It is widely accepted that certain metal ions are essential to sustaining biological life. A major component in their biological relevance is that many enzymes require metal cofactors for their catalytic activity (Bertini et al., 2006). One such enzyme is Bovine Intestinal Alkaline Phosphatase or BIAP, a member of the wider enzyme classification of alkaline phosphatases. Alkaline phosphatases are dimeric metalloenzymes, enzyme proteins with metal ion cofactors directly bound to the protein, containing two Zn2+ and one Mg2+ metal-binding sites in each active site region (Kim et al., 1991). They function to catalyze the hydrolysis and transphosphorylation of phosphate monoesters. The presence of these metal ions in the enzyme structure are …show more content…
As one would expect, the deprotonation of the hydroxyl group requires a general base to accept the proton. An Mg2+ ion in coordination with a nearby water molecule can function as this general base, accepting the released proton and allowing for the deprotonation of the serine residue(Stec et al., 2000).
It is well documented that the binding of Mg2+ onto the alkaline phosphatase metal binding site is a relatively slow process when compared to other metal ions. This allows it to be outcompeted for the position by other metal ions such as Zn2+ and Co2+ (Hung et al, 2000 & Chan et al., 2002). From this ion competition, we can assume that the binding of Mg2+ is not a permanent institution; it sometimes dissociates and then reassociates with the enzyme. Therefore, basic probability tells us that if there is a higher concentration of Mg2+ ions in the enzyme solution, it will be more likely for an Mg2+ ion to be nearby to, and subsequently able to bind to, the vacated metal-binding site. Since the Mg2+ returns the enzyme to a catalytically favorable conformation, enzyme solution with a higher concentration of Mg2+ ions, should have a higher catalytic rate.
We hypothesize that the concentration of Mg2+ ions is positively correlated with the rate of hydrolysis of para-Nitrophenyl Phosphate (pNPP) by Bovine Intestinal Alkaline Phosphatase (BIAP). Our approach to test this hypothesis is to measure the absorbance of solutions after the hydrolysis of pNPP by BIAP at
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In this lab or experiment, the aim was to determine the following factors of enzymes: (1) the effects of enzymes concentration the catalytic rate or the rate of the reaction, (2) the effects of pH on a particular enzyme, an enzyme known and referred throughout this experiment as ALP (alkaline phosphate enzyme) and lastly (3) the effects of various temperatures on the reaction or catalytic rate. Throughout the experiment 8 separate cuvettes and tubes are mixed with various solutions (labeled as tables 1,3 & 4 in the apparatus/materials sections of the lab) and tested for the effects of the factors mentioned above (concentration, pH and temperature). The tubes labeled 1-4 are tested for pH with pH paper and by spectrophotometer, cuvettes 1a-4a was tested for concentration and cuvettes labeled 1b-4b was tested for temperature in four different atmospheric conditions (4ºC, 23ºC, 32ºC and 60ºC) to see how the enzyme solution was affected by the various conditions. After carrying out the procedures the results showed that the experiment followed the theory for the most part, which is that all the factors work best at its optimum level. So, the optimum pH that the enzymes reacted at was a pH of 7 (neutral), the optimum temperature that the reactions occurs with the enzymes is a temperature of 4ºC or
Tables 2,3,4,5 and 6 show that as duration increased the absorbance also increased for each pH. The solution in the conical flask became darker (yellow) in time this is because the substrate, p-nitrophenyl phosphate was catalysed by acid phosphatase, releasing Nitrophenolate anion. It was the Nitrophenolate anion giving off the yellow colour; the presence of this feature increases the absorbance rate. The addition of sodium hydroxide distorted the shape of the enzyme making it no longer effective in its function.
These results shown from this experiment led us to conclude that enzymes work best at certain pH rates. For this particular enzyme, pH 7 worked best. When compared to high levels of pH, the lower levels worked better. The wrong level of pH can denature enzymes; therefore finding the right level is essential. The independent variable was the amount of pH, and the dependent being the rate of oxygen. The results are reliable as they are reinforced by the fact that enzymes typically work best at neutral pH
= PH changes affect the structure of an enzyme molecule and therefore affect its ability to bind with its substrate molecules. Changes in pH affect the ionic bonds and hydrogen bonds that hold the enzyme together, which naturally affects the rate of reaction of the enzyme with the substrate. On top if this, the hydrogen ions neutralise the negative charges of the R groups in the
The purpose of this experiment is using Compleximetric titration and EDTA to determine the concentration of Mg2+ in solution; and also calculating the percent by mass of MgO in the unknown sample. This procedure results no significant deviations.
Enzymes are biological catalysts that facilitate specific chemical reactions (Raven, et al., 2014). Enzymes do their job by
The independent variable in this investigation is pH. Each individual enzyme has it’s own pH characteristic. This is because the hydrogen and ionic bonds between –NH2 and –COOH groups of the polypeptides that make up the enzyme, fix the exact arrangement of the active site of an enzyme. It is crucial to be aware of how even small changes in the
After passing through the esophagus, which absorbs much of the salt ions in the swallowed saltwater, and the gut the luminal fluid is isosmotic with the plasma. The intestines continue to absorb salt (sodium through chloride co-transport proteins and the chloride through the sodium co transport proteins and anion exchange protinis) which is followed by an uptake of water. More chloride is absorbed than sodium which creates an electrogradient in the cell (the cell being more positive and the plasma more negative). The anion exchanger intakes chloride all while excreting HCO3- into the intestinal lumen. The intestinal fluid is highly alkaline, high in HcO3- and high in calcium (from the environment), this allows for CaCo3 to be precipitated in the
The last factor that increases rate of reaction is the surface area. Grinding up the magnesium into a powder increases the surface area, so the acid has more space to react on. This means the larger surface area, the quicker reaction time. Magnesium reacts very strongly with hydrochloric acid. In a high concentration of acid it can take just a few seconds for magnesium to furiously bubble and finally completely dissolve with no trace except for hydrogen gas.
The main objective of this experiment is to carry out qualitative analysis to identify metal cations in unknown solution 1.
Acid phosphatase removes phosphate groups from a variety of molecules under slightly acidic conditions. Wheat germ has been found to be a particularly good source of acid phosphatase. Wheat germ acid phosphatase catalyzes the hydrolysis of phosphate groups from phytins, ATP, protein phosphates, and nucleic phosphates (Phosphatase, 2015) that are stored in the wheat seed. The growing wheat embryo uses the freed phosphate in germination and growth (Joyce et al, 1960). Para-Nitrophenyl phosphate (pNPP), a colorless compound, was used as a substrate in this experiment. The hydrolysis products are para-nitrophenol and
The purpose of this lab was to understand how different solutions played a role in the digestion protein. By looking at different variables, such as temperature, and pH we’re capable of understanding just how certain substances functioned and when they didn’t. The data for all labs are clear and concise and give a clear understanding of what solutions work best. All three labs were placed in a warm water bath set at 37’C to stimulate the reaction as if it were taking place within the human body. This gives us a more accurate reading on how they would react at that set temperature. We concluded why certain tubes changed to the color they did and further explained it. This lab focuses primarily on two crucial
~0 mV versus Normal Hydrogen Electrode (Rodgers and Sligar, 1991) suggests that electron transfer to ferric P450 (redox potential ~300 mV vs. NHE) is unfavorable. Hence it was suggested that the redox function of cyt b5 involved electron transfer to the ferrous dioxygen intermediate which has a redox potential near 0 mV (Lipscomb et al., 1976) thus providing the “second electron” in the normal monooxygenase stoichiometry. In an attempt to differentiate between these two roles, Coon and co-workers reconstituted apo cyt b5 with manganese protoporphyrin IX (Morgan and Coon, 1984). They found cytochrome P450 reductase (CPR) and NADPH could not reduce the manganese substituted cyt b5, whereas iron cyt b5 was rapidly reduced. Hence Mn b5 is incapable of any electron transfer to the P450. They concluded that cyt b5 effects depend on the specific P450 in question, the substrate being examined, and molar ratio of CPR to P450. This suggested that their observations could not be explained solely by a simple electron transfer role and some effects may also be caused by possible conformational changes caused by cyt b5 binding.
The titration curve of the unknown exhibited many characteristics, such as equivalence points, pKa of ionizable groups, isoelectric point, and buffer regions, that are particularly distinct to lysine. For unclear reasons, the pH during the titration did not reach the pH for pure 0.2 M NaOH nor 0.2 M HCl and normal equivalence points expected at two extreme ends of the titration curves for all amino acids were not observed. The titration of a phosphate buffer showed that the buffer capacity is directly proportional to the molarity of the buffer. However, our results showed that although the initial pH of the phosphate buffer was less than the pKa value, the measured buffer capacity was higher towards acid than base. The accuracy of the pH meter and calibration process was questioned under assumptions that the pH of the prepared phosphate buffer was actually above pKa.