Exp 4 Enzymology Part 2 Determination of Enzyme's Activity

BIO 462 EXPERIMENT 4 ENZYMOLOGY PART 2: DETERMINATION ACTIVITY

NAME: NURFADHILAH BINTI JAAFAR STUDENT ID: 2016675256 PROGRAM: AS 246 GROUP: 4B DATE OF THE EXPERIMENT: 12 TH APRIL 2017 DATE OF SUBMISSION: 19 TH APRIL 2017 LECTURER: MADAM AZANI BINTI SALEH

OF ENZYME’S

PROCEDURE -

Refer to Biochemistry Laboratory Manual page 25 until 29 -

RESULTS 1. Absorbance for BSA

Concentration

OD

OD

OD

Mean ± SD

of BSA

(replicate 1)

(replicate 2)

(replicate 3)

0.2 mg/ml

1.345

1.340

1.346

1.304± 0.003

0.4 mg/ ml

1.437

1.444

1.435

1.439±0.004

0.6 mg/ml

1.433

1.425

1.451

1.436± 0.013

0.8 mg/ml

1.495

1.493

1.494

1.494± 0.001

1.0 mg /ml

1.845

1.840

1.833

1.835± 0.006

Test tube 6

1.149

1.150

1.151

1.150 ± 0.001

2. Enzyme Activity

Acetylcholinesterase

OD

OD

OD

(replicate 1)

(replicate 2)

(replicate 3)

0.831

0.833

0.833

3. Specific Activity of AChE R = Absorbance/t x 60x 1000/ £ x 1/c R= specific activity of AChE t= incubation time (30 minutes) £ = extinction coefficient (1.96 x 10 4 M-1 cm-1) c= protein concentration

R = 0.832 nm/ 30 min x 60x1000/ 1.34 x 10 4 x 1/ 0.27 = 0.124 x 1/ 0.27 = 0.459

Mean ± SD

0.832 ± 0.0012

DISCUSSIONS In the laboratory we conducted the experiment to determine the protein concentration of  AchE from the standard curve, the enzyme activity and the specific activity of AChE. Enzymes are specialized protein that serves as biocatalyst for nearly all the chemical reactions on the living organisms. The primary cholinesterase in the body is Acethylcholinesterase (AChE). It is an enzyme that catalyzes the breakdown of acetylcholine and some of other choline esters that function as neurotransmitters. It is the primary target of inhibition by organophosphorus compounds such as nerve agents and pesticides. Several chemical and techniques can be used for the detection of AChE and in this experiment we determine the AChE spectrophotometrically. From the standard curve plotted we identified the protein concentration of AChE with value of 0.47 mg/mL.

The standard curve

absorbance against the concentration of is directly proportional and was plotted on the graph sheet. It shows the best fit line, however a linear trend could not be identified that may be due to some possible causes. The enzyme activity for Acetylcholinesterase is determined by the absorbance of the spectrophotometer with average value of 0.832 nm. The specific activity of AChE is determined by the formula shown on the data sheet. From the calculation it shows that the specific activity for AChE is 0.459. The possible errors that may cause the inaccurate results might be due to the instrumental error of the spectrophotometer which was defective or not sensitive enough for the measurement of the absorbance. Scattering can have various origins, some due to the instability of the instruments or measurements in turbid solutions like cell homogenates, while others, like contaminations, turbidity 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 actually 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 disrupt 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. CONCLUSIONS 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 0.47 while the enzyme activity resulted with the average value of the absorbance is 0.832 nm. Lastly, the specific activity of the AChE is determined by a formula from Ellman and resulted with value 0.459.