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Practical 4 experiment
Concept of Biology (BIO400)
Universiti Teknologi MARA
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BIO462Preview text
COURSE CODE BIO462 – BIOCHEMISTRY
GROUP AS2571A
EXPERIMENT EXPERIMENT 4:
ENZYMOLOGY
PART 2: DETERMINATION OF ENZYME’S ACTION
LECTURER’S NAME DR HISYAM BIN ABDUL HAMID
STUDENT’S NAME NUR DANIA BINTI MOHD YUSOFF
(2022484662)
DATE OF EXPERIMENT /12/
DATE OF SUBMISSION /12/
LABORATORY REPORT
INTRODUCTION
Enzymes are a class of protein with function as a biological catalyst. They speed up the metabolic reactions that occur in the body. Most enzymes are globular proteins.
Acetylcholinesterase (AChE) is an enzyme that has indispensable role in synaptic transmission by catalysing the hydrolysis of the acetylcholine neurotransmitter (Paraoanu and Layer, 2007). This enzyme can be found in mammals as well as insects: AChE belongs to the type ' B ' esterase which is susceptible to insecticides (Aldrige, 1953).
Similar to butyrilcholinesterase (BuChE), AChE has a molecular weight of 50,000 to 500,000 dalton. This enzyme can be found in so called 'globular' and asymmetric forms and exist as polymers of catalytic subunits. It exists in 12 subunits and the molecular weight varies depending on the number of subunit as well as the bonding with carbohydrate, glycolipids, and collagen (Kennedy, 1991).
ACHE hydrolyses acetylcholine (ACh) and is inhibited by relatively low concentration of organophosphorus (OP) (10 M). AChE has the same hydrolytic reaction or metabolism towards OPS. OPS mimic the gross molecular shape of the natural substrates of ACh (Matsumura, 1985 Hence it is a good inhibitor for AChE.
AChE has two active sites, esterase site and ionic site (Wilson and Bergman, 1950; Matsumura, 1985). Within ChE, the active site serine is phosphorylated by OPs. When phosphorylation occurs, OP will destroy the enzyme activity by reacting with a specific serine within the catalytic centre of toe enzyme to produce O, dialkyl phosphoserine (Aldridge and Reiner, 1972: Fest and Schmidt, 1978; Eto, 1979; Mineau, 1991). The enzyme is unable to hydrolyse choline esters and this will lead to inhibition of AChE (Kennedy, 1991).
Materials
Spectrophotometer
Acetylcholinesterase ACTHI (Acetylthiocholine iodide)
DTNB (Dithiobis Nitrobenzoic Acid)
Phosphate buffer (pH 7)
R = (Absorbance/t) x (60x1000/ε) x 1/c
R= specific activity of ACHE
t= incubation time (30 minutes)
ε = extinction coefficient (1 X10 M cm)
c = protein concentration the unit for the specific enzyme activity for AChE is mM / minute / ug protein
RESULTS
a. Table 1. Absorbance for BSA Concentration of BSA
OD
(Replicate 1)
OD
(Replicate 2)
OD
(Replicate 3)
Mean +- S. D 0 mg/ml 0 0 0 0. 0 mg/ml 0 0 0 0. 0 mg/ml 0 0 0 0. 0 mg/ml 0 0 0 0. 0 mg/ml 0 0 0 0. 0 mg/ml 0 0 0 0. 1 mg/ml 0 0 0 0.
b. Table 2. Enzyme activity OD (Replicate 1)
OD
(Replicate 2)
OD
(Replicate 3)
Mean +- S. D Acetylcholinesterase 1 1 1 1.
0 blank
Graph 1: Standard Curve of BSA
CALCULATIONS
R = (Absorbance/t) x (60x1000/ε) x 1/c
R= specific activity of AChE
t= incubation time (30 minutes) ε = extinction coefficient (1 x 104 M-1 cm-1)
c= protein concentration
R = (1 nm/ 30 min) x (60x1000/1 x 10 4 ) x 1/ 0. = 0 x 1/ 0. = 0.
y = 0 + 0.
1 = 0 + 0.
X = (1 – 0) / 0.
= 67 mg/ml
00 0 0 0 0 1 1.
0 0
f(x) = 0 x + 0 0.
OD against Concentration of BSA (mg/ml)
Concentration of BSA (mg/ml)
OD
caused by weakly soluble substances, soiling, dust or air bubbles can at least be reduced by careful handling. Scattering is also can be reduced if only the observed component (substrate or product) produces the signal for instance an absorption while the other components show no signal (no absorption) in the observed range, so that the reaction starts at zero and any change in the signal indicates the ongoing reaction.
Systematic error may occur due to the incorrect experimental steps that cause the result to be skewed in the same direction each time. These are usually due to a procedure that fails to control for outside variables. For instance, the freshness of the enzymes might be disrupted and the enzyme source is decreased in amount which could lead to less functional enzyme to be present for the experiment to be occurred for the next several days. The poor standard curve may occur due to the inaccurate pipette and as the corrective action make sure to check and calibrate the pipettes. The low reading of OD value may also occur because of the improper sample storage, improper sample collection and preparation and the low quantity of the analyte in the sample. In order to prevent this to occur, store the sample properly and use the fresh sample, take proper sample collection and preparation method, and use a new sample and repeat assay.
CONCLUSION
In conclusions, the protein concentration of AChE can be determined by a standard curve constructed, the enzyme activity and the specific activity of the AChE can also be determined in the experiment by a spectrophotometer. The protein concentration of AChE based on the graph is 67, while the enzyme activity resulted with the average value of the absorbance is 1 nm. Lastly, the specific activity of the AChE is determined by a formula from Ellman and resulted with value 0.
Practical 4 experiment
Course: Concept of Biology (BIO400)
University: Universiti Teknologi MARA
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