Abdulkareem Almazied

CHEM 464L

Dr. Rashid Sayed

December 5, 2016

Abstract:

Lactate Dehydrogenase (LDH) is an enzyme which catalyzes the

reaction of the substrate pyruvate to lactate with the help of the cofactor

nicotinamide adenine dinucleotide (NADH). The purpose of this experiment is

to isolate LDH from rabbit muscle then purify it using ammonium sulfate

precipitation and affi gel blue affinity chromatography followed by

determination of LDH kinetic characterization and lastly determining the type

of the unknown inhibitor. Calculating Km and Vmax of LDH kinetics have

helped to determine the type of inhibition caused by the unknown inhibitor.

The total activity of B-1 has been calculated to be 4.52 LDH unit/ml and 1.08

LDH unit/mL for B-11. The specific activity was determined to be 28.25U/mg

for B-1 and 435U/mg for B-11. The purification factor is 15.39 and the

recovery percentage of the affi gel chromatography is 47.8%. The Km value

is 0.0959mM and Vmax is 0.257 A/min in the absence of inhibitor. In the

presence of the inhibitor Km is 0.116mM and Vmax is 0.212 A/min. the type

of the inhibition caused by the unknown inhibitor is mixed inhibition.

Introduction:

The purpose of this experiment is to isolate LDH from rabbit muscle

then purify it using ammonium sulfate precipitation and affi gel blue affinity

chromatography followed by determination of LDH kinetic characterization

and lastly determining the type of the unknown inhibitor.

The homogenization of rabbit muscle extract is the first part of the

procedure then an extraction and partial purification of LDH using

ammonium sulfate precipitation which is a commonly used method to isolate

and fractionate protein based on their polarity and solubility (2). Ammonium sulfate

is a salt that separates proteins from water ion which makes the protein precipitates

after they separate from water. This method is preformed more than once by adding

more salt each time to assure a complete separation of protein. Each protein needs

different levels of ammonium sulfate saturation where the most polar protein will

precipitate last. LDH needs around 75% saturation of ammonium sulfate to

precipitates. After LDH is collected from precipitation a purifications steps is

followed to remove any impurities.

Affi-gel blue chromatography is a method to further purify LDH. Affi-gel blue

is similar to the structure of NADH which enables LDH to bind to it. Affi-gel blue is

also a good visualization method since it is blue so when it forms a pellet in bottom

of the tube it can be viewed and can be concluded that the LDH is in the bottom

with it (1). To further purify an addition of a specific elution buffer that contains

NADH is applied to separate LDH from affi-gel blue since LDH has a higher affinity

towered NADH. The supernatant will contain the LDH purified solution.
 LDH assay involves some further dilution of the LDH affi gel

purified and un purified samples in order to minimize their activity to a level

where it can be measurable. This assay enables us to measure the

purification factor at the end by dividing the specific activity of affi- gel blue

purified LDH by the specific activity of the unpurified LDH

Enzyme kinetics using pyruvate as the substrate enables us to

calculate Km and Vmax in the presence and absence of an inhibitor to

determine the type of inhibition is caused by the obtained unknown inhibitor.

Materials and Methods:

Extraction and purification of LDH:

First, a homogenization of a rabbit muscle was done by a waring

blender in 0.03M KOH. The homogenate was then filtered through six layers

of cheesecloth and the volume was estimated and 1.5 volumes of KOH was

added. Another three-layered filter of cheesecloth was followed then an

addition of an equal volume of ammonium sulfate was applied. For

contamination removal, a purifications steps was then applied. the left

ammonium sulfate suspension of LDH was measured and labeled then stored

in a refrigerator.

Affi-gel Blue and Batch purification:

Affi-gel blue was prepared by transferring 1 ml of the affi-gel blue (AGB)

suspension to a centrifuge tube. The ammonium sulfate LDH suspension was

taken out and diluted 1 to 5 using phosphate buffer. This solution was then

transferred to the tube containing the AGB and centrifuged then supernatant

was discarded, another 2 ml of buffer was added, centrifuged and then

supernatant was discarded again. AGB was lastly washed with spacific

elution buffer containing NADH. This solution is called AGB purified LDH

solution ( B-11).

LDH assay:

For LDH the B-1 solution (1 to 5diulted AS suspention) was diluted 1 to

40 with buffer this makes the C-2 solution. B-11 solution was diluted 1 to 10

with buffer this makes the C-2 solution. Kinetic node spectrophotometer was

used at wave length 340nm to measure the activity of C-1 and C-2 where

100 ul was used for both with 1 ml of water, 1ml of buffer, 0.7 ml of pyruvate

and 0.2 of NADH.

Protein determination:

A known concentration of BSA was used to make six slandered curve

0.025, 0.05, 0.075, 0.1, 0.125 mg/ml of BSA. The unknown concentrations

proteins AGB purified LDH, the elution buffer, 1 to 25 dilution of B-1 and 1 to

50 dilution of B-1 absorbance were measured at wavelength 660nm after

Lowery and Folin reagents has been used then all tubes were heated for 15

minutes.

Km and Vmax determination:

 Two sets of six measurements has been performed with and without

an inhibitor where kinetic mode spectrophotometer was used and a graph for

both measurements was obtained. First set of six measurements was done

with o.2 ml of NADH, 1 ml of buffer, 0.1 ml of LDH and a fixed volume of Di

water and the substrate pyruvate. The second sets of measurments was

carried out using 0.2ml of NADH, 1 ml of buffer, 0.1 ml of 10mM unknown

inhibitor and a fixed volumes of water and pyruvate. The total volume in both

sets was 3 ml

Results:

The concentration of B-1 calculated from the BSA standred curve

(Figure-1) is 0.16mg/ml which is the average of two counts (table-1). The

concentration of B-11 solution is 0,00248mg/ml (Table-1). The total activity of

B-1 is 4.52 LDH unit/ml and its spacific activity is 28.25U/mg. the total

activity of B-11 is 1.08 LDH unit/ml and its spacific activity is 435U/mg.

The purification factor is 15.39 and the percent recovery 47.8%. The total

units of LDH activity in the original ammonium sulfate suspention is 72.32U.

In (Table-2) the kenetic charchtrization of LDH is shown for both the

presence and absence of the inhibitor. The Vmax and Km is estimated to be

around 0.25 A/min and 0.08mM with the absence of inhibition and around

0.17A/min and 0.05 mM in the presence of inhibition (Figure-2). In

Lineweaver-Burk plot the Vmax and Km is measured to be 0.257A/min and

0.0959mM in the absence of inhibitor (Figure-3). In the presence of inhibitor

Vmax is measured to be 0.212A/min and Km is 0.116mM (figure-3). The

type of inhibition is determined from graphs to be a mixed inhibition.

BSA standred curve

1.2

1
f(x) = 7.53x + 0.06
0.8 R = 0.99

A660 0.6
0.4

0.2

0
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14

mg/ml BSA

(Figure-1) the graph is showing the mg/ml of BSA protein and the absorbance

value at 660nm

mg/ml
Tube Sample A660 protein Column1
1 std 0 0
2 std 0.2666 0.025
3 std 0.4777 0.05
4 std 0.66 0.075
5 std 0.795 0.1
6 std 0.965 0.125
average=0.16m
7 B-1 1 to 25 0.1778 0.0086 g/ml
8 B-1 1 to 50 0.1322 0.002
9 B-11 0.2833 0.00248
10 elution buffer 0.1477
 11 corrected 9 0.1356 0.00248

(Table-1) the table is showing the standed curve points and the result of
protein concentration for B-1 and B-11 protein
Pyruva Column With no Column With Column
te 1 Inhibitor 2 Inhibitor 3
[So] 1/[so] Vo 1/Vo Vo 1/Vo
0 0 0 0 0 0
0.083 12 0.1215 8.2 0.0867 11.5
0.167 6 0.1571 6.4 0.1292 7.7
0.25 4 0.176 5.7 0.1522 6.6
0.42 2.4 0.2218 4.5 0.1756 5.7
0.67 1.49 0.229 4.36 0.1719 5.8
0.92 1.1 0.2345 4.26 0.1768 5.7
(Table-2) this table shows the result of LDH characterization in the presence
and absence of inhibition.

Michaelist-menten plot Vo VS [So]

0.25

0.2

0.15
Vo(A/Min)
0.1

0.05

0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

[So](mM)

(Figure-2) this figure shows the concentration of substrate and the

absorbance value for the presence and absence of inhibitor.
 Lineweaver-Burk 1/Vo VS 1/[So]
14
12
10 f(x) = 0.55x + 4.71
R = 0.98
8
f(x) = 0.37x + 3.89
1/Vo(1/A/Min) 6 R = 0.98

4
2
0
0 2 4 6 8 10 12 14

1/[So](1/mM)

(Figure-3) this graph shows Lineweaver-Burk plot of 1/[So] Vs 1/Vo

Discussion:
In summary, inhibition type was determined to be a mixed inhibition

and the Km and Vmax values was measured to be 0.0959mM and

0.257A/min in the absence of inhibition and 0.116mM and 0.212 A/min in

the presence of inhibition. The present recovery from affi-gel blue was

measured to be 47.8% with a 15.39 purification factor.

The Vmax and Km estimated from (figure-2) is 0.25A/min and

0.08mM in the absence of inhibitor and 0.17A/min and 0.05mM with

inhibitor while in Lineweaver-Burk plot (figure-3) the Km and Vmax was

measured to be 0.0959mM and 0.257A/min with no inhibitor and 0.116mM

and 0.212A/min with an inhibitor. In (figure-2) Vo is dependent on the

amount of substrate added [S] since everything else is constant including the
enzyme. Increasing [So] is going to make Vo increase causing a sigmoid

graph until it reaches a plateau line where Vo is going to equal Vmax, Km

can be measured as the [So] when Vo is equal to Vmax. From this type of

graph Vmax and Km hard to be measured so it is estimated instead. The

value of Vo and [So] can be transferred to a different plot called Lineweaver-

Burk plot where there is going to be a linear graph and can easily measure

Vmax and Km more accurate using the Lineweaver-Burk equation.

In (figure-3) graph km has increased in the presence of inhibitor

while Vmax has decreased moreover, the measured y- axis of the two graphs

are 3.89 and 4.71. based on these data we can conclude that the inhibition

type in this experiment is a mixed inhibition so the unknown inhibitor# 60 is

a mixed inhibitor.

This experiment is a long experiment and have to be delt with very

carefully because it is easy for a person to produce an error while preforming

this experiment. One possible source of error expected is dealing with NADH

due to its spontaneous oxidation. Another source of error can be while

preforming dialysis because it is really hard to deal with the small bags

especially when applying the solution to them a person can loss all the

solutions due to leakage if not carful. A possiple source of error that might

have been expected is while preforming the protein determination assay due

to not correctly making the reagent which will not give an accurate result.
 To conclude, the type of the obtained unknown inhibitor is a mixed

inhibitor and that was determined after looking at the Vmax and km values

of the graphs with and without inhibition.

Acknowledgment:

I would like to thank professor Rashyed for helping us and also would like to

thank my lap partners Elroma David and Marlein for for their assistance

References:

Nelson, D.L. and Cox, M.M. Enzymes. In Lehninger: Principles of

Biochemistry, 6th ed.; W.H. Freeman and Co.: New York, 2012; pp 1144-
1145.

(1) Wilson, J. E. Rapid purification of Mitochondrial Hexokinase from rat

brain by a single affinity chromatography step on Affi-Gel blue.
Preparative Biochemistry 1989, 19 (1) (Jan), 1321.

(2) Peng, F.; Bian, J.; Peng, P.; Xiao, H.; Ren, J.-L.; Xu, F.; Sun, R.-C.
Separation and characterization of Acetyl and Non-Acetyl
Hemicelluloses of Arundo donax by ammonium Sulfate Precipitation.
Journal of Agricultural and Food Chemistry 2012, 60 (16) (April 25),
40394047.