The aims of this experiment were to separate and locate the enzyme N-acetyl-β-D-Hexosaminidase from protein sample #15 and determine the specific activity of the enzyme within the sample. DEAE cellulose anion exchange chromatography was used to separate proteins from within sample #15. The initial separation was performed through the addition of Tris/HCl buffer pH 7.2 and yielded a peak consisting of positively charged proteins. Salting out was performed with Tris/HCl pH 7.2 1.0 M salt buffer and eluted negatively charged proteins. Peaks also represented the highest amount of a negatively and positively charged protein respectively. It was determined that sample #15 did not contain N-acetyl-β-D-hexosaminidase. Sample #15 concentration was determined to be 4.7 mg/mL. INTRODUCTION Aside from the cellular role proteins have numerous uses ranging from cosmetics to clinical applications (1). To successfully add on to the current uses of proteins, successful separation and location of proteins must be performed. One method to achieve this is through column chromatography. Column chromatography is a biochemical technique, which can achieve …show more content…
In initial elution using 25 mM Tris/HCl pH 7.2 buffer test tube #10 was determined to have the highest peak with an absorbance of 0.12. In elution with 25 mM Tris/HCl pH 7.2 1.0 M salt buffer test tube #20 was determined to have the highest peak with an absorbance of 0.4. These peaks represent the amount of protein present within the fraction. The first peak may include proteins with many positive amino acid residues. The second peak contained proteins with negatively charged amino acid residues. Due to the differences in absorbance of test tubes #10 and #20, elution using the 25 mM Tris/HCl pH 7.2 1.0 M salt buffer eluted more proteins from the column than the 25 mM Tris/HCl pH 7.2 buffer. The concentration of the protein was determined to be 4.37
The addition of the extra catechol was supposed to have sped up the reaction with the enzyme and force the inhibitor out of the enzymes active site. If there was more time allowed to observe any possible color changes the results would have been more conclusive and our results more accurate. In other
SOD activity was calculated in terms of units/mg protein. Total protein thiols (TPT) - This assay is based on the principle of formation of relatively stable yellow color by sulphydryl groups with DTNB (Moron et al., 1979). Briefly, 0.2 ml of liver homogenate was mixed with phosphate buffer (pH 8.0), 40 µl of 10 mM DTNB and 3.16 ml of methanol. This mixture was incubated for 10 min and the absorbance was measured at 412 nm against appropriate blanks.
The three genes are lac Z, lac Y, and lac A encode the enzymes beta-galactosidase, permease, and transacetylase respectively. In this experiment, we will measure the enzyme activity of beta-galactosidase which was induced when lactose was added to a 50ml log phase E. coli culture grown with glycerol. Another E. coli mixture grown with glycerol and glucose was also used. Eight tubes were labeled with 0, 5, 10, 15, 20, 25, 30, and G-30.
Starch solution is then placed into the test tube at a quantity of 5 mL. 5 drops of Lugol’s Iodine solution is added to the test tube. If the color changes, then it is known that starches are present in the solution. Proteins are next tested. In order to do this, 5 mL of gelatin solution is added to the test tube. 10 drops of Biuret’s reagent are added to test for protein.
: The hypothesis was a failure because the absorbency was not consistently increasing or decreasing as the pH level increased. The absorbency kept decreasing and increasing at random pH levels. When the characteristics of the enzyme reaction were tested, test tube one was given ten drops of enzyme and ten drops of substrate as well as distilled water. The contents then turned from a clear to dark yellow when mixed with absorbency (A400) of 1.370, the highest absorbency out of all tests. The reactant that was lacking in the control reaction was the enzyme.
Colorimetric determination of total protein in serum is based on the biuret reaction. The serum protein reacts with copper sulphate in the presence of sodium hydroxide. The Rochelle salt (K-Na-tartarate) contained in the biuret reagent is utilized to keep the formed cupric hydroxide in solution which gives the blue colour. The absorbencies of the sample (A sample) and of the standard (A standard) were read against the reagent blank in the spectrophotometer at a wavelength of 545nm. The total serum protein concentration (C) was calculated as follows:
In test tube three: 15uL of DNA 2, and 15uL of enzyme 1 were added. In test tube four: 15uL of DNA 2, and 15uL of enzyme 2 were added. The tubes were shaken and then put in a waterbath at 37o C to incubate for 50 minutes. After the incubation was finished, 5uL of solution to stop the reactions was added to all four tubes and they were stored in the refrigerator Casting and Loading a Gel for Electrophoresis
Introduction Enzymes are highly selective catalytic proteins which control and regulate all biochemical processes in the body. They are produced by living cells in order to accelerate both the rate and specificity of metabolic reactions. Enzymes are highly specific in their function because each enzyme is programmed to carry out one special task. Several million enzymes mediate chemical reactions occurring in a living system. Microbial enzymes play a major role in the diagnosis, curing, biochemical investigation, and monitoring of many dreaded diseases.
The concentration rate hypothesis was supported by the results that the enzyme absorption does increase as the concentration increases.
After 72 h, crude enzyme was extracted from the culture medium. The fibrinolytic enzyme was purified from the crude sample by various steps: ammonium sulfate precipitation, dialysis, ion exchange chromatography, and casein-agarose affinity chromatography. All purification steps were performed at 4°C until otherwise stated. The crude enzyme was precipitated at 70% saturation of ammonium sulfate, and the protein was collected by centrifugation (10,000×g for 10 min). It was suspended in minimal volume of double-distilled water.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis also known as SDS-PAGE is one of the methods for determining the molecular weight of unknown proteins. SDS is an anionic molecule which denaturizes proteins and brings it back to its’ primary structure and it also provides a negative charge to the uncharged molecule. The SDS-PAGE enables the separation of proteins based on their sizes. The larger the size of the protein, the harder it is to travel through the gel thus heavier proteins stay near the cathode side of the gel. For this experiment, a software named Gel Analyzer was used in order to obtain the molecular weight of the unknown proteins with the help of a protein ladder with known molecular weight and protein concentration.
Along with being found in plants, they are also present in liver cells, kidney cells, leukocytes and erythrocytes. For the concentration of enzyme experiment, the hypothesis was if the concentration of an enzyme increases, then the enzyme activity will increase as well. The hypothesis was proven to be true, because there are more enzymes to react with substrates. For the enzyme—factors affecting, the hypothesis concluded was if the temperature increases, than the enzyme activity will increase. This however was proven wrong, because enzymes become unstable at higher temperatures.
Introduction Enzymes regulate the biochemical processes in various organisms. The enzymes catalyze reactions and at times help with the generation of the ATP, which is an energy source. Among the enzymes of biological importance is the succinyl CoA synthetase. The essay focuses on the structure, functions, and relations of succinyl CoA synthetase.
The reaction that occurs can be investigated via the adding of the liver extract which contains the arginase to urea concentrations and distilled water. The amount of urea formed is determined via spectrophotometric analysis α-INPP. The urea produced was known via the color reaction with the α-INPP, it is the reagent used for the colorimetric determination of urea. (Barry J, et al. 1984). The red color formed when the α-INPP is reacted with the urea, is sensitive to light thus it is photo labile.
Western blotting is a procedure used to detect specific proteins in a given sample. Gel electrophoresis is used to separate the proteins which can be observed as thick and thin bands on the electrophoresis gel. In this experiment we use SDS-free polyacrylamide gel. Sample proteins used in this case are bovine serum, human serum, goat serum, chicken serum and horse serum. Since the SDS is negatively charged, sample proteins move to the positively charged anode through the gel.