The Effect of Salicin on Enzyme Elastase

By: Sumona Banerjee

Niles North High School

GRAPHS

STATISTIC
S
Absorbance vs. Concentration of Salicin

Neg Control

Elastatinal

500uM

250uM

1.0818182

0.872727273

0.5545455

0.7909091

2.2545455

1.827272727

0.6 0.8727273

1.6681818

1.349999798

0.5772727 0.8318182

100uM

50uM

10uM

1uM

0.6181818 0.6909091

0.727273

0.95091

1.0636364 0.6454545

1.190909

1.690909

Trial 1

Trial 2

Average
0.8409091

0.6681818

0.959091

1.320909

Absorbance vs. Substrate Concentration

Substrate
Concentration
.00173 (A)
.00186 (B)
.00199 (C)
.00213 (D)

Blank

Control

Trial 1 with
Salicin

Trial 2 with
Salicin

Trial 3 with
Salicin

Elastatinal Trial

0.52818182

0.39090909

0.509090909

0.509090909

0.518181818

0.518181818

0.536363636

0.450505051

0.5363636364

0.5354545455

0.5181818182

0.5354545454

0.554545455

0.495959596

0.5545454545

0.5436363636

0.5545454545

0.5323232323

0.681818182

0.532323232

0.6747474747

0.6818181818

0.6818181818

0.6272727273

DISCUSSION

DATA ANALYSIS
For determining the most effective concentration of salicin on constant concentrations of salicin,
it was necessary to compare them to two control groups, the negative control, which contained
no inhibitor, and the elastatinal trial. During trial one, 500 uM showed the least absorbance of
color, while 1 uM showed the most.
50 uM absorbed less than 100 uM, 250 uM, 10 uM, and 1 uM but it absorbed similarly to 500
uM.
1 uM showed the most similarity to the elastatinal trial. There was an increase in absorbance
from 500 uM to 1 uM, except for the data for 50 uM.
Due to 1 uM of salicin having a similar absorbency as elastase inhibiting elastatinal, it was
chosen as the most effective concentration.
After establishing 1uM to be the most effective concentration of salicin, it was used to determine
its efficiency in inhibiting elastase from breaking down different concentrations of the substrate.
For .00173 M (A) concentration of the substrate the control showed a .39091 OD\min
absorbency and the blank showed a .52818 absorbency. The average absorbency with
salicin was .51212 and for elastatinal it was .51818. Both salicin and elastatinal absorbed more
than the control and similarly to the blank. On average, salicin absorbed about .00970 less
than the blank and .17576 more than the control. Elastatinal absorbed .00364 less than the
blank and .12424 more than the control. The average difference in absorbance between
salicin and elastatinal was .00606 OD\min
For .00186 M (B) concentration of the substrate the control showed a .53636 OD\min
absorbency and the blank showed a .45051 absorbency. Wells treated with salicin showed
an average absorbency of .52999 and the absorbency with elastatinal was .53545. For this
concentration too, both salicin and elastatinal absorbed more than the control and similarly
to the blank. Wells with elastatinal absorbed .08495 more than the control and .00009 less
than the blank. On average, wells with salicin absorbed .07798 more than the control and .
00609 less than the blank. The average difference in absorbance between salicin and
elastatinal was .00546 OD\min.
For .00199 M (C) concentration of the substrate, the control absorbed .49596 OD\min and
the blank absorbed .55455. For this concentration, as well, both salicin and elastatinal
absorbed more than the control and similarly to the blank. Wells treated with salicin
absorbed .55091 OD\min on average, and with elastatinal .53232 was absorbed. Wells with
elastatinal absorbed .03636 more than the control and .02222 less than the blank. On
average, wells with salicin absorbed .05494 more than the control and .01091 less than the
blank. The average difference in absorbance between salicin and elastatinal was .01859
OD\min.
Lastly, for .00213 M (D) concentration of the substrate, the control absorbed .53232 OD\min
and the blank absorbed .68182. On average, wells treated with salicin absorbed .67946
OD\min and wells with elastatinal absorbed .62727. For this concentration, as well, both
salicin and elastatinal absorbed more than the control and similarly to the blank. Wells
treated with salicin absorbed, on average, .14713 more than the control and .00236 less than
the blank Wells with elastatinal absorbed .09495 more than the control and .05455 less than
the blank. The average difference in absorbance between salicin and elastatinal was .05219
OD\min.

From the data collected during this experiment, several things can be
concluded. In order to determine the efficiency of salicin inhibiting elastase,
it was first necessary to determine which concentration of salicin was the
most effective, if any, in inhibiting elastase.
Since the wells with 500 uM and 50 uM of salicin showed about the same
absorbance, it was not clear if the inconsistency was due to because the
concentrations from 500 to 50 uM of salicin were denaturing the enzyme.
Nevertheless, the results also showed that the absorbance of 1 uM and the
control inhibitor, elastatinal, were about the same. This meant that salicin of
1M was inhibiting elastase as much as elastatinal was. Since elastatinal has
already been scientifically proven as elastase inhibiting, the 1 uM of salicin
was chosen to be most effective, so it was used to test the efficiency of
salicin on different concentrations of the substrate.
For this test, the data showed that salicin was able to inhibit elastase as well
as elastatinal was. The blanks only contained the substrate and buffer with
no enzyme. Thus, the data for the blanks showed how the substrate was
before it was broken down by the enzyme. The control wells contained the
enzyme, substrate and buffer. Thus, this data showed how the substrate
was after being broken down. A lower absorbance means that theres more
substrate in the well. More substrate can be explained by the enzyme
breaking down the substrate.
Wells treated with salicin absorbed similarly to the blank. This meant that
salicin was able to maintain the substrates concentration for all four
concentrations tested. The data shows that salicin was able to inhibit
elastase from breaking down the substrate and consequently decreasing
the amount of absorbance.
Compared to elastatinal, salicin inhibited elastase better for the substrate
concentrations C and D, the higher two concentrations. On average,
however, salicin and elastatinal inhibited elastase very similarly.

EXPERIMENTAL ERROR
During this experiment, there were several areas that error could have
occurred in. Since microliters is such a small unit, it was hard to see and
verify if the correct amounts were going into each well. Measurements
such as .4 uL were difficult to verify if they were properly transferring from
the pipet tip to the well. If these small measurements were not transferring
correctly, then this may have caused an overall difference in the amount of
absorbance. For example, if .1 uL of the substrate did not transfer properly
from the pipet tip to the well, then a lower absorbance would have
showed up for that well.
To avoid this error in the future, the use of a pipet that says how much
it has pipetted and released would be helpful to verify the correct
transfer of material.
Also, when measuring 0.028 grams of salcin for the stock solution, the
actual measurements were a little above or below 0.028 (ie. 0.0289 or
0.0285). This may have caused the solutions to be either a higher or lower
molar concentration than indicated. In addition, the salicin solutions were
made 8 hours prior to testing.
Although, it is not known for sure, this 8 hour waiting period may have
diminished the effects of salicin. Thus, if the solutions were made
immediately prior to testing, then the effects may have been a bit stronger.
In order to eliminate this error in the future, solutions should be made
immediately before testing to make sure that the effects of salicin
arent being diminished by a waiting period.
Another source of error may have occurred when the vials in the
Neutrophil Elastase Colorimetric Drug Discovery Kit were being defrosted.
For each trial, there wasnt a set time indicated that the components would
defrost until. The components were placed in the incubator at 37oC and
they were checked on periodically to see if they felt warm enough to test
with. Consequently, for each trial the amount of defrosting time may have
been different. This may have resulted in the components being more
active in some trials than others.
In order to make sure that all the components are functioning at the
same level for all trials, in the future it would be necessary to indicate
how much time the components should defrost for.