$ In theory you could buy some blood from a local butchers' so it's not that bad of an idea. But you'd have to check with health and safety due to the risk of blood borne diseases and religious concerns. Surely if you can dissect kidneys in class, you can use a test tube of blood though $\endgroup$ – Jam Sep 27 at 12:44

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