POST LAB: PARTIAL PURIFICATION AND CHARACTERIZATION OF YEAST INVERTASE(lab4/5)

NAME: LAB PARTNER:

ID# DATE:Monday/02/4/2012

COURSE: ADVANCED GENERAL BIOCHEMISTRY CODE:BIOL 2364 9(AGB)

RESULTS:

TABLE 1 CALIBRATION CURVE VALUES OF AVERAGE ABSORBANCE READINGS AT 510nm AND MICROMOLES OF STANDARD GLUCOSE SOLUTION

VOLUME OF STANDARD 4mM GLUCOSE SOLUTION (mL)

MILLIMOLES OF GLUCOSE (mM)

MICROMOLES OF GLUCOSE (µMol)

ABSORBANCE @ 510nm

AVERAGE ABSORBANCE @ 510nm

0.00 0.0000 0.00 0.000 0.000

0.05 0.0002 0.20 0.167 0.169

0.05 0.0002 0.20 0.171

0.10 0.0004 0.40 0.345 0.345

0.15 0.0006 0.60 0.511 0.511

0.20 0.0008 0.80 0.634 0.669

0.20 0.0008 0.80 0.705

0.25 0.0010 1.00 0.860 0.868

0.30 0.0012 1.20 1.018 1.018

TABLE 2 SHOWING THE EFFECT OF ENZYME CONCENTRATION ON INITIAL VELOCITY

Volume of fraction 4 (ml)

Abs at 510nm

Corrected Abs at 510nm

mg of protein

µmol reducing sugar

(v)µmol/min

µmol/min/ml

0.02 0.212 0.078 0.000141 0.0914 0.00914 457.00.05 0.340 0.206 0.000353 0.2287 0.02287 457.40.10 0.486 0.352 0.000706 0.4129 0.04129 412.90.20 0.686 0.552 0.001412 0.6475 0.06475 323.50.40 0.994 0.860 0.002824 1.0087 0.10087 252.10.60 0.052 0.082 0.004236 0.0961 0.00961 16.00.60 0.145 0.011 --- ---- ---- ----

Sucrose blank 0.2

0.134 0.134 ---- --- ---- ----

Glucose blank0.2

0.025 0.025 ---- ---- ----- ----

Glucose standard (0.2)

0.528 0.528 ----- ----- ----- ----

Table 3 showing absorbance at 510nm vs. time

Tube Time (min) Absorbance @510 µmol reducing sugar1 0 0 0.002 1 0.022 0.203 2 0.089 0.204 4 0.107 0.405 8 0.168 0.606 10 0.172 0.807 12 0.186 0.808 15 0.264 1.009 20 0.672 1.20

Table 4 showing controls and zero t ime tubes

Tube Time(min) Absorbance@510nm µmol reducing sugar10 20 0.697 0.0011 10 0.00 0.2012 10 0.582 0.20

Table 5 showing substrate concentration and the velocity of the enzyme in the absence of urea

[S  ] M

[S ]mM

1/[S ] Absorbance @510nm

Corrected absorbance @510nm

Vµmol/min

1/v

0.00 0.00 0.00 0.000 0.00 0.00 0.000.01 10 0.100 0.082 0.0695 0.0100 100.00.02 20 0.050 0.092 0.0671 0.0097 103.00.03 30 0.030 0.124 0.0866 0.0125 80.00.04 40 0.025 0.185 0.1351 0.0196 51.00.05 50 0.020 0.218 0.1557 0.0255 39.00.10 100 0.010 0.288 0.1634 0.0236 42.00.20 200 0.005 0.403 0.1538 0.0225 44.0

Table 6 showing substrate concentration and the velocity of the enzyme in the presence of urea

[S  ] M

[S ]mM

1/[S ] Absorbance @510nm

Corrected absorbance @510nm

Vµmol/min

1/v

0.00 0.00 0.00 0.0010 0.0000 0.0000 00000.01 10 0.100 0.001 0.00064 0.000090 11111.00.02 20 0.050 0.002 0.00128 0.000185 5405.00.03 30 0.030 0.005 0.00392 0.000560 1785.00.04 40 0.025 0.006 0.00456 0.000660 1515.00.05 50 0.020 0.004 0.00300 0.000434 2304.00.10 100 0.010 0.003 0.0000 0.000 0.00.20 200 0.005 0.081 0.07400 0.01070 93.0

Table 7 showing the zero time, glucose blank, standard and their corresponding absorbance values

Tubes Absorbance @510nm9 (zero time control) 0.12110( zero time control) 0.25111 (glucose blank) 0.01412 (glucose standard) 0.668

Calculations

Millimoles of standard glucose solution used

1000mL= 4mM0.05mL= 4/1000m*0.05= 0.0002mmoles

Micromoles of the standard glucose solution used1mL = 1000µmoles0.0002mmoles = 0.0002mmoles * 1000/1 = 0.2µmols

Corrected absorbance = Absorbance of sucrose(0.134) – Absorbance’s

Using Tube 4

0.212 - 0.134=0.078

Calculating Mg of protein (using tube 4)

0.01ml 10ml

0.2ml

0.02 x 0.01 = 0.0002ml

1ml = 0.706mg

0.002ml = 0.706/ 1 x 0.0002

=0.0001412mg

µmol of reducing sugar (using tube 4)

0.01ml 10ml

0.02ml 10ml

From calibration curve y =0.8525x

Y = 0.078

X = 0.078/ 0.8525 = 0.0914µmol

µmol/min of reducing sugar

0.0914 is in 10 minutes

In 1min= 0.0914/10 = 0.00914µmol/min

µmol/min/ml (tube 4)

0.02ml = 0.0914

10ml= 0.0914/0.02 x 10

= 45.7µmol

0.01ml = 45.7

1ml = 45.70 / 0.01 x 1 = 4570µmol/ml

10min = 4570

1min = 4570/ 10 x 1 = 457 µmol/min/ml

Substrate concentration (tube 2)

Sucrose 0.5M

Volume 0.02

[S] = 0.5 x 0.02 = 0.01

mM = 0.01 x 1000 = 10mM

Correction for absorbance’s (session 5)

From the graph of volume of sucrose (0, 0.2 and 0.4ml) vs. Absorbance

Y = 0.623x

Using tube 2 (0.02ml)

0.623 x 0.02 = 0.01246

Absorbance = 0.082

0.082 – 0.01246 = 0.0695(corrected absorbance)

Velocity (v)/µmol/min

From calibration curve y = 0.6906x

Using tube 2

Absorbance= 0.0695

X = 0.0695/ 0.606 = 0.1006µmol

In 10 minutes =0.1006 µmol

In 1 min = 0.1006/10 = 0.01006

Calculating Km and Vmax using the Michaelis Menton Curve

Without inhibitor

Vmax = 0.025µmol/min

Km= 12mM

With inhibitor

Vmax=0.010µmol/min

Km= 70mM

Calculating Km and Vmax of enzyme using the Lineweaver burke plot

Absence of inhibitor urea

Y=mx + c

M is the slope and c the intercept

Therefore from the equation of the graph y=880.95x + 30.946

Slope 880.8 and intercept 30.946

Determining the V max

The Vmax is the reciprocal of the intercept

Inhibitor absent

1/30.946= 0.0323Vmax = 0.0323µmol/min

x = - c / m= - 30.946/880.95= -0.035Km= -1/-0.035 = 28.57mM

Inhibitor present

Y= 116045x-704.72 Vmax = 1/ 704.72= 0.0014µmol/min

-704.72/116045=-0.0061

Km= -1/-0.0061 = 163.9mM

Dilution of fraction 4

F4= 0.248mg/ml

1ml1mg

1ml= 1000µL

50µl= 1/1000 x 50 = 0.05mg

0.05mg --> 1/0.248 x 0.05 = 0.2m0.2ml=200µL

0.02ml through the spin column

0 0.2 0.4 0.6 0.8 1 1.2 1.40

0.2

0.4

0.6

0.8

1

1.2

f(x) = 0.852527472527473 x

Calibration curve of absorbance @510nm vs micromoles of glucose

µmol of glucose

Abso

rbac

e@51

0nm

0 5 10 15 20 250

0.2

0.4

0.6

0.8

1

1.2

1.4

graph showing micromoles of glucose vs time

Time(min)

µmol

of g

luco

se

0 0.2 0.4 0.6 0.8 1 1.2 1.40

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8f(x) = 0.690604395604396 x

Calibration curve 2 showing absorbance @510nm micromoles of glucose

µmol glucose

Asor

banc

e@51

0nm

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.450

0.05

0.1

0.15

0.2

0.25

0.3

f(x) = 0.623 x

Graph showing the absorbance vs the volume of sucrose in the absence of urea

Y-ValuesLinear (Y-Values)

volume(ml)

Abso

rban

ce@

510n

m

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.450

0.001

0.002

0.003

0.004

0.005

0.006

0.007f(x) = 0.018 x

Graph showing the absorbance vs the volume of sucrose in the presence of urea

volume of sucrose(ml)

Abso

rban

ce@

510n

m

Graph showing 1/v vs 1/S in the absence of inhibit

0 0.02 0.04 0.06 0.08 0.1 0.120

20

40

60

80

100

120f(x) = 880.952380952381 x + 30.9464285714286

1/S

1/V

Graph showing 1/V vs 1 / S in the presence of an inhibitor or urea

0 0.02 0.04 0.06 0.08 0.1 0.120

2000

4000

6000

8000

10000

12000

f(x) = 116044.897959184 x − 704.72193877551

Series2Linear (Series2)

1/S(mM-1)

1/V

Discussion

Throughout this experiment the enzyme invertase has been isolated, purified and characterized by

various methods. In these final two processes the enzyme kinetics was investigated using fraction 4

which through the various methods of purification and characterization had the purest form of the

enzyme as opposed to the fractions 1 through 3(Ahmed,2005). In the fourth session the effects of

enzyme concentration on the initial velocity was investigated. This process was achieved by increasing

the concentration of faction four (which contained the enzyme) whilst the substrate (sucrose)

concentration remained constant as well as the other reagents. Ten tubes were set up , one tube served

as a glucose standard, one as a glucose blank, another as the zero time control and one a sucrose blank

whose absorbance were to be subtracted from the other tubes to attain the corrected absorbance’s.

From table 1 it can be seen that the velocity generally increased with increasing amount of the enzyme,

as the volume increased from 0.4 to 0.6ml the velocity decreased from 0.10087 to 0.00961. At the point

where the volume was increased to 0.6ml it can be deduced that the enzyme was fully saturated with

substrate therefore it was not expected for the velocity to increase further. A tube was set up as a zero

time control this also contained 0.6ml fraction 4, 1ml of Nelsons reagent was added to this tube before

the substrate sucrose was added this was done to ensure that there was no enzymatic formation of

reducing sugar (Murray et al.2003). In this session the effect of increasing incubation time on product

formation was also investigated, it was observed that as the incubation time increased so did the rate by

which the product/reducing sugar was formed. This trend is further emphasized by the plot of µmol of

reducing sugar vs. the varied incubation times, it is seen that as the time of incubation increased so did

the amount of product formed. The shape of the graph shows that at the time 1 to 10 there was a

steady increase in the product formed( this is the straight part of the graph ) beyond 10 minutes the

graph began to curve slightly, nevertheless increasing. It can therefore be deduced that beyond 20

minutes may not have lead to an increase in product.

In the second session the effects of increasing substrate concentration on the velocity of the enzyme

was investigated this time keeping the enzyme concentration constant throughout. The trend observed

in table 5 showed that as the concentration of the substrate increased from 10 to 50 Mm so did the

velocity, at 100 and 200mM sucrose the velocity was observed to decrease. The absorbances were

corrected since sucrose spontaneously hydrolyses to reducing sugar; hence the absorbance value

obtained from the sucrose blank was subtracted from the absorbances obtained.

This experiment was repeated with urea as well as the substrate; this was done to investigate the

effects of the inhibitor on the velocity of the enzyme. The Vmax ( the maximal velocity) and Km (the

concentration by which Vmax is half) values were determined for both experiments with and without

the inhibitor using the Michaelis-Menten curve (V vs.[S ]) as well as the lineweaver burke plot (1/vs.

1/[S ]). The Vmax was found to be 0.025µmol/min without the inhibitor and 0.010µmol/min with the

inhibitor from the Michaelis-Menten curve whereas the Vmax from the lineweaver burke plot was found

to be 0.0323 and 0.0014 in the absence and presence of the inhibitor respectively. The Km in the

absence and presence of the inhibitor was found to be 12 and 70mM respectively from the Micaelis-

Menten curve,in contrast the Km values obtained from the Lineweaver burke plot was 163.9mM with

inhibitor and 28.57mM without the inhibitor.

It was expected that the velocity of the enzyme would have increased as the amount of enzyme

increased; this is so because as the amount of enzyme increases the probability of an enzyme/substrate

complex being formed also increases therefore the amount of product formed will increase, if all the

enzyme is bound to a given amount of substrate then increasing the amount of enzyme will not affect

the velocity of reaction until the product has been formed hence allowing the enzyme to bind to other

substrate molecules. This is seen in the experiment as the amount of enzyme is increased so is the

velocity, this trend are observed to the point where although the amount of enzyme was increased the

velocity did not.

This trend is also seen when incubation time is increased, there was a general increase in the amount of

product formed. The longer an enzyme is incubated with its substrate, the greater the amount of

product that will be formed. However, the rate of formation of product is not a simple linear function of

the time of incubation, as was seen in the graph.

All enzymes may suffer denaturation, and hence loss of catalytic activity, with time(Nelson,2008).Some

enzymes, especially in partially purified preparations, may be noticeably unstable, losing a significant

amount of activity over the period of incubation. If the activity of the enzyme is such that much of the

substrate is used up during the incubation, then, even if the concentration of substrate added was great

enough to ensure saturation of the enzyme at the beginning of the experiment, it will become

inadequate as the incubation proceeds, and the formation of product will decrease(Hansen2010). As

seen in the graph of µmol of glucose vs. time the amount of product formed increased as the incubation

time increased, if the incubation time had increased beyond 20 minutes the amount of product formed

may have remained constant or decrease from the previous value obtained.

The binding of an enzyme to its substrate is an essential part of the enzyme-catalyzed reaction, at low

substrate concentrations, the active sites of the enzyme may not be saturated by substrate. As the

concentration of the substrate increases, the sites are bound to a greater degree of the substrate until

saturation occurs, this is where no more sites are available for substrate binding. At this saturating

substrate concentration, the maximum velocity (Vmax) of the reaction is seen. (Murray et al.2003)

The Vmax in the absence of an inhibitor was found to be 0.025µmol/min from the Michaelis Menten

curve whereas with the inhibitor it was found to be 0.01 µmol/min, whereas the Km values were 12mM

and 70mM with and without the inhibitor respectively. Urea must therefore be a mixed inhibitor since it

increases the Km and decreases Vmax. This was also seen in the Lineweaver burk plot were the Vmax

values were found to be 0.0323µmol/min and 0.0061µmol/min without and with the inhibitor

respectively. In addition the The Km values were 163.9mM -1 and 28.57mM-1 with and without the

inhibitor respectively. This type of inhibition is due the inhibitor binding to an allosteric site (a site

different from the active site).

Gel electrophoresis was carried out on all the fractions using polyacrylamide as the gel medium, as seen

in the sketch of the electrophoretogram obtained. Electrophoresis through polyacrylamide gel leads to

enhanced resolution of sample components because the separation is based on both molecular sieving

and electrophoretic mobility (Boyer, 2000). The larger molecules do not move through the gel medium

easily therefore the rate of movement is the smaller molecules followed by the larger molecules.

In these sessions the effects of varying the enzyme amount was investigated, it was found that as the

amount of enzyme increased so did the velocity that is until saturation occurred. The effect of increasing

the incubation time has on the formation of the product was also observed, it was found that as the

incubation time increased so did the amount of product formed. Finally the concentration of the

substrate was increased in the absence and presence of an inhibitor urea, from the results it was

deduced that increasing the sucrose concentration increased the velocity of the reaction until saturation

(Vmax).The inhibitor urea acted as a mixed inhibitor decreasing the maximal velocity whilst increasing

the Km.

References

Ahmed, Hafiz. 2005. Protein Extraction, Purification and Charcterization, 2nd Edition, New York .CRC press

Boyer, Rodney.2000.Modern Experimental Biochemistry.3rd Edition,San Francisco;Carlifornia. Longman

Pblishers

Hansen, P.J .2010. “Lowry protein assay” .Dept. of Animal Sciences, University of South Florida, accessed

March 21st,2012. http://www.animal.ufl.edu/hansen/protocols/lowry.htm

Murray K. Robert, Granner.K Daryl, Mayes.A Peter, Rodwell.W Victor.2003. Harpers Ilustrated

Biochemistry. 23rd Edition, USA. Lange Medical.

Nelson. David, Cox. Michael. 2008. Leninger Principles of Biochemistry 5th Edition, New York. Mc Millan

press.