Measurement Of Alkaline Phosphatase Activity And 4-Nitrophenol Production In Serum

Explaining clearly your reasoning and showing all necessary working.

(a)Do your results indicate that the rate of production of 4-nitrophenol is constant?  Explain how you have reached your answer.

(a)What is the rate of the reaction (in terms of absorbance change per minute)?

Use the Beer-Lambert Law to convert your answer in (b) into a rate expressed in terms of the change in concentration of 4-nitrophenol per minute.  To do this you need to know that a 1 mM solution of 4-nitrophenol has an absorbance at 400 nm (A400) of 11.5 under the conditions used in the experiment.

Determination of elevated alkaline phosphatase activity in patient serum samples

Elevated levels of alkaline phosphatase activity in serum are used as an indicator of a number of clinical disorders, including liver disorders. You will measure the alkaline phosphatase activity present in the serum of patient A, B or C and determine the clinical significance.

Insert an Excel graph of these results as previously and from this, calculate the rate of the alkaline phosphatase reaction (in absorbance change per minute).

Express rate in terms of the change in concentration of 4-nitrophenol per minute.

Under the assay conditions detailed here, the change in concentration of 4-nitrophenol per minute is 0.5 M for the serum from a healthy patient. Do your results indicate that your patient is suffering from a possible liver disorder? Explain clearly.

The Beer-Lambert Law

Alkaline phosphatase is a very widely distributed enzyme.  As the name implies it catalyses (speeds up) the hydrolysis of phosphate esters (e.g. glucose phosphate, ATP etc.) under alkaline conditions, i.e. at pH values above 7.  Although the physiological function of the enzyme is not properly understood, the assay of the enzyme in samples of serum is very commonly performed in biochemistry laboratories in hospitals.  This is because the presence of substantial amounts of the enzyme in serum indicates certain types of disease (including jaundice and a number of bone diseases).  

In this experiment you will assay (measure) the activity (rate of reaction) of the alkaline phosphatase towards the substrate 4-nitrophenylphosphate.  The reaction products are 4-nitrophenol and phosphate.  

4-nitrophenol has a strong yellow colour whereas 4-nitrophenylphosphate is effectively colourless, so it is easy to monitor the course of the reaction by the increase in absorbance of light of a suitable wavelength using a spectrophotometer.  For this experiment, 400 nm (at the violet end of the visible spectrum) is the appropriate wavelength.  The amount of the product formed will be measured at one minute intervals over a period of five minutes.

The Beer-Lambert Law

This Law states that the absorbance (A) of a fixed wavelength of light by a solution is proportional to the concentration (c) of that component of the solution which is responsible for the absorption.  (It should be noted that the path length of the solution through which the light travels should be constant for the Law to apply).

Thus,   A µ c  (the symbol µ means “is proportional to”, ie if c doubles, then A doubles).  Expressed another way, A = k.c, where k is a constant. The constant k is particular to individual chemicals absorbance at specific wavelengths, and has been determined for a vast array of compounds including 4-nitrophenol.

The skills you will develop by completing this experiment successfully are:-

to pipette defined volumes of solutions accurately

to organise your work within a defined time scale

to plot your results in Excel and following the guidelines given in box 50.1 (pp308 ) of Practical Skills in Biology (3rd edition) Jones, Reed and Weyers, or in Bryson, Foundation Mathematics for Biosciences

to determine the slope of a graph and

to use the Beer-Lambert Law to convert changes in absorbance to changes in concentration

Methods

• Measuring alkaline phosphatase activity

1. Set up 'quench tubes'

 Pipette 500 mL of sodium carbonate solution (15 %, w/v) in each of 5 eppendorf tubes; mark these Q1, Q2, Q3, Q4 and Q5.

1. Set up reaction tube

Pipette 3 mL of substrate solution (2 mM 4-nitrophenylphosphate in 50 mM glycine buffer, pH 9.5) into a test tube.

1. Start reaction

 Add 25 mL enzyme solution (0.2 mg/mL) to this substrate solution, mix the contents quickly by inversion using a piece of parafilm and immediately start the clock.

1. Stop reaction at 1 minute intervals

At times 1, 2, 3, 4 and 5 minutes after starting the reaction withdraw 500 mL samples and add in turn to the quench tubes Q1, Q2, Q3, Q4 and Q5 respectively.  Mix the contents thoroughly (close eppendorf lid and invert 4-5 times) as quickly as you can.  The sodium carbonate solution is quite dense, so vigorous mixing is necessary.  The very alkaline nature (high pH) of the sodium carbonate denatures the enzyme and thus stops the reaction at the time the sample has been withdrawn and quenched.

1. e)  Finally read the absorbances of all the tubes at 400 nm relative to a water blank.

Results

• Record your results in the Table:-

Time of sample (min)	A400
1	0.310 / 0.297
2	0.497/0.339
3	0.629/0.400
4	0.740/0.463
5	0.812

• Using Excel, plot a graph of the results in the form Absorbance against time of sample.  Make sure you know which variable should be plotted on which axis (see Practical Skills in Biology for guidance, and the instructions for doing this in Excel available on Canvas).
• Answer the following questions, explaining clearly your reasoning and showing all necessary working.
• Do your results (i.e. the raw data above) indicate that the rate of production of 4-nitrophenol is constant?  Explain how you have reached your answer.

Yes, the results indicate a constant rate of production of 4-nitrophenol. This is ecause the graph of the absorbance against time drawn is a straight line.

• What is the rate of the reaction (in terms of absorbance change per minute)?

                The rate of the reaction is the gradient of the graph=0.1247 absorbance per minute

• Use the Beer-Lambert Law to convert your answer in (b) into a rate expressed in terms of the change in concentration of 4-nitrophenol per minute.  To do this you need to know that a 1 mM solution of 4-nitrophenol has an absorbance at 400 nm (A₄₀₀) of 11.5 under the conditions used in the experiment.

According to Beer Lambert’s law: A=kC

                 Where A=absorbance

                k= constant

                C=concentration

                1Mm=11.5A(for k)

     But we used 2mM= 2x11.5= 23A

    Our rate in terms of absorbance change per minute =

our rate in absorbance change per minute

        =0.1247/23=5.4217x10^-3 Mm per minute

Determination of elevated alkaline phosphatase activity in patient serum samples

Your demonstrator will supply you with a sample of serum from a patient (A, B or C) suspected of suffering from a liver disorder. Elevated levels of alkaline phosphatase activity in serum are used as an indicator of a number of clinical disorders, including liver disorders. You will measure the alkaline phosphatase activity present in the serum of patient A, B or C and determine the clinical significance.

Method

Repeat steps 1-4 for measuring alkaline phosphatase activity detailed in the first part of the practical, however, rather than adding enzyme solution in step 3, add 25 mL of patient's serum ( A, B or C).

IMPORTANT: ensure that you use fresh cuvettes, or thoroughly wash out the previous cuvettes in water

Sample A, B or C?

Record your results

Time of sample (min)	A400
1	0.158
2	0.148
3	0.156
4	0.164
5	0.155

• Insert an Excel graph of these results as previously and from this, calculate the rate of the alkaline phosphatase reaction (in absorbance change per minute).
• Express rate in terms of the change in concentration of 4-nitrophenol per minute.

           Rate= gradient =0.001 absorbance per minute

          According to Beer Lambert’s law: A=kC

          Where A=absorbance

          k= constant

           C=concentration

          Remember our k=23A

          Rate in terms of concentration=0.001/23=4.3479x10^-5 mM per minute

• Under the assay conditions detailed here, the change in concentration of 4-nitrophenol per minute is 0.5 mM for the serum from a healthy patient. Do your results indicate that your patient is suffering from a possible liver disorder? Explain clearly.

  Change in concentration from the assay results= 5x10^-4  mM per minute(from a healthy person)

 Change in concentration from my experimental results= 4.3479x10^-5  mM (from patient)

From the results above, i conclude that the person is suffering from liver disorder this clinical significance arises because the reduced rate of concentration of the 4-nitrophenol in the patient which depicts a lower absorbance value rate than that of a healthy person. Hence the person is possibly suffering from Liver disorder.