An Experimental and Statistical Analysis of Sdh Activity as Compared in Liver, Kidney, and Heart Homogenates of the Bos Taurus

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Jeffrey Shenfeld

An Experimental and Statistical Analysis of SDH activity as compared in Liver, Kidney, and Heart Homogenates of the Bos taurus

Methods

The three tissues being analyzed in this experiment, those of the kidney, heart and liver, were taken from the animal Bos taurus. The tissue homogenates used were made by adding 1 gram of tissue to 20 ml of sucrose phosphate buffer. The buffer was composed of 250 mM sucrose, 50 mM NaPO4, with a pH of 7.4. This mixture was homogenized with a high-speed blender for 3 pulses for a duration of 10 seconds each. The homogenates were then filtered through a cheesecloth and stored at -70° C.

The homogenate used in this experiment was liver homogenate. Five different samples and a blank were made in order to test SDH activity in the three homogenates. The samples were prepared using varying amounts of 50 mM NaPO4 pH 7.4, which was added to all samples. 3000 μl of solution for the assay was prepared with final concentrations of 83.3 μM of 2,6-dichloroindophenol (DCIP), and 10 mM succinate in 50 mM NaPO4 pH 7.4 The final concentrations of malonate used were: 1mM, 2mM, and 3mM and were used in tubes 3-6. SDH activity was measured against malonate activity, since malonate was expected to inhibit SDH activity. The control used in this assay was used to determine the background amount of SDH activity, using the same setup bit no succinate in 50 mM NaPO4 pH 7.4 or malonate was added to the control. After the samples were prepared, 250 μl of homogenate was added to each tube successively.

Homogenate was added right before analysis in spectrophotometer. Samples were analyzed via spectrophotometer at 600 nm, and the spectrophotometer was set to 1.00. The tube without malonate was used as a blank.

Reactions were then incubated at 30°C for 10 minutes, and then placed on ice for 5 minutes. After incubation and cooling, the absorbances of the various samples were analyzed via spectrophotometry, and the final absorbance values were subtracted from initial absorbance values, and subtracting from this the control final and initial absorbance difference. This calculation was multiplied by 4 to yield the final amount of SDH activity. This figure was then divided b previously calculated protein concentrations to yield SDH activity/mg protein. The spectrophotometer was reset and the absorbances were again measured with the spectrophotometer set to 600 nm.

Reduced DCIP due to succinate was calculated for samples 2-5 the calculations were done as below:
(reaction initial A600 – final A600) – [control (sample 1) initial A600 – final A600) This figure was multiplied by 4 to get the SDH activity/ml.

Using previously calculated protein concentrations, SDH activity/mg protein was calculated for all reactions to yield enzyme activity/mg protein. This was done using liver homogenate figures (Table 5).

The class averages for SDH activity in various homogenates were then statistically analyzed and the hypotheses were quantitatively analyzed.

A statistical analysis was done comparing the SDH activity in the different homogenates analyzed. T-tests were don’t to compare enzyme activity between liver-kidney, liver-heart, and kidney-heart

Results

In this experiment, DCIP acted as an electron-acceptor, and underwent a color change as it became reduced. The oxidized form of DCIP is blue, while the reduced form is colorless. DCIP replaced DCIP was used to replace FAD, a coenzyme of SDH. DCIP was used to accept electrons instead of SDH, becoming an alternate electron acceptor. The rate at which the color change took place was measured via spectrophotometry and thus used to determine SDH activity. Reduced DCIP due to succinate dehydrogenase activity:

The control reaction did not contain malonate, and was thus used to determine SDH activity when it is not inhibited. This was used as a sort of benchmark to compare the rest of the samples with. The control was prepared because we...
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