Cytochrome P450

This Lab Report is an analysis of the results of a two-part experiment. In the first part, we used a gel filtration column to separate the components of a mixture composed of protein and non-protein molecules. By doing so we hoped to obtain fractions that contained single components of the mixture, while also gaining insight into the relative molecular weight of each component compared to each other. We would then plot these fractions onto nitrocellulose paper in order to determine which fractions had protein. In the second part, we would use the fractions which we had determined had protein to conduct an SDS-PAGE. By doing so we hoped to determine an estimate on the molecular weight of the proteins present in each fraction by comparing it to a tracker dye composed of a variety of molecules of differing molecular weight.

Gel electrophoresis method. Qualitative analysis shows protein concentrations in kidney, heart, and liver. 1-6 are kidney tissue. 7-14 are liver and 15-19 are the heart tissue.

Second, in order to further confirm the information about characteristics and function of the targeting protein that we have

Promoting or inhibiting the transcription of genes that code for the synthesis of cellular proteins intracellular second-messenger system are correct

Western blotting - In Western blotting first, the macromolecules have to be separated via gel electrophoresis. The molecules now separated by electrophoresis are blotted onto either a nitrocellulose or a polyvinylidene difluoride (PVDF) membrane (a second matrix). To inhibit the binding of nonspecific antibodies to the membrane surface it is subsequently blocked. Then a complex is formed (a probe) from the protein that was transferred and an enzyme linked with an antibody. The enzyme is supplied a substrate then the 2 together should create a product e.g. chromogenic precipitate that can be detected. Detection methods with most sensitivity use chemiluminescent substrate because light is a by-product of the reaction between the substrate and the enzyme. The output of the light can be measured using a CCD camera or on the other hand, antibodies that have been tagged with fluorescents that are detected with a fluorescence imaging system can be used (Thermo-Fisher Scientific 2015).

Figure 3. Protein expression and purification protocol is shown in the schematic in (A). To confirm expression of the P278L mutant, a western blot was performed. Note the presence of Kif5A in all fractions, and enriched in the E1 and E2 eluate.

Xenobiotic metabolism is usually divided to phase I and II, where polar function groups are unmasked or introduced to the chemical structure (phase I or activation), followed by the conjugation between the xenobiotic and endogenous molecules, notably glucuronic acid, to further improve hydrophilicity (phase II or conjugation) (Golan 2012). Hepatic microsomal P450 are the enzymes that dominate the catalysis of phase I metabolism of xenobiotics (Table 1 and 2) (Anzenbacher and Anzenbacherova 2001). Therefore,

The mammalian cytochrome P450 CYP belongs to superfamily and mainly encodes enzymes involved in the metabolism of pharmaceuticals, foreign chemicals and pollutants [1]. Several mutations were observed in CYP genes which are responsible for inborn errors of metabolism leading to multiple diseases. It was observed from previous studies, gene nomenclature was simpler before arrival of complete genome. This is due to the fact that detailed information about gene position on the chromosome did not affect the gene names. Therefore, in the past names were assigned only on the basis of sequence similarity and evolutionary divergence [4, 5]. But now genes occur in clusters, with several related genes, and many pseudogenes. Also, genes in subfamilies are found to be clustered with genes of other subfamilies [6]. Pseudogenes are found to be manily involved

The aromatase enzyme CYP19A1 is so named because it is responsible for catalyzing the conversion of 19-carbon androgens to 18-carbon aromatic estrogens . The CYP19A1 is complex bound to the SER of the cells, and is localized in the ovaries, testis, placenta, and fat-, brain-, and bone tissues.

Traditional reagent strip testing for protein uses the principle of the protein error of indicators to produce a visible colorimetric reaction. The protein (primarily albumin) accepts hydrogen ions from the indicator causing change in color. (Susan King Strasinger, 2008)

The testing of various proteins was performed by comparing the molecular weight of proteins using SDS PAGE. The molecular focus in the lab was the testing of proteins, which are macromolecules consisting of amino acid monomers linked through chemical bonds. These proteins have a hierarchy of structure that consists of folding that determines the direct function of each protein.. The molecular weight of these proteins were measured using SDS PAGE. SDS PAGE stands for sodium dodecylsulfate polyacrylamide gel electrophoresis. SDS is an anion detergent that binds with the protein structures and causes them to separate due to the change in bonding charge. SDS and heat are how the proteins are denatured. The process of denaturing a protein is breaking

After purifying the proteins, the students will be able to compare the molecular weights of the samples by running a denatured SDS polyacrylamide gel (Purification & Size Determination of GFP & BFP, EDVOTEK). This will show students which proteins different from one another and how denaturing can affect the proteins. Background:

The size was also determined by this method. The buffer chambers were filled with SDS-PAGE (Sodium Dodecyl Sulfate polyacrylamide gel electrophoresis), which helped add negative charges to the proteins and ran at 120 v for 30-50 minutes and then 100v for an hour. The samples were loaded in the order of ladder, S1, P1, P1 med salt, P1 high salt, S2, P2, P2 med salt, and P2 high salt. Western Bolt

Pyranose 2-oxidase catalyzes the oxidation of aldopyranoses by using molecular oxygen as an electron acceptor to yield the corresponding keto-aldoses and hydrogen peroxide. This enzyme belongs to the Glucose-Methanol-Choline (GMC) oxidoreductase superfamily and contains flavin adenine dinucleotide (FAD) as a cofactor. P2O catalyzes regio-specific oxidation at the C2 position. Here, the hydride transfer reaction from glucose to oxidized flavin catalyzed by pyranose 2-oxidase (P2O) was investigated by density functional theory calculations and transient kinetics. Our findings suggest that the first step of the P2O reaction is a hydride transfer from C2 position of glucose to N5 of the flavin. Then, the proton abstraction occurs by the conserved

Our findings demonstrate that all five biguanide compounds tested inhibited oxidative phosphorylation (OP) through an interaction with complex I in the electron transport chain (ETC). Through Electron Paramagnetic Resonance (EPR) analysis we have shown that biguanides do not inhibit the movement of electrons within complex I due to the normal activity of FeS clusters in the presence of the biguanide compounds. We subsequently ruled out competitive inhibition of the ubiquinone-binding site as a possible mechanism, by showing altered Michaelis-Menten kinetics in the presence of decylubiquinone. These data indicate that inhibition is likely a result of an altered catalytic function due to the interaction between the compounds and complex I. Biguanide-dependent inhibition of complex I isolated from mammalian, yeast, and bacterial sources indicates a conserved target of action. We hypothesized that biguanide inhibition may be occurring at the enzymatic moiety of the matrix-facing ND3 subunit of complex I; where NADH oxidation occurs facilitating the transmembrane transfer of hydrogen and the inter-ETC-complex electron exchange. A specific residue, Cys39, located in an amphipathic region between the redox and proton-transfer domains is particularly important in determining the functional confirmation of the protein. The presence or absence of substrate is responsible for either the ‘closed’ active confirmation, or the ‘open’