Succinate Dehydrogenase Lab Report

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Enzyme assay analysis of succinate dehydrogenase to resolve Km and Vmax values and to determine the affects of different variables on the oxidation of succinate to fumerate
Introduction
Enzyme kinetics studies chemical reactions where enzymes are involved; rate of reaction and other factors that affect it such as increasing substrate concentration, enzyme concentration, presence of an inhibitor, deviation of temperature and pH. This will highly aid the information about mechanism of an enzyme and its effect on the substrate, subsequently, acquiring a deeper understanding of the system in which enzyme acts upon on; e.g. how metabolism is controlled.
The mitochondria contain enzymes that are involved in cellular respiration where amino acids, …show more content…

This was done quantitatively by monitoring the reduction of an artificial electron acceptor, DCIP. As DCIP is initially coloured blue, the reduction gradually turns this to transparent colourless as the reaction continues. Absorption values were recorded at set intervals and used to construct a substrate concentration- velocity graph. This is used to find the Vmax which is the maximum velocity in which the enzymes present can work. The Michaelis constant, Km is the substrate concentration at 1/2Vmax is also calculated from this. Using these value along with the Michaelis Menten model, will aid the understanding of the mechanism by providing a measure of affinity of succinate dehydrogenase (SDH) with its substrate, succinate and subsequently, the efficiency of the reaction.




Results





δAbs at 5 min δAbs at 10 min δAbs at 15 min δAbs at 20 min δAbs at 25 min δAbs at 30 min
Tube A 0.042 0.323 0.592 0.717 0.830 0.892
Tube B 0.114 0.917 0.949 0.955 0.956 0.957
Tube C 0.093 0.543 0.625 0.919 0.947 0.952
Tube D 0.169 0.234 0.410 0.416 0.438 0.445
Tube E 0.044 0.057 0.106 0.115 0.201 0.227
Tube F 0.039 0.044 0.129 0.133 0.134 0.133
Tube G 0.045 0.046 0.064 0.105 0.134 0.134

Table 1 shows that the difference in absorption increases as time progresses. …show more content…

Tube B has the highest increase in absorbance due to the great amount of mitochondrion present whereas tube A and C has less. This would mean that there are more succinate-SDH complexes; in return, more succinate being oxidized to fumerate, thus the reduction of E-FAD to E-FADH2 (DCIP) is greater in tube 2. Malonate is a compound which acts as a competitive inhibitor, it’s chemical structure is similar to succinate, allowing it to bind to the active site of SDH, its presence is seen in tube 4; this restricts the formation of enzyme-substrate complexes being made and consequently, less reaction of succinate to fumerate resulting a significantly less increase of the absorbance recorded over time compared to tubes A,B and

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