TITLE

LAB REPORT: QUANTITATIVE DETERMINATION OF PROTEASE

ACTIVITY

INTRODUCTION
Determining the quantity and pace of protein degradation by proteases in a variety of
materials, including physiological fluids, tissues, cells, and dietary items, is the main
goal of quantitative protease activity measurement. Peptide bond hydrolysis in
proteins is catalyzed by proteases, which are enzymes. Numerous physiological
functions, including blood coagulation, immunological responses, cell signaling, and
digression, depend on protease activity ( Bach et al., 2012, Rao et al., 1998, Sumantha
et al., 2006). But disorders including cancer, inflammation, and neurodegeneration
can also be brought on by excessive or uncontrolled protease activity (Judith S. Bond,
2019). Protease activity must thus be measured and continuously observed in a variety
of materials, including biological fluids, tissues, cells, and dietary items.
Understanding the function, regulation, and significance of proteases in health and
disease can be aided by quantifying their activity.
Measuring the quantity or rate of protease-mediated protein breakdown
activity in a sample is known as quantitative analysis of protease activity. Depending
on the kind, source, and specificity of the protease as well as the substrate's
characteristics and accessibility, many approaches and procedures can be used to
measure protease activity.

According to Bill S. Hosker et al. (2018), spectrophotometric methods for identifying

protease activity include colorimetric or fluorogenic substrates that release a
chromophore or fluorophore following proteolytic cleavage. Protease activity is
directly correlated with the amount of colour or fluorescence generated in the sample.
According to Toni A. Trumbo, Emeric Schultz, Michael G. Borland, Michael Eugene
Pugh et al. (2013), the theory takes into account the kinetics of enzyme-substrate
reactions, the Beer-Lambert law, emission and excitation spectra of fluorescence, and
factors that affect absorbance and fluorescence intensity, such as pH, temperature,
concentration, and interference. These techniques are straightforward, sensitive, and
appropriate for high-throughput screening; nevertheless, they may be hampered by
other compounds present in the environmental sample.
MATERIAL
Protease
Azocasein
10% Trichloroacetic acid (TCA)
0.1 M Tris- HC pH 9
Microcentrifuge tube 2mL
Cuvette
Spectrophotometer
Microcentrifuge
Micropipette Pipettes (100uL, 1mL)
Water bath shaker
1M NaOH

METHOD
In 0.1 M Tris-HCl (pH 9) buffer, 0.9 ml of 0.5% Azocasein was added. After that,
three separate microcentrifuges were filled with 0.5% Azocasein (0.9 ml) and an
enzyme sample (0.1 ml), and they were all placed in an incubator set to 70 °C for 30
minutes. All three microcentrifuges were filled to capacity with 10% Trichloroacetic
acid (TCA) (1:1) after incubation, and 10 minutes were spent at room temperature.
Three other microcentrifuges were filled with 0.1 ml of sample at the same time, and
they were incubated for 30 minutes at 70 °C. Following incubation, each of the three
microcentrifuges received 0.9 ml of 0.5% azocasein and an equivalent volume of 10%
Trichloroacetic acid (TCA) (1:1), which were then allowed to sit at room temperature
for ten minutes. Next, for ten minutes, all six microcentrifuges were centrifuged at
13,000 rpm. Finally, 1 ml of sodium hydroxide (NaOH) was combined with 1 ml of
supernatant from each microcentrifuge, and the absorbance at 450 nm was measured.
Table 1 is a tabulation of all the data.
RESULT
Table 1: Protease activity

ABS PROTEASE
AVERAGE
VALUE ACTIVITY
VALUE
(450nm) (U/mL)
Control
1 0.043
2 0.028 0.027±0.013
3 0.010
Sample 94.67
1 0.298
2 0.331 0.311±0.014
3 0.304

0.311− 0.027
Protease Activity =
0.001× 30 ×0.1 ml

= 94.67
DISCUSSION (1)

The process of measuring the concentration of protease enzymes in a sample or

control in order to evaluate their enzymatic activity is known as quantitative
determination of protease activity. Proteases effectively perform a common chemical
reaction called the hydrolysis of peptide bonds. While specific proteases perform
slightly different actions, most proteolytic enzymes break down α-peptide bonds that
bind naturally occurring amino acids. More than merely isolated catalytic enzymes
searching for substrates to hydrolyze are known as proteolytic enzymes.
Consequently, several proteases link a diverse array of specialised functional modules
or domains to their catalytic domains, which impart substrate selectivity, control
cellular localization, modify kinetic properties, and change susceptibility to
endogenous inhibitors.
Tricholoroacetic acid (TCA) is an incredibly effective protein-precipitating
agent when it comes to the precipitation of proteins from diluted solutions.
Trichloroacetic acid (TCA) can be used to precipitate proteins in order to concentrate
protein samples or remove contaminants. The first crucial stage in assessing the
overall calibre of the proteome output is sample preparation for proteomic analysis.
The data shows that while the protease is inconsistent for a number of reasons,
the sample is growing. Utilising trichloroacetic acid (TCA) in reaction mixes
containing proteases might lead to fluctuations or inconsistencies in the activity of the
enzyme due to differences in protease activity. The pH variations can have an impact
on the protease activity. This is a result of the potent acidity and protein denaturant
found in trichloroacetic acid (TCA). Every protease enzyme has an ideal pH range
whereby its catalytic reaction rate reaches its maximum. The reaction rate will
decrease with a small departure from the ideal pH value.
CONCLUSION (1)
The assessment of protease activity qualitatively is an essential component of
biological and biochemical research. Assays for qualitative protease activity can help
determine if protease activity is present in a sample or not. Through this experiment,
we are able to determine each sample's ABS value at 450 nm with success. Thus, in
this experiment, the amount of azocaseinase activity that causes changes in
absorbance of 0.001 per minute at 450 nm at 80˚C is defined as one unit (U) of
azocaseinase activity.
REFERENCES

JOURNAL/ARTICLE

Dakshinamurthy Rajalingam, Charles Loftis, Jiashou J Xu, and Thallapuranam

Krishnaswamy S Kumar. (2009 May 16). Trichloroacetic acid-induced protein
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intermediate. Journal of Biotechnology Information. 18(5): 980–993.

J Biol Chem. (2008 Nov 7). Proteases: Multifunctional Enzymes in Life and Disease.
Journal of Biological Chemistry. 283(45): 30433–30437.

Laura Koontz. (2014). Trichloroacetic acid (TCA) precipitation. Journal of

Biotechnology Information. (10.1016/B978-0-12-420119-4.00001-).

BOOK

P. Novák, V. Havlíček. (2016). The handbook of Protein Extraction and Precipitation.

2nd ed. Proteomic Profiling and Analytical Chemistry. Pages 51-62.

WEBSITE

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Production and application in obtaining protein hydrolysates. Food Research
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and disease. Journal of Biological Chemistry.
https://doi.org/10.1074/jbc.tm118.004156
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Applied spectrophotometry: Analysis of a biochemical mixture. Biochemistry and
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