Assay of Alkaline Phosphatase in Serum Samples - Practical Schedule

SCIU1LS Practical Schedule for practical 6
The laboratory assay II: Alkaline phosphatase in serum
Registration Number
Lab Session - Tues pm

Practical 6 SCIU1LS
Assay of the activity of the enzyme alkaline phosphatase present in
serum samples
NOTE- this practical will be assessed and contributes 10% of the grade
for the module. This sheet must be completed, together with
embedded Excel graphs (instructions for this are included in the
materials on Canvas) and must be submitted to Canvas by 12:00 noon
on 26/11/2018
Aims of practical
(i) measure activity of alkaline phosphatase
(ii) determine presence of elevated levels of alkaline phosphatase in serum samples
Essential reading
(i) Campbell (9th Edition), Chapter 8, pp198-203 ( Enzymes)
(ii) Practical Skills in Biological & Environmental Sciences (2011)
Chapter 21 Working with liquids
Chapter 22 Basic laboratory procedures
Chapter 50 Calibration
Page 247-248 Types of assay
Chapter 59 Basic Spectroscopy
(iii) Foundation Mathematics for Biosciences (Bryson)
Chapters on Enzyme kinetics and Graphs, Trendlines and Equations
Introduction
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.
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4-nitrophenylphosphate + water 4-nitrophenol + 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  c (the symbol  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
+
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