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DETERMINATION OF Km – Biochemistry (Practical Report))

DETERMINATION OF Km – Biochemistry (Practical Report)) In this practical you will determine the Km of the enzyme alkaline phosphatase. To determine the Km it is necessary to set up reaction mixtures containing a range of substrate concentrations. Alkaline phosphatases have group specificity, catalyzing the (generalized) reaction; Orthophosphate ester + H2O  à corresponding alcohol + orthophosphate   Using your results, or class data provided, you will determine the values of Vmax and Km using a Michaelis-Menten plot (V against [S]) and a Lineweaver-Burk plot (1/v against 1/[S]).

Method

Tris Buffer CautionHARMFUL, IRRITANT MgCl2  CautionHARMFUL, IRRITANT Enzyme (in Tris Buffer) Caution- HARMFUL, IRRITANT Refer to COSHH data in red folder (with Technical staff) for procedures and precautions in case of spillage/contact with skin/eyes. PPE (Laboratory coats, gloves and eye-protection must be worn).   Set up the following reaction mixtures as follows (in this order- all volumes in cm3); Accurate pipetting is essential if you are to obtain usable data. Revise your pipetting technique see; http://www.youtube.com/watch?v=weBuqelKwP4  
Reagent                      Tube 1 2 3 4 5 6 7
200mM Tris buffer pH 8.5 0.1 0.1 0.1 0.1 0.1 0.1 0.1
33mM MgCl2 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Distilled water 3.6 3.5 3.4 3.2 2.8 2.0 1.2
Enzyme (2.0 mg cm-3) 0.2 0.2 0.2 0.2 0.2 0.2 0.2
  Add the enzyme after all the other reagents have been added- (WHY?). Mix by inversion (gloves) and incubate the tubes at 37oC for 10 minutes.   PTO   Then add the substrate (Glucose-1-phosphate) as follows;  
Reagent Tube   1 2 3 4 5 6 7
4mM G-1-P 0 0.1 0.2 0.4 0.8 1.6 2.4
  Incubate the tubes for exactly 30 minutes at 37oC. To achieve this you will need to stagger the start of each reaction. 30 second intervals should be sufficient- after addition of the substrate mix the contents of the tube by inversion.   While you are waiting make up another set of seven “stop” tubes containing 3.8 cm3 water and 0.5 cm3 of acid molybdate. (Caution HARMFUL, CORROSIVE). At the end of the 30 minute incubation period, withdraw 0.5 cm3 from each of the seven reaction tubes and add to the “stop” tubes. This will immediately stop the enzymic reaction (why?) and it is essential to stagger these additions at 30 second intervals in the same order as you started. Finally add 0.2 cm3 of reducing agent (Caution HARMFUL, CORROSIVE) (no particular timing of addition here) mix by inversion and allow to stand for a minimum of 10 minutes. Between them these two reagents produce a coloured complex with orthophosphate. Measure the absorbance at 700nm as soon as possible, once the 10 minute colour development period is over.  

Results

Using an appropriate blank, complete the table (below) showing substrate concentration ([S] mM) per tube and absorbances (@700nm) for each tube. Calculate 1/[S] and 1/V- (assuming that absorbance is a good proxy for the rate of reaction) (10 marks).  
Tube [S] mM Abs (V) 1/[S]mM 1/V
1
2
3
Etc.
  By assuming that the reaction velocity is directly proportional to the absorbance at 700nM, determine the value of Km from Excel plots of;
  • Rate v substrate concentration (5 marks)
  • A double reciprocal plot i.e. 1/absorbance v 1/substrate (10marks). To get full marks you need to show all numerical
 

Discussion

 
  1. Which graph is likely to give the most accurate value for Km and why (5 marks)?
 
  1. Is it reasonable to assume that the reaction velocity is directly proportional to absorbance? CAREFULLY explain your reasoning. How could you determine whether this was the case? (15 marks)
 
  1. If p-nitrophenyl phosphate had been used as a substrate would the same Km have been obtained? Explain your answer (5 marks).
 
  1. Elevated blood plasma levels of alkaline phosphatase (hyperphosphatasaemia) are an indication of a number of pathologies. Briefly comment on the use of alkaline phosphatase assays as a diagnostic aid for TWO named common medical conditions. Also explain the requirement and possible nature of follow-up/confirmatory tests (10 marks).
 
  1. You are characterising the properties of a new enzyme that you have just isolated. In the absence of this enzyme, the rate of hydrolysis of its substrate is about 100 molecules per year. In the presence of the enzyme, it’s about 10 molecules per second. What is the increase in the rate of the catalysed reaction, compared with the uncatalysed? (5 marks).
 
  1. A mutation that changes an alanine residue in the interior of your enzyme to a valine residue is found to lead to complete loss of activity. However, some activity is restored when a second mutation at another position, close by, changes an isoleucine residue to a glycine residue. How might this second mutation lead to some restoration of activity? (5 marks)
Total Marks 70   Up to 15 marks will be deducted for poor presentation, poor use of English and incorrect referencing (see overleaf for checklist summary- which you must fill in and append to the back of your assignment (– 5 marks if you do not).   You MUST attach a copy of this schedule to you work. Automatic 5 mark deduction if you do not do this. Word Count max. 1000- excluding tables, diagrams, graphs, reference list.   You must run your completed assignment through “Turnitin” prior to submission and note your Turnitin receipt number on the Assignment Coversheet.]]>

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