km(Michaelis-Menton ) - page 1
Keywords: 500
By mickcy on 04/04/2011
Level: Bachelor Honours Degree (BA, BEng, BSc etc)
Page Number: 1 of 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. Incubate the tubes at 37oC for 10 minutes.
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
Then 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. At the end of the 30 minute incubation period, withdraw 0.5 cm3 and transfer to another tube containing 3.8 cm3 water and 0.5 cm3 of acid molybdate. (Caution HARMFUL, CORROSIVE) This will stop the enzymic reaction. It will again be necessary to stagger these additions at 30 second intervals.
Then add 0.2 cm3 of reducing agent (Caution HARMFUL, CORROSIVE) (no particular timing of addition here) 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 minutes is over.
Using an appropriate blank, complete the table (below) showing substrate concentration ([S] mM) per tube and absorbances for each tube, 1/[S] and 1/V (15 marks).
Tube [S] mM Abs (V) 1/[S] 1/V
By assuming that the reaction velocity is directly proportional to the absorbance at 700nM, determine the value of Km;
(I) from a simple plot of rate v substrate concentration (5 marks)
1. Which is likely to be the most accurate value for Km and why (5 marks)?
2. Is it reasonable to assume that the reaction velocity is directly proportional to absorbance? Explain your reasoning. How could you determine whether this was the case? (5 marks)
3. If p-nitrophenyl phosphate had been used as a substrate would the same Km have been obtained? Explain your answer (5 marks).
4. Elevated blood plasma levels of alkaline phosphatase (hyperphosphatasaemia) are an indication of a number of conditions. Briefly comment on the use of alkaline phosphatase assays as a diagnostic aid for TWO named situations. Also explain the requirement and nature of “follow-up/confirmatory tests
(10 marks).
Last 5 comments…
pls answers are needed to all the quesions and the data s that were used to answer the quesions
davis (14-Apr-2012)