LDH Purification lab Report

The normalized SDH activity of two homogenates can be compared by looking at the class statistics for the Liver and Kidney homogenate samples in the data sheet attached. The kidney exhibited higher enzyme SDH activity than the liver. This was in agreement with the proposed hypothesis. Comparing the same two homogenates in which malonate was present, it can be seen that the kidney exhibited higher SDH activity than the liver. Thus, both homogenates did in fact have a decrease in enzyme activity, as malonate inhibits the activity of SDH. In successive experiments more malonate was used, and class statistics, not the activity itself, showed lower amounts of enzyme activity/mg protein as reaction number increased and a greater significance. Thus, malonate’s effect did increase proportionally to its concentration. There was a significant difference in SDH activity between the liver and kidney homogenates (p=0.0001)0.05). (Figure 1)

The homogenates provided were made by homogenizing tissues in a sucrose phosphate buffer in a 1:20 ratio. The protein concentration in bovine cells was measured by diluting the homogenate with a 1:5 ratio; 50 microliters of homogenate and 200 microliters of water. Then 5 known protein concentration samples which were 0.4, 0.8, 1.2, 1.6, 2.0 mg/ml of bovine serum were used to determine absorbance with a spectrophotometer. Two additional samples were made; one was blank and the other was for the specific homogenate sample. Then 3 microliters of bradford assay reagent, which indicates the amount of protein present

This experiment looked at how substrate concentration can affect enzyme activity. In this case the substrate was hydrogen peroxide and the enzyme was catalase. Pieces of meat providing the catalase were added to increasing concentrations of hydrogen peroxide in order to measure the effect of hydrogen peroxide concentrations on the enzyme’s activity. The variable measured was oxygen produced, as water would be too difficult to measure with basic equipment.

Each standard and unknown sample was individually placed into a square cuvette that was then placed into the spectrometer, and the absorbance for each sample was recorded individually. The absorbance for the standard samples was then graphed against the concentration for each sample. The slope of this line was used to produce a concentration for each unknown sample, given their

The absorbance is measured using a Plate reader and a Standard curve is generated. Also, the different types of pipetting techniques are assessed in this Assay.

Leucine. This amino acid is unique in its ability to promote protein production in the skeletal muscle system. It is more anabolic in nature than other amino acids, which means it is a potent builder of muscle and protein synthesiser.

Succinate Dehydrogenase and the Inhibitory Effect on its Specific Activity by Malonate By: Danielle Santiago, Nikita Shah, Zachary Voigt, Roseline Oyebanji Introduction This experiment is used to determine the level of succinate dehydrogenase (SDH) activity in a variety of reactions. Mitochondrion from a sample of cow liver is isolated via differential sedimentation and a Bradford Assay is performed to determine the concentration of SDH in the sample (Week 1 Recitation, 2013-2014).

If oxaloacetate is used as a reagent in this lab, then the succinate dehydrogenase enzyme’s activity will be limited.

Multiple samples with known properties can be measured and graphed, which then enables the same properties to be determined for the unknown samples, in this case the concentration, by interpolating the graph which depicts the relationship between the absorbance and concentration.

The effect different temperatures have on the rate that enzymes work was tested by this lab. The concentration of the enzyme catalase in different tissues was also tested. It was tested by pouring 5 ml of hydrogen peroxide over liver in different conditions and over a piece of meat and potato.

Lactate dehydrogenase (LDH) is an enzyme that catalyzes the reaction of glycolysis. It is responsible for the metabolism of carbohydrates, principally glucose, catalyzing interconversion of lactate and pyruvate with nicotinamide adenine dinucleotide as coenzyme (Heinová, Kostecká & Csank, 2015). Under aerobic conditions, pyruvate arrives the mitochondrial matrix and will be transformed into acetyl-CoA (Wei, Wei, Li, Xu & Wei, 2015). LDH can be found in both plants and human. LDH has two unique subunits, LDH-H (heart) and LDH-M (muscle), different subunits can combine to form different LDH isoenzymes according to their electrophoretic mobility from anode to cathode as LDH 1, LDH 2, LDH 3, LDH 4, and LDH 5 (Heinová, Kostecká & Csank, 2015). Combinations are

Enzymes mediate almost every single biochemical reaction, process or metabolic event in the body. Overall, enzymes are proteins whose primary function is to catalyze, increase the rate of the biochemical reactions (Champe et al., 2005). Enzymes are not only efficient in increasing the rate or velocity in a biological reaction, but also are incredibly accurate at recognizing other biochemical structures to create specific products. Taking into consideration how diverse the properties of enzymes are and their importance in the biological process of the human body, it is not a surprise how enzymes can be used as invaluable tools in the diagnosis of certain diseases, therapeutic applications and innumerable cases of clinical trials and lab analysis (Devlin, 2011). One example of the many application of enzymes in the medical field is how Proteases Pronase (Hydrolytic enzyme) and RNases are being used to remove adhesions in contaminated

The objectives of this experiment were to gain the knowledge of how to perform an enzyme assay, compare the LDH activity in three species, and to associate the LDH activity values with the capacity for anaerobic metabolism. My group originally thought that all three of these organisms would have higher LDH levels due to their daily activities. However due to more in depth background information about their behaviors, my group hypothesized that croakers would have the highest LDH activity levels due to their continuous migration between multiple habitats. This constant movement requires a lot of energy. Our results showed that lizardfish had the highest LDH activity values as opposed to our

An enzyme solution was prepared by mixing one mL saliva with nine mL distilled water andthirty mL 0.5% sodium chloride. Two mL of the enzyme solution was put in a large test tube andlabeled as 4°C. Two mL of the buffered starch solution (1% stach in phosphate buffer pH 6.7)was added in a separate large test tube. Both the test tubes were incubated for ten minutes inan ice bath (4°C). The solutions were immediately mixed. Three drops of the mixture was takenquickly and two drops of the 0.001 M iodine solution was added simultaneously onto a spotplate (first well). This was the zero minute. After one minute interval (incubation continued),three drops of the mixture was taken again and two drops of the iodine solution was addedsimultaneously

An important aspect of studying proteins is understanding their properties. The hypothesis of this experiment aimed to test whether the enzyme in question, N-acetyl-β-D-Hexosaminidase, has an overall negative charge and will elute from the DEAE column in the first protein peak. The experiment was done in two parts. The absorbance values obtained from conducting the Lowry protein assay were used to extrapolate the total amount of protein and calculate the concentration of the protein. The total amount of protein in the sample was 120µg and the concentration of the protein was 24.0 µg/µl, which was within the allowed range of error. An enzyme assay with paranitrophenol was conducted to determine the specific activity of the enzyme. The specific activity of the isozyme HEX B was calculated to be 0.00009 units/mg and the specific activity of the isozyme HEX A was 0.00004 units/mg. At the conclusion of this experiment, the data obtained from the ion-exchange chromatography indicated that the unknown protein sample contained two isozymes of the enzyme N-acetyl-β-D-Hexosaminidase. It was observed that isozyme HEX A had an overall negative charge and therefore eluted last from the DEAE column, whereas HEX B had an overall positive charge and did not bind to the column, hence eluted first.