POST LAB: PARTIAL PURIFICATION AND CHARACTERIZATION OF YEAST INVERTASE(lab4/5) NAME: ID# COURSE: ADVANCED GENERAL BIOCHEMISTRY LAB

PARTNER: DATE:Monday/02/4/2012 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 (Mol)

ABSORBANCE @ 510nm

AVERAGE ABSORBANCE @ 510nm

0.00 0.05 0.05 0.10 0.15 0.20 0.20 0.25 0.30

0.0000 0.0002 0.0002 0.0004 0.0006 0.0008 0.0008 0.0010 0.0012

0.00 0.20 0.20 0.40 0.60 0.80 0.80 1.00 1.20

0.000 0.167 0.171 0.345 0.511 0.634 0.705 0.860 1.018

0.000 0.169 0.345 0.511 0.669 0.868 1.018

TABLE 2 SHOWING THE EFFECT OF ENZYME CONCENTRATION ON INITIAL VELOCITY

Volume of fraction 4 (ml) 0.02 0.05 0.10 0.20 0.40 0.60 0.60 Sucrose blank 0.2 Glucose blank 0.2 Glucose standard (0.2)

Abs at 510nm

Corrected Abs at 510nm 0.078 0.206 0.352 0.552 0.860 0.082 0.011 0.134

mg of protein

mol reducing sugar 0.0914 0.2287 0.4129 0.6475 1.0087 0.0961 ------

(v) mol/min

mol/min/ml

0.212 0.340 0.486 0.686 0.994 0.052 0.145 0.134

0.000141 0.000353 0.000706 0.001412 0.002824 0.004236 ------

0.00914 0.02287 0.04129 0.06475 0.10087 0.00961 -------

457.0 457.4 412.9 323.5 252.1 16.0 -------

0.025

0.025

----

----

-----

----

0.528

0.528

-----

-----

-----

----

Table 3 showing absorbance at 510nm vs. time Tube 1 2 3 4 5 6 7 8 9 Time (min) 0 1 2 4 8 10 12 15 20 Absorbance @510 0 0.022 0.089 0.107 0.168 0.172 0.186 0.264 0.672 mol reducing sugar
0.00 0.20 0.20 0.40 0.60 0.80 0.80 1.00 1.20

Table 4 showing controls and zero t ime tubes Tube 10 11 12 Time(min) 20 10 10 Absorbance@510nm 0.697 0.00 0.582 mol reducing sugar
0.00 0.20 0.20

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

[S ] mM

1/[S ]

Absorbance @510nm

Corrected absorbance @510nm

V mol/min

1/v

0.00 0.01 0.02 0.03 0.04 0.05 0.10 0.20

0.00 10 20 30 40 50 100 200

0.00 0.100 0.050 0.030 0.025 0.020 0.010 0.005

0.000 0.082 0.092 0.124 0.185 0.218 0.288 0.403

0.00 0.0695 0.0671 0.0866 0.1351 0.1557 0.1634 0.1538

0.00 0.0100 0.0097 0.0125 0.0196 0.0255 0.0236 0.0225

0.00 100.0 103.0 80.0 51.0 39.0 42.0 44.0

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

[S ]

[S ] mM

1/[S ]

Absorbance @510nm

Corrected absorbance @510nm

V mol/min

1/v

0.00 0.01 0.02 0.03 0.04 0.05 0.10 0.20

0.00 10 20 30 40 50 100 200

0.00 0.100 0.050 0.030 0.025 0.020 0.010 0.005

0.0010 0.001 0.002 0.005 0.006 0.004 0.003 0.081

0.0000 0.00064 0.00128 0.00392 0.00456 0.00300 0.0000 0.07400

0.0000 0.000090 0.000185 0.000560 0.000660 0.000434 0.000 0.01070

0000 11111.0 5405.0 1785.0 1515.0 2304.0 0.0 93.0

Table 7 showing the zero time, glucose blank, standard and their corresponding absorbance values Tubes 9 (zero time control) 10( zero time control) 11 (glucose blank) 12 (glucose standard) Absorbance @510nm 0.121 0.251 0.014 0.668

Calculations Millimoles of standard glucose solution used 1000mL= 4mM 0.05mL= 4/1000m*0.05= 0.0002mmoles

Micromoles of the standard glucose solution used 1mL = 1000moles 0.0002mmoles = 0.0002mmoles * 1000/1 = 0.2mols Corrected absorbance = Absorbance of sucrose(0.134) Absorbances
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

mol 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 = 0.0914mol mol/min of reducing sugar

0.0914 is in 10 minutes In 1min= 0.0914/10 = 0.00914mol/min mol/min/ml (tube 4) 0.02ml = 0.0914 10ml= 0.0914/0.02 x 10 = 45.7mol 0.01ml = 45.7 1ml = 45.70 / 0.01 x 1 = 4570mol/ml 10min = 4570 1min = 4570/ 10 x 1 = 457 mol/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 absorbances (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)/mol/min From calibration curve y = 0.6906x Using tube 2 Absorbance= 0.0695 X = 0.0695/ 0.606 = 0.1006mol In 10 minutes =0.1006 mol In 1 min = 0.1006/10 = 0.01006 Calculating Km and Vmax using the Michaelis Menton Curve Without inhibitor Vmax = 0.025mol/min Km= 12mM With inhibitor Vmax=0.010mol/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= 0.0323 Vmax = 0.0323mol/min x = - c / m = - 30.946/880.95 = -0.035 Km= -1/-0.035 = 28.57mM

Inhibitor present Y= 116045x-704.72 Vmax = 1/ 704.72= 0.0014mol/min -704.72/116045=-0.0061 Km= -1/-0.0061 = 163.9mM

Dilution of fraction 4
F4= 0.248mg/ml 1ml1mg 1ml= 1000L 50l= 1/1000 x 50 = 0.05mg 0.05mg --> 1/0.248 x 0.05 = 0.2m0.2ml=200L 0.02ml through the spin column

Calibration curve of absorbance @510nm vs micromoles of glucose

1.2 y = 0.8525x

Absorbace@510nm

0.8

0.6

0.4

0.2

0 0 0.2 0.4 0.6 0.8 mol of glucose 1 1.2 1.4

graph showing micromoles of glucose vs time

1.4 1.2 mol of glucose 1

0.8 0.6 0.4 0.2 0 0 5 10 Time(min) 15 20 25

0.9 0.8 0.7 Asorbance@510nm 0.6 0.5 0.4 0.3 0.2 0.1 0 0

Calibration curve 2 showing absorbance @510nm micromoles of glucose

y = 0.6906x

0.2

0.4

0.6

0.8

1.2

1.4

mol glucose

Graph showing the absorbance vs the volume of sucrose in the absence of urea
0.3 0.25 Absorbance@510nm 0.2 0.15 0.1 0.05 0 0 0.1 0.2 0.3 0.4 0.5 volume(ml) Y-Values Linear (Y-Values) y = 0.623x

Graph showing the absorbance vs the volume of sucrose in the presence of urea
0.008 0.007 Absorbance@510nm 0.006 0.005 0.004 0.003 0.002 0.001 0 0 0.1 0.2 0.3 0.4 0.5 volume of sucrose(ml) y = 0.018x

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

150 y = 880.95x + 30.946 100 50 0 -0.4 1/V -0.3 -0.2 -0.1 -50 -100 -150 -200 -250 -300 0 0.1 0.2

1/S

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

15000 y = 116045x - 704.72 10000 5000 0 -0.25 1/V -0.2 -0.15 -0.1 -0.05 -5000 Series1 -10000 -15000 -20000 -25000 -30000 1/S(mM-1) Linear (Series1) 0 0.05 0.1 0.15

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 absorbances. 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 mol 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 0.025mol/min without the inhibitor and 0.010mol/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 MicaelisMenten 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 mol 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 0.025mol/min from the Michaelis Menten curve whereas with the inhibitor it was found to be 0.01 mol/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 0.0323mol/min and 0.0061mol/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.