Section A – Answer ALL questions A 1 month old baby is failing to thrive, with poor feeding and growth, and frequent irritability. After becoming ill with a severe cold, he is admitted to hospital with abdominal tenderness and yellowing of the whites of the eyes, and is diagnosed with jaundice. Clinical investigation reveals elevated serum bilirubin, splenomegaly, and a low level of haemoglobin.   Whole blood extract containing 200mg of red blood cells were resuspended in 1mL saline stabilised with 1,8-beta-mercaptoethanol and EDTA. The activity of “enzyme X” was measured in the sample by adding 100µL of this suspension to 0.9 mL of Tris-HCl EDTA buffer (pH 8.0) containing 0.1M MgCl2, 2.0mM NADH, 50mM phosphoenolpyruvate (PEP), 30mM ADP and lactate dehydrogenase at 60units/mL.   The decrease in absorbance at 340nm was followed over a period of 10 minutes and compared with a blank sample containing all components except the ADP. The results are shown in the table below:   Time/min Absorbance   With ADP Without ADP 0 0.53 0.092 2 0.44 0.087 4 0.34 0.082 6 0.23 0.062 8 0.12 0.03 10 0 0   Draw an appropriate graph and calculate the initial rate of reaction due to enzyme X in absorbance units/min/100µL of extract.

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Section A – Answer ALL questions

A 1 month old baby is failing to thrive, with poor feeding and growth, and frequent irritability. After becoming ill with a severe cold, he is admitted to hospital with abdominal tenderness and yellowing of the whites of the eyes, and is diagnosed with jaundice. Clinical investigation reveals elevated serum bilirubin, splenomegaly, and a low level of haemoglobin.

 

Whole blood extract containing 200mg of red blood cells were resuspended in 1mL saline stabilised with 1,8-beta-mercaptoethanol and EDTA. The activity of “enzyme X” was measured in the sample by adding 100µL of this suspension to 0.9 mL of Tris-HCl EDTA buffer (pH 8.0) containing 0.1M MgCl2, 2.0mM NADH, 50mM phosphoenolpyruvate (PEP), 30mM ADP and lactate dehydrogenase at 60units/mL.

 

The decrease in absorbance at 340nm was followed over a period of 10 minutes and compared with a blank sample containing all components except the ADP. The results are shown in the table below:

 

Time/min

Absorbance

 

With ADP

Without ADP

0

0.53

0.092

2

0.44

0.087

4

0.34

0.082

6

0.23

0.062

8

0.12

0.03

10

0

0

 

Draw an appropriate graph and calculate the initial rate of reaction due to enzyme X in absorbance units/min/100µL of extract. 

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