1) You should be able to correctly construct and use an accurate calibration graph and derive concentrations of unknown samples using the graph.
2) You should be able to plot kinetic data in an appropriate format
and obtain relevant kinetic parameters from the plots including initial rates vo, the maximal rate Vmax and the Michaelis constant (Km, apparent affinity) using freeware such as hyper 32 (free to download from the internet or using thegraphing functions within EXCEL
Allows you to plot v vs. [S] and 1/v vs 1/[S] graphs
3) You should be able to transform data using simple algebraic methods.
4) You should be able to calculate the degree of saturation of an enzyme knowing the substrate concentration.
5) You should have some knowledge of the role of phosphatases within the human body in both health and disease.
Where there is text in red you should replace the red text with the relevant units etc as described in the red text. Please delete the red text prior to submission
1) Abstract
Please type a short abstract relating to this practical (maximum size 200 words, please note number of words used)
2) Calibration Graph
a) At what wavelength (nm) is the absorbance measured? 410 nM
b) Calculation of the concentration of paranitrophenol (PNP) in µM
1 mM= 1000 µM
Tube 1: 0 µM Tube 2: 4 µM Tube 3: 8 µM
Tube 4: 12 µM Tube 5: 16 µM Tube 6: 20 µM
Details of calculations (These MUST be shown to the lecturer/demonstrator prior to proceeding to the next part of the practical).
Table 1:
Tube Volume of PNP Stock (mL) Conc. of PNP
Abs. 1 at
410 nm Abs. 2 at
410 nm Mean Absorbance at 410 nM
1
0 (blank) 0 0.00 0.00 0.00
2
0.2 0.004 mM 0.072 0.072 0.072
3
0.4 0.008 mM 0.144 0.142 0.143
4
0.6 0.012 mM 0.213 0.219 0.216
5
0.8 0.016 mM 0.283 0.283 0.283
6
1.0 0.02 mM 0.354 0.354 0.354
c) Please electronically paste a fully labelled calibration graph/standard curve (drawn using a computer ,not a hand drawn graph) for PNP
(para nitrophenol) in this space.
3) Measurement of Initial rates (ΔA.min-1)
a) Please complete the table showing the concentrations of substrate used.(remove red text on completion)
Table 2: Enter a title
Cell
(Experiment number) Volume of PNPP used Final Concentration of PNPP in cell
1
1.3 ml 4.3 mM
2
1.1 ml 3.66 mM
3
0.9 ml 3 mM
4
0.65 ml 2.16 mM
5
0.3 ml 1 mM
6
0.1 ml 0.33 mM
b) Please show FULL details of one of the calculations converting PNPP volume to PNPP concentration.
Concentration (Final) = Concentration (Initial) x Volume (Initial)
Volume Final
Concentration (Final) = 10 mM x 1.3 ml
3 ml
= 4.3 mM
c) What are the units of PNPP concentration?
Millimoles
4) Please enter a scan or photograph (generated via e.g your smart phone and saved to your computer as e.g a JPEG file) of your original data sheet from the practical to be used for the calculation of initial rates into this space.
5) Please paste fully labelled electronic graphs of your initial rate plots (Abs@410 nm vs time) into this space. ( you should enter no more than two graphs into this space
6) What, if any, are the units used on the axes of the graphs used to determine initial rates?
y-axis:___________________
x-axis:___________________
7) Electronically enter graphical plots for:
a) A saturation (hyperbolic) plot of initial rate (vo) vs. substrate concentration [S]. (use a freeware programme such as Hyper 32 to construct the graph) (note if you use a computer with a mac based operating system you will need to download a different programme as hyper 32 will only work with a Windows OS.
b) A Lineweaver-Burk (double reciprocal) plot. (use either Excel or Hyper 32 to construct the graph) into this space.
Please indicate which programme you have used to draw the graphs and obtain values for Vmax and Km
c) Determine both the Michaelis constant (Km) and the maximal rate (Vmax) from each of the two plots. You should include the units for each parameter (note you may use a screen save function and paste th4e saved screen into this space)
Hyperbolic Plot Lineweaver-Burk plot
Km
Vmax
d) Using the calibration graph convert the Vmax values above into Vmax in units of μMmin-1 from the estimate you obtained using:
(i) The hyperbolic plot:
(ii) The Lineweaver-Burk plot:
e) Indicate strengths and weaknesses of each of the above types of plot.
8) Questions
a) Using the equation:
If Km = 3.5 mM and [S] is 2.68 mM
Calculate the percentage of enzyme having substrate bound at the above concentration? Type your calculation in full into this space.
b) Discuss the role, function and cellular/tissue location of both acid and alkaline phosphatases in an organism. (You will find the answer to this question in any standard biochemistry textbook/Google search) Please indicate the source of your information
c) How many buffer compounds would you need to cover the pH range pH 4.5 to pH 8.5? Give examples of compounds that would allow you to cover this range (You will need to consult a standard text on Biochemistry or to consult Google Scholar for the answers to this question).