Enzyme assay

Malachite Green Assay: The reaction that AGPase catalyzes produces PPi. The resulting PPi that is produced is hydrolyzed by an inorganic pyrophosphatase (Sigma Aldrich) to generate phosphate (Pi). Each reaction for the assay was conducted on a 96 well plate and the reaction mixture was 50µL and contained Pre-Mix which contained 50mM HEPPS, pH=8.0 (MP Biomedicals), 10mM MgCl2, 1.5mM FBP (Sigma Aldrich), and 0.2mg/mL Bovine Serum Albumin (Sigma Aldrich), and 1.5mU/µL yeast inorganic…

Chapter 4 - Discussion A standard curve of p-nitrophenol was drawn at the start of the project, before any enzyme assay of the fractions. The standard curve is set up so the equation of the line can be used to measure the enzyme activity in each sample fraction. P-nitrophenol shows a linear relationship with acid phosphatase. An Enzyme assay gives the activity levels of acid phosphatase in each fraction. The results from each fraction were placed into the equation of the line from the standard…

introduce the basics of enzymes. The topic is important because they are in every living cell and because of enzymes chemistry is allowed in the cell. They do this by providing the energy needed for chemical bonds to break down and lets molecules interact with each other. For part 1 of the lab my hypothesis was if you increase the concentration, then the absorbance will also increase. For part 2 section A my hypothesis was if you increase the temperature, then the enzyme reaction time will also…

supernatant served as source of enzyme for peroxidase assay. The reaction mixture consisted of 2.1 ml distilled water, 0.32 ml 100 mM potassium phosphate buffer (pH 6), 0.32 ml 5% freshly prepared pyrogallol solution, and 0.16 ml 0.5 % H2O2. Reaction mixture was mixed properly and incubated at room temperature for 10 minutes. Optical density of mixture duplicate was measured at 420 nm in a spectrophotometer (UV-Visible Spectrophotometer, Shimadzu, Japan). 100 µl of enzyme was added in the…

Km and Vmax determination of glucocerebrosidase towards p-NPG The Michaelis constant (Km) is a measure of the affinity of the enzyme towards the substrate, with smaller values representing greater affinity. Km and the maximum rate (Vmax) of leukocyte GCase were obtained through the Lineweaver-Burk plot (Figure 4) with artificial substrate p-NPG in concentrations from 0.71 mM to2.50 mM. Km and Vmax values for leukocyte GCase using p-NPG as substrate was found to be 12.6 mM and 333 U/mg…

succinate dehydrogenase protein derived from the mitochondria of cauliflower cells. Succinate dehydrogenase, once active, is known to reduce the coenzyme flavin adenine dinucleotide (FAD) to FADH2. Succinate dehydrogenase and its coenzyme creates an enzyme complex (E-FADH2) which transfers electrons to coenzyme Q during the Krebs cycle. In this experiment, sodium azide will be added to the mitochondrial solution to prevent the transfer of electrons to natural electron acceptors, such as coenzyme…

Enzymes are biological catalysts that increase the rate of a reaction by reducing the activation energy required for a reaction. Many enzymes are proteins but not all are; enzymes possess high levels of substrate specificity which they bind to through their active sites. Enzymes display optimal activity at a certain pH level and temperature. Temperature and pH level have drastic effects on enzyme activity; the enzyme will denature if the pH or temperature has changed from its optimal level.…

1. Introduction Hydroxynitrile lyases (HNL, EC 4.1.2.x) are enzymes which mediate the release of hydrogen cyanide (HCN) and aldehyde or ketone from cyanohydrins [1]. These HNL catalyse enantioselective synthesis and cleavage of cyanohydrins [2]. The cyanohydrins and their derivatives are finding wide applications in industries like pharmaceuticals, agrochemicals and cosmetics since these can be readily converted into other important molecules like α-hydroxy carboxylic acids, α-hydroxy ketones…

it was achieved by denaturing the non-recombinant E.Coli proteins. The Second step was much more beneficial to the total purification but the combination of the two steps achieved an even better purification of 4.6 fold. From this assay we can see that the correct enzyme has been successfully purified. Considering the SDS-PAGE results we can confidently say that the purification steps were effective at removing a large proportion of E.Coli proteins. However, not all E.Coli proteins have been…

shown in Figure 1. The metabolically active coenzymes of riboflavin are riboflavin -5-phosphate or Flavin Mononucleotide (FMN) and flavin adenine dinucleotide (FAD) that participate in many vital oxidation reduction processes. Catalytic sites of some enzymes are covalently bound with riboflavin as prosthetic group. FAD is not strictly referred as a dinucleotide because the ribityl moiety is not attached to the isoalloxazine ring by glycosidic linkage.In FMN, the phosphoric acid is attached to…