Different Assays to Detect Presence of Activity of Enzymes in Specific Organelle

11042575

N05

Biology Department

De La Salle University

ABSTRACT

INTRODUCTION

Researchers biochemically study and analyze the structure, function and molecular composition

of subcellular organelles obtained from the well-fractionized samples of cells or tissues. To be able to

study the subcellular organelles, the specific organelle to be studied should be present on the sample or it

may be just a waste of time and money. Marker enzyme is a technique to trace the activity of enzymes of

a specific organelle. This must be done carefully because contamination may happen.

There are different assays which includes: Alkaline Phosphodiesterase (APDE) Assay – traces the

activity of enzymes in the vacuoles found in microsome fraction; Peroxidase Assay – traces the activity of

enzymes in the peroxisomes that can also be found in microsome fraction; Acid Phosphatase (ACP)

Assay – determine the presence of the activity of enzymes of lysosomes which can also be found in

microsome fraction; Mitochondrial Reductase/ Dehydrogenase (Alamar Blue) Assay – identify the

presence of activity of the enzymes of mitochondria, also found in microsome fraction; Protease Activity

Assay – tests the activity of enzymes of the proteins that are present in soluble fraction; and DNAse

Activity Assay – traces the enzyme activity of the DNA in the nuclei fraction.

In this experiment, the fractions used are the fractions from the previous experiment to check the

success of the homogenization and fractionation of cellular components. This experiment aims to trace

and check the enzyme activities present on specific samples. It is expected that the APDE Assay,

Peroxidase Assay, ACP Assay, and Alamar Blue Assay are positive in microsome fraction; Protease

Assay positive in soluble fraction; and DNAse Assay positive in nuclei fraction.

MATERIALS AND METHOD

NOTE: All reagents in this experiment (substrates and fluorescent dyes) were EXTREMELY

EXPENSIVE that it was used with DISCRETION and the REAGENTS were NOT CONTAMINATED

after experiment. Only three assays (Peroxidase, Alamar Blue, and Protease Assays) were done in this

experiment due to lack of reagents, however, the other four assays procedure were also written and

discussed.

Alkaline Phosphodiesterase (APDE) Assay

Microfuge tubes should be labeled according to cell fractions (C, N, S, M) and blank. 100ul

Buffer D should be added to each microfuge tubes and 50ul Substrade D should also be added to each

tubes. Then 100ul of right fractions should then be added to the tubes and PBS to the blank. The starting

time for the reaction must be noted. The tubes must be incubated in 37°C for 30 minutes and color change

should be observed.

Peroxidase Assay

Microfuge tubes were labeled according to the cell fractions (C, N, S, M) and a blank. Fifty (50)

ul of the fractions were pipetted into the proper labeled tubes and PBS was added for the blank. Then 50ul

of tetramethyl benzidine (TMB) was added to each tube and the tubes were incubated at room

temperature for about 15 minutes and color changes were observed.

Acid Phosphatase (ACP) Assay

Microfuge tubes must be labeled according to the cell fractions (C, N, S, M) and a blank. One

hundred (100) ul of Buffer C should be added on the tubes as well as 50ul Substrate C or Substrate D.

1:10 dilution of C, N, S, M should be prepared by adding 100ul of fractions and 100ul of ice-cold PBS,

then 100ul of the 1:10 dilution of the appropriate fraction must then be added to the microfuge tubes.

Then it must be incubated at 37°C for 30 minutes and color change should be observed.

Alamar Blue Assay for Mitochondrial Reductases

Fifty (50) ul of each cell fractions (C, N, S, M) were pipetted into diferrent microfuge tubes and

50ul of PBS to a blank. It was then carefully added 5ul of Alamar Blue and then incubated for 30 minutes

at 37°C. Color changes were observed.

Protease Activity Assay

Mastermix was prepared first, and this mix was good for 6 separate reactions. This was done

through mixing the reagents below (Table 1):

Table 1. Preparation of the Mastermix for Protease Activity Assay

Volume (μl) x 6 Total (μl)

PBS 44.50 (given) x 6 267.00Albumin 0.50 x 6 3.00Flamingo Pink

5.00 x 6 30.00

Total 50.00 x 6 300.00

The albumin was calculated via:

0.1 μgμl

x 50μl

10 μgμl

=0.50 μl

Where:0.1 μg

μl is the concentration of the albumin needed;

10 μgμl

is the concentration of the albumin sample;

50 μl is the total volume needed per reaction.

The Flamingo Pink was calculated via:

50 μl x1 X10 X

=5.00 μl

Where: 1X is the concentration of FP needed;

10X is the concentration of the original FP;

50μl is the total volume needed per reaction.

Fifty (50) ul of mastermix were pipetted into PCR tubes (C, N, S, M) and a blank, then 10ul of

appropriate fractions were added, but PBS was added for the blank. Then it was placed carefully in a

Rotor-Gene cycler that recorded the data of every 5°C increment from 25°C to 40°C with equilibrium of

3 minutes. Fluorescence level per fraction was observed via computer software.

DNAse Assay

Final concentration of 50pM pUC18 or lambda DNA, 2.5mM MgCl2, 1x SYBR green and cell

fraction to fill 20uL must be pipetted to a 0.2mL PCR tubes. These tubes should be placed in a Rotor-

Gene 3000 cycler and results will be recorded for every 5°C increment from 25°C to 40°C with

equilibrium of 3 minutes and fluorescence level per fraction then now be observed via computer software.

RESULTS

The three assays results were:

Table 2. Three assays showing color observed after reaction with positive or negative results of the fractions tested

Fraction Alamar Blue Assay Peroxidase Assay Protease Assay , 60th cycle (uv)Crude Cloudy red (+) Brownish clear (-) 0.02Nuclei Cloudy blue (-) Yellowish clear (-) -0.015Soluble Transparent blue (-) Transparent light blue

(+)0.07

Microsome Violet-ish red (+) Transparent clear (-) 0.02Blank Transparent blue (-) Transparent clear (-) 0

The crude and the microsome change its color from blue to red (Figure 1) after adding Alamar

Blue reagent in about 10 minutes. In peroxidase assay, only the soluble fraction changed in color where it

turned to transparent light blue color. In protease assay, the fluorescence levels of the fractions were

recorded having soluble as the highest and nuclei has the lowest (Figure 2).

Figure 1. Alamar Blue Assay shows that the crude and the microsome has a positive result where color changes were observed

Crude Nuclei Soluble Microsome

Figure 2. Protease Assay showing the result read from the Rotor-Gene cycler via computer softwareDISCUSSION

In Alkaline Phosphodiesterase Assay, Substrate D was consist of Para-nitrophenol thymidine 5’

monophosphate and the cleavage of thymidine 5’ monophosphate generates para-nitrohenol, a yellow

compound, that when done, it should be practically show a positive result in microsome fraction because

this detects the activity of the alkaline phosphodiesterase enzyme in the vacuoles (Oyong, 2012).

Microsome fraction was composed of membrane bound organelles from Endoplasmic Reticulum that

account for the half of all cell membranes (Cooper, 2000).

Peroxidase Assay tests the presence of the peroxidase enzyme activity in peroxisomes which

detoxifies the cells against free radicals formed mainly from generated H2O2. H2O2 was converted into

water (H2O) and O2 by peroxisomes (Figure 3). The generated O2 in turn oxidizes reduced TMB which

blue appearance was observed (Oyong, 2012) in soluble fraction (Table 2) which should be in the

microsome fraction. This happened maybe because there was not enough organelles amount in

microsome because it was diluted four times than the other fractions. Or maybe the peroxisomes were

included in soluble fraction because it was located originally on cytoplasm of the cell.

Figure 3. Conversion of H2O2 to H2O and O2 and produces blue color change after the reaction of TMB and O2 (Liu S. et. al., 2012)

Crude

Nuclei

Soluble

Microsome

Blank

Acid Phosphatase Assay was an assay that was used to trace the activity of enzymes in

lysosomes. Yellow compound was produced when the phosphate group was cleaved and the para-

nitrophenol was formed (Figure 4). This happened when the para-nitrophenol phosphate (pNPP) was

added to the fractions (Oyong, 2012). This usually has a positive result in microsome fraction because

lysosomes were membrane bound organelles.

Figure 4. The formation of the para-nitrophenol when the phosphate group was cleaved and transforming to a yellow compound after having a reaction with the Buffer C (G Biosciences, 2013).

Alamar Blue Assay showed a positive result in microsome and crude with a red color appearance.

This was detected by reducing blue resazurin or the Alamar Blue into red resofurin as shown in Figure 5

(Oyong, 2012). This tells that the mitochondrial activity was present in the microsome and crude. In

crude, there was a positive result because mechanical procedure was done when homogenizing the liver

cell tissue and this may cause the breakage of the cell membrane releasing the mitochondria and then

detected by the Alamar blue. It was expected that the microsome will have a positive result since

mitochondria were membrane bound organelles like the peroxisome and lysosomes.

Figure 5. Mitochondrial activity was observed as the blue resazurin or the Alamar Blue was reduced into red resofurin (AbD Serotec, u.d.).

Protease Activity Assay was used to determine if proteins were present in the different fractions.

There was presence of fluorescence as the Flamingo pink denaturize the proteins (Figure 6) and

proteolytic products binds with it (Oyong, 2012). As expected, soluble fraction has the highest

fluorescence level with 0.07 uv (shown in Table 2 and Figure 2). Proteins were found in soluble because

protein synthesis happens outside the nucleus of the cell (Hardin, 2012). Followed by the crude and the

microsome fractions were both has 0.2 uv at the end of the 60 th cycle. Like the Alamar Blue Assay,

probably the crude has proteins because of the ruptured cell membrane while microsome has also a result

because mitochondria have DNA that also encodes for proteins. The nuclei has a negative result (-0.015

uv) maybe because of nucleus does not have proteins in it, purely nucleic acids only.

Figure 6. Presence of fluorescence as the Flamingo pink binds to the proteolytic products

DNAse Assay was used when detecting DNAses which were known to be one of the key

enzymes released during the apoptosis and these damage the DNA (Oyong, 2012). When added with

SYBR green, this attaches to the end of the DNA fragments then produced a green fluorescence (Figure

7) which the fluorescence were read via computer software and this fluorescence can be found in the

nuclei fraction.

50 pM pUC18, + 2.5mM Mg Cl2 + 1X SYBR green

Figure 7. Presence of fluorescence as the SYBR green binds to the ends of DNA fragments and no fluorescence if there was no damaged DNAs (Integrated DNA Technologies, u.d.)

CONCLUSION AND RECOMMENDATION

Almost all the results were expected as the Alamar Blue, positive in microsome as well as crude,

the protease assay, with the highest fluorescence level among the fractions, except from the peroxidase

assay because soluble has the positive result and negative to microsome. However this may be acceptable

since the peroxisomes might have been included because of its location and mechanical work was done

for homogenization. For the following experiments, possible homogenization should be done using other

different techniques other than using blender to have more accurate results and to make sure that the

membranes will not be ruptured and the organelles will not be mixed up.

REFERENCES

AbD Serotec. Unknown Date. Alamar Blue. Retrieved from http://www.abdserotec.com/alamarblue-assay-cell-proliferation-cytotoxicity.html

Cooper G. 2000. The Endoplasmic Reticulum. Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK9889/

G Biosciences. 2013. Phosphatase Assay. Retrieved from http://www.gbiosciences.com/ResearchProducts/PhosphataseAssay-desc.aspx

Hardin J, et.al. 2012. Becker’s World of the Cell.Integrated DNA Technologies. Unkown Date. Retrieved from www.idtdna.comLiu S. et. al. 2012 Fast and Sensitive Colorimetric Detection of H2O2 and Glucose: A Strategy Based on

Polyoxometalate Clusters. Retrieved from http://onlinelibrary.wiley.com/doi/10.1002/cplu.201200051/abstract

Oyong G. 2012. Basic and Advance Techniques in Cell and Molecular Biology. URCO.Sittampalam GS et. al. 2004. Assay Guidance Manual. Retrieved from

http://www.ncbi.nlm.nih.gov/books/NBK92006/figure/proteaserev.F1/?report=objectonly