Introductions: Enzymes are proteins that speed up the rate of reactions that would otherwise

happen more slowly. They are the proteins that acts as biological catalyst. Catalase are the
antioxidant enzymes that catalyze the conversion of H2O2 (hydrogen peroxide) water and
molecular oxygen. The balanced reaction for the breakdown of hydrogen peroxide is 2 H2O2 →
2 H2O + O2. This reaction is important to cells because hydrogen peroxide (H2O2) is produced as
a byproduct of many normal cellular reactions. If the cells did not break down the hydrogen
peroxide, they would build up toxic levels of this chemical and potentially die. Catalyze is
located in all major sites of H2O2 production in the cellular environment (such as peroxisomes,
mitochondria, cytosol and chloroplast) of higher plants. Superoxide dismutase (SOD) and
catalase are enzymes that protect cells from radical attack. These are the only antioxidant
enzyme that scavenges the superoxide anion by converting this free radical to oxygen and
hydrogen peroxide (Fei Wang, Yu-Qing Zhang, 2015). All aerobic organisms containing
cytochrome systems were found to contain both superoxide dismutase and catalase.
Aerotolerant anaerobes, which survive exposure to air and metabolize oxygen to a limited
extent but do not contain cytochrome systems, were found to be devoid of catalase activity but
did exhibit superoxide dismutase activity (Joe M. McCord, Bernard B. Keele Jr., and Irwin
Fridovich). In this report, we will look over the levels of Catalase in two Bell Pepper samples;
Green Pepper and Yellow Pepper. Green and Yellow are the same organisms. In these species
what matters the most is ripening, it is characyerised as by important visual and metabolic
changes. Green Pepper is the less ripened and matured than the Yellow pepper (Nichols and
Rhoades, 2019). Pepper’s are mainly characterized by its high vitamin C and A content, it is one
of the most consumed vegetables in the world. During fruit ripening, chlorophyll breakdown
and synthesis of new carotenoids and anthocyanins take place in an intense metabolism
characterized by the emission of volatile organic compounds associated with respiration,
change to acidic taste, pH and astringency changes, pectin formation, new protein synthesis,
cleavage of existing proteins, conversion of starch to simple sugars, flavor accumulation, and
cell wall softening, among other events (Palma, J.M.; Sevilla, F.; Jiménez, A.; Del Río, L.A.;
Corpas, F.J.; Álvarez de Morales, P.; Camejo, D.M. Physiology of pepper fruit and the
metabolism of antioxidants: Chloroplasts, mitochondria and peroxisomes. Ann. Bot. 2015).
Based upon this research, I hypothesized that Younger Green Pepper contain a higher
concentration of catalase than that of the more mature yellow pepper.

M+M: To measure the reaction rates of Catalase standards, we will use the floating disc method.
In this lab, we will be using small discs (6mm in diameter) of filter paper. We will briefly soak the
filter paper in either a standard solution or an experimental solution, then we will carefully drop
the disc into a beaker containing a 1% solution of H2O2 (hydrogen peroxide). The catalase
absorbed by the filter paper disc will immediately begin to convert the H 2O2 molecules into
water and oxygen and this will allow the paper disc to rise. The higher and faster the rate of
reaction, the less time it will take for the filter paper to rise.
In our experiment we will use a sample that contains no catalase as our negative control and
Instead of a single positive control we will use multiple catalase samples of increasing amounts
of enzyme activity. Starting with a purified catalase stock of 400U/ml, four experiment standards
varying of concentrations are created by diluting catalase stock with pH 7 buffer. The Final
Catalases concentrations used are 0U/ml,20U/ml, 200U/ml, and 400U/ml in Table 1. And For the
concentration of Yellow Pepper Extracts (YPE) and Green Pepper Extracts (GPE) mentioned in the
study was prepared by the extraction of plant catalase by homogenizing 10 g of yellow or green
bell pepper with 100 ml distilled water for 1 minute and filtering it through cheesecloth and then
into several 1.5 ml flip-cap tubes.

To perform the rest of the experiment, first we will start by punching out 18-20 discs from the
filter paper and cut it in a small piece as the server to the blotter for removing excess solution
from the soaked discs. With the help of forceps, a single filter paper discs is then dipped half-way
into a 100 ml of H2O2 and it should completely sink reaction condition and capillary action is used
to allow the reaction to cover the disc. Now, to measure the rate of reaction for each condition
we need a stopwatch. A stopwatch will be used to time the moment as soon as the disk touches
the surface of 1% H2O2. It will rise to the surface, when the bubbles of O2 accumulate on the disk
in sufficient quantity. And we will stop the watch and record the time in seconds to the nearest
0.1 second in our data table, as soon as the disk reaches the surface of 1% H2O2 and record the
time in seconds to the nearest 0.1 second in your data table.

For each experimental condition this reaction for controls will be performed repeat this
measurement 3 times and average, standard deviation and standard errors will be calculated for
each data set which is (Table 1 and 2). If a reaction does not occur within 3 minutes (180 seconds),
then the reaction condition will be described as “No Reaction” and will be assigned the value of
180 seconds. Reaction times were measured in seconds and converted to rate (B/s) for data
analysis.

Results: After performing the Filter paper disc we will evaluate the Catalase activity, we see a
pattern here by which we can say that the reaction rate increases as concentration increases
(Figure 1). The average rate of reaction for the 0U/mL condition was 180 B/s. The average rate
of reaction for the 20U/mL condition was 86.33333333 B/s. The average rate of reaction for the
200U/mL condition was 28 B/s. Finally, the average rate of reaction for the 400U/mL condition
15 B/s. All Catalase Standard data reported in this study, including statistical analysis, can be
found in Table 1.

We then measured the rate of reaction for Green Pepper Extracts and Yellow Pepper Extracts
Samples. The average rate of reaction for the GPE was 30.66666667 B/s and the average rate of
reaction the YPE was 57.33333333 B/s. All Plant extract data reported in this study, including
statistical analysis, can be found in Table 2.
To calculate the number of Catalase units per gram for Yellow and Green Pepper Extracts, a
standard curve was constructed using Catalase Concentration which is Figure 1. The slope of the
trendline was calculated and used to find the approximate number of Catalase Units (Figure
1).The slope of the trendline was calculated and used to approximate the number of Catalase
Units per gram of tissue (Figure 1). We find that the GPE had an approximate concentration of
14825.440 U/g for GPE and an approximate concentration of 8312.336 U/g for YPE (Table 2).

Table 1. The Rate of Reaction of catalase Standards are examined in this study. The rate of
each concentration is used performed to measure the accuracy of our results, to construct the
Standard Curve shown in Figure 1.
Catalase Trial Trial Trial Average Standard Standard
Concentration 1 2 3 Deviation Error
(U/mL) (B/S) (B/S) (B/S)
0 180 180 180 180 0 0
20 92 83 84 86.33333333 4.932882862 2.466441431
200 25 31 28 28 3 1.5
400 12 14 19 15 3.605551275 1.802775638

Table 2. The Rate of Reaction of Green and Yellow Pepper Extracts. Final concentration of
Catalase in 1 gram of respective pepper extracts are reported.

Experimental Trial 1 Trial 2 Trial 3 Average Standard Standard

Samples (seconds (seconds (seconds Deviation Error
(U/mL) ) ) )
Green Pepper 33 28 31 30.6666666 2.51661147 1.77951304
Extract 7 8 2
Yellow Pepper 61 53 58 57.3333333 4.04145188 2.85773803
Extract 3 4 3
Bar Graph 1. This Graph shows the average of Ctalase Concentration with Time on y- axis and
Catalase concentrations on x-axis

Average of Catalase Concentrations

3
Time(s)

0 50 100 150 200 250 300 350 400 450

Catalase Concentration

Catalase Concentration (U/mL) Average

Average of Experimental Samples

Average

Experimental samples(U/mL)

0 1000 2000 3000 4000 5000 6000 7000 8000 9000

Series2 Series1

Bar Graph 2. This Graph shows the average of Experimental Samples.

 Rate of RXN Vs. Experimental Samples
0.1

y = 0.0207x - 0.022
0.08

0.06
Rate of RXN (1/s)

0.04

0.02

0
0 20 200 400

-0.02
Experimental Samples(U/mL)

Figure 1. Rate of RXN Vs. Experimental Samples produced from mean data in Table 1. As
concentration of Catalase increases, the rate of reaction also increases. Equation of the line and
Correlation coefficient are reported.

Discussion: In this report, we show that on average, Green Pepper Extracts has higher
concentration of Catalase than that of Yellow Pepper Extracts. This data also support the
hypothesis that Green Pepper Extracts will have higher concentration because of the following
like maturity in the pepper, ripening, chlorophyll, which also helps in photosynthesis. However,
support for this statement is that a Catalase activity in species like Solanum melongena and
Capsicum annuum has much higher catalase concentration in green than in red pepper fruits
and this was coincident with the lower amount of this enzyme in the red fruits, as shown by
immunoblot analysis carried out with an antibody against (Mateos et al., 2003)
Also, for more support the decrease of catalase in red fruits implies a lower capacity to
scavenge H2O2, thus promoting the lipid peroxidation already reported in ripe pepper fruits
(Marti et al., 2011) This is why the Younger Green Extracts have the highest concentration than
the Red ones.
Fei Wang, Yu-Qing Zhang, in Advances in Protein Chemistry and Structural Biology, 2015

Joe M. McCord, Bernard B. Keele, Irwin Fridovich, Proceedings of the National Academy of
Sciences May 1971

Martí MC Camejo D Vallejo F et al. . 2011. Influence of fruit ripening stage and harvest period on
the antioxidant content of sweet pepper cultivars.

Mateos RM León AM Sandalio LM Gómez M del Río LA Palma JM . 2003. Peroxisomes from
pepper fruits (Capsicum annuum L): purification, characterization and antioxidant activity.
Journal of Plant Physiology160: 1507–1516.

Nichols, Wade A, and Rhoades, Nicholas A. “Lab 1: Determination of Catalase Enzymatic Activity
in Plant Tissue.” BSC 197 Lab Manual, pp. 1–5.

Palma, J.M.; Sevilla, F.; Jiménez, A.; Del Río, L.A.; Corpas, F.J.; Álvarez de Morales, P.; Camejo,
D.M. Physiology of pepper fruit and the metabolism of antioxidants: Chloroplasts, mitochondria
and peroxisomes. Ann. Bot. 2015