Assay for Different Marker Enzymes within Chicken Liver

Introduction

Cell Theory claims that living organism contain skin cells which will be the basic product of life. These skin cells contain certain organelles that perform specific functions. In order for Scientists to review these compartments, they isolated these organelles through the process of Cell Fractionation and certain enzymes are present in these organelles for it to be recognized from one another. These enzymes are called Marker Enzymes and give us clues in to identify a certain small fraction. Marker enzymes function by looking through an enzyme's function that may be found in a certain organelles. It also gives out information about the purity of a specific fraction. Examples of marker enzymes are Succinate dehydrogenase resides in the Mitochondria, Acid phosphatise in Lysosomes and Alkaline phosphodiesterase in Contractile vacuoles. The event of unwanted marker enzyme in different fractions may only be considered a sign of contamination during the preparation of the fractions.

Objectives

  1. To identify the identity of the organelles in the various fractions using marker enzymes
  2. To determine the purity of the fractions using marker enzymes

Methodology

Results and Discussion

Figure 1. 0

Fraction

Alkaline Phosphodiesterase (APDE) Assay

Peroxidase Assay

Alamar Blue Mitochondrial Assay for Mitochondrial Reductase

Blank

Crude

+

-

+

Nuclei

+

-

+

Soluble

+

-

-

Microsome

-

-

-

Figure 1. 1

Fraction

Protease Activity Assay

Blank

0. 85

Crude

2. 2

Nuclei

1. 20

Soluble

1. 6

Microsome

0. 75

Different substrates were used for the detection of certain marker enzymes to be able to detect certain organelles. Inside the Alkaline Phosphodiesterase (APDE) Assay that was used for the detection of phagocytic vacuoles. For the Peroxidase Assay, this was used to detect for the presence of peroxisomes by using a substrate which is Tetramethyl benzidine (TMB). Alamar Blue Mitochondrial Assay for the diagnosis of Mitochondrial Reductase by reducing blue resazurin to red resofurin. Proteins assay for the diagnosis of denatured proteins in the cell which binds to the Flamingo Red which will lead to a enhance fluorescence.

Based on the group's results found on Physique 1. 0, the existence phagocytic vacuoles were generally present in the Crude, Nuclei and Soluble. Crude, this in essence contains every one of the proteins and is known as to be the portion with the highest protein content, Nuclei which contains the DNA/histones and the mitochondria and is known as to be second to the Crude fraction which has the highest protein content and the Soluble portion which is the most diluted fraction and contains enzyme and contractile vacuoles reacted with the given substrate of the APDE Assay which is the para-nitrophenol thymidine 5' monophosphate and yielded a response. For the Peroxidase Assay, all fractions yielded no reaction with the substrate used which is Tetramethyl Benzidine (TMB). The fractions Crude and Nuclei were diagnosed to really have the Mitochondrial Reductase using the Alamar Blue Mitochondrial Assay. On this Assay, the Nuclei small percentage may or may well not yield a response since this fraction contains the DNA/histones and the mitochondria which Assay is used for the recognition of Mitochondrial Reductase which is often found in the Crude, Microsome fraction (which is following the Nuclei fraction where most of the Mitochondrial Reductase can be found) and the Soluble portion. Inside the Protease Assay for the diagnosis of denatured protein, based on the group's results, it runs from 0. 75 to 2. 2. Soluble being the small fraction with the lowest denatured proteins and Crude having the lowest denatured proteins.

Based on the group's results, some fractions contain a few of the organelles that should not be present in these fractions. These mistakes may be considered a cause during Cell Fractionation wherein the organelles did not separate completely plus some of the reagents used may be contaminated.

Conclusion and Recommendations

Based on the test done, different marker enzymes can be found in different fractions including different organelles with the own specific functions. These enzymes perform different reactions to identify the purity of any small fraction. Some fractions contain organelles which should not be within that fraction plus some fractions yielded a negative effect for an Assay. These is actually a cause of a few of the organelles not properly separated from each small fraction by using Centrifuge. It might also be a reason behind the reagents being polluted.

It is further advised that centrifugation should be done on a higher centrifugation speed for the organelles to separate properly also to get the exact data needed for the experiment. It is also advisable to execute this experiment in an environment where there is less contamination and these fractions should be stored where there may also be no pollutants present.

References

Answer to Guide Questions

  1. Based on a report, alkaline phosphodiesterase can regulate the intracellular degrees of cyclic AMP and cyclic GMP and can show role in multiple cellular functions (Levy, et al. 2011). For the enzyme protease, sre involved with a wide range of biological processes such as cell routine, cell signalling, cell proliferation and cell death and proteases are also involved in many individual diseases (Deu, et al. 2012). Acid phosphatase, are indicated in cells of monocytes and phagocytes and deficiency of this enzyme may result to mild phenotypes which may result to shared complentation (Suter, 2011). Deoxyribonuclease II was uncovered that these were very important to the engulfment-mediated of DNA degradation credited to apoptotic-cell nuclei (Evans and Aguilera, 2003).

2.

Succinate dehydrogenase, just like Acid phosphatise, is commonly within the mitochondria as a marker enzyme. It catalyzes the oxidation result of succinate to form fumarate that will be found in the Krebs circuit. In addition, it feeds electrons to the respiratory chain ubiquinone (Rustin, et al. 2002).

Alkaline Phosphatase can be used to know the potency of pasterurization on different milk products such as dairy and cheese (Sigma- Aldrich, 2014).

  1. Based on the results from the experiment that can be found on Physique 1. 0, a few of the marker enzymes were within their appropriate fractions and some were not. A good example of this is actually the fraction which has the Crude. Because the Crude is considered to be the fraction with the highest protein content, it ought to be present in every one of the Assays that were done during the laboratory experiment. In our obtained results, it had not been within the Peroxidase Assay that was done for the detection of peroxisomes. There could be a contaminants in the used reagents for the Assay that is why the Crude Portion did not produce any effect. Another example is the Soluble Small percentage wherein it should be positive in all the Assay done through the laboratory experiment since after centrifugation, it will contain enzymes and contractile vacuoles and based on our results, it did not yield any effect in the Peroxidase Assay and Alamar Blue Mitochondrial Assay. Previous but not the least is the Microsome Portion wherein it ought to be positive in the Peroxidase Assay since it includes the peroxisomes after it was centrifuged and the Alamar Blue Mitochondrial Assay for the detection of Mitochondrial Reductase. Since Microsome is the next fraction after the Nuclei, which provides the DNA/histones and the mitochondria, if not fractionated properly, Microsome may have a chance of having Mitochondria.

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