William Qu

Thursday 8:30Am
March 1st 2012
Matt Ballinger

Lab #4
Enzyme
Kinetics

II. Objective: Our objective is to demonstrate the effect of different

quantities of lysosome( in samples 1-8 and in the HEW, load and carb
sample) on the absorbance of micrococcus bacteria. With this data we
sought to create a standard curve. With the standard curve we sought
to quantify the amount of lysozyme in our purified sample from our ion
exchange chromatography lab based on the lysozymes enzymatic
activity.
III. Procedure- See lab manual
IV.Results
Figures 1 and 2 are on the next page.
Table 1 is our slope vs lysozyme data set
Concentrat
ion
Slope
Samples
0
0.002
0
0.5
0.099
5
1
0.163
4
2
0.31
3
5
0.598
2
10
0.432
1
Figure 3: Note our sample 6 is missing because my lab partner and I
accidently discarded the piece of paper that the sample 6 data set was
on as it printed on another piece of paper and we thought we already
had all the data sets.
The figure shows a definitely relationship between lysozyme protein
concentration vs slope.

Slope vs lysozyme Concentration

0.8
0.6
Slope

0.4

Slope

0.2
0
0

10

12

Lysozyme concentration(mg/ml)

Table 2:
Sample

8X dil
HEW
HEW
Load
Carb 1

Dilution
factor
before
taking
100 ul

MeasureSlo
pe ( delta
Abs/Min at
450nm)
after 100ul> 3ml

Diluted
Lysozyme
mg/ml
before
taking
100ul

Undilute
Lysozyme
mg/ml

Ml of
sample

Mg lysozyme

Mg proteins
Of all types

28.8
0.0
21

917.58
731.25
6.375

48
8
1
1

-0.107
-0.004
-0.22

0.6
0.0
1.4

4.8
0.0
1.4

6
48
15

V. Questions
1) The total number was quite similar, as the amount of lysozyme in
HEW was 28.8mg, while the amount of lysozyme in carb 1 was 21 mg. I
ended up with 72.9% yield recovered in carb 1 from HEW.
21mg of lysozyme in Carb X 100% 72.9%
28.8mg of lysozyme in HEW
2) The purity for this would 329.5% purity from the carb 1 data in table
2 divided by the 3.10% purity from the HEW, which would be 106.3%
purity. This is clearly wrong. Since you cant have over 100% purity.
VI. Conclusions and Discussions
From figure #3, one can tell that there is a positive linear relationship
between lysozyme concentration and slope. The higher the

concentration of lysozyme the faster the degradation of micrococcus

bacteria. However there in figure 3 the 10mg/ml lysozyme
concentration did not kill off the bacteria as fast as the 5mg/ml solution
of lysozyme. This could arise because since the 10mg/ml solution of
lysozyme was our 1st test trial with bacteria in it, we could have spent
too long mixing the mixture of the bacteria and lysozyme, and some of
the bacteria could already have been killed leading to a smaller slope
as much of the bacteria degradation had already occurred as oppose to
the solution of 5mg/ml. Also while the data of sample #6 is not
available, from the figure 1 graph, sample #6 with .2mg/ml of
lysozyme clearly has a smaller slope(as it is less steeper) than sample
#5, which has .5mg/ml of lysozyme and thus would fit into the linear
relationship between concentration of lysozyme and slope.
In addition our figure 2 and table 2 showed us that our results of
HEW,load and Carb 1 are indeed pretty pure. The HEW was able to
degrade the enzyme since it already had lysozyme while the load did
not have any effect do to the fact that it had only negative or neutral
proteins and thus did not have lysozyme which is positive at pH of 7. In
addition our Carb which mainly consisted of the lysozyme had a much
higher slope or fasting killing of the bacteria than HEW which had
lysozyme mixed with other proteins. Our HEW had about 3% lysozyme,
while out Carb 1 had 329.5% lysozyme. This was due to the fact that in
lab 3 we calculated that we had only 6.375 mg of carb1, while we
derived 21mg of lysozyme from the slope readings from the
spectrometer. Our low carb 1 amount was due to the low spectrometer
absorbance reading from lab 3., which could have been due to a smear
of protein on the outside of the reference tube.
Overall we were able to see that there was a positive relationship
between lysozyme concentration and slope as seen in figure 3 which
was our standard curve. With figure 3 we were able to quantify the
amount of lysozyme in the HEW, load and Carb from the slopes of our
HEW, Load and Carb, which were measured by the spectrometer and
recorded in figure 2. Thus we were able to demonstrate lysozymes
presence based on its reaction with its substrates based on enzymatic
activity.