LSM 1101- Lab Report (Practical 3: Enzyme Kinetics) Group 1- Bench 6 Date of Experiment: 11th Sept 2009 Members

name: Karthig S/O R.Kunasakaran (U0804997) Kang Bo Han Abraham (U0900023)

Practical 3 Q3.1.3 Part 1 Absorption Values Wavelength(nm) 400 380 360 340 330 320 300 280 260 NAD+ 0.026 0.027 0.039 0.035 0.040 0.030 0.343 1.506 0.906 NADH 0.036 0.127 0.396 0.548 0.449 0.214 0.377 1.383 0.762

Graph of Absorbance against Wavelength(nm)

Part 2 NADH has much higher absorbance than NAD+ over the 300-400nm range. NAD+ only has a higher absorbance when both have peaks in the graph at 280nm.

Part 3 The wavelength of choice for measuring NADH in a mixture containing both NAD+ and NADH is 340nm. Both NAD+ and NADH peak at 280nm but only NADH peaks at 340nm.

Thus in order to differentiate between the 2 solutions in a mixture 340nm will be the wavelength of choice. Q3.2.3 Part 1 Final concentration of NADH(mM) 0.00 0.01 0.05 0.10 0.15 0.20 Volume of 1mM NADH used(ml) 0.00 0.03 0.15 0.30 0.45 0.60 Volume of Buffer used(ml) 3.00 2.97 2.85 2.70 2.55 2.40

A340 0.000 0.044 0.215 0.502 0.878 1.185

Part 2

Part 3 Beer-Lambert Law: A = cl. Where A = Absorbance, = Molar extinction coefficient, c = concentration of solution and l = light path length. Curvette length is 1cm therefore light path length = 1cm Concentration of solution used in calculation = 0.15mM = 1.5x10-7 A = cl 0.878 = (1.5x10-7)(1) = 5.85x10-6

3.3 Determining Km and Vmax of lactate dehydrogenase Results:

Tube No. 1

10mM NAD+ (ml) 0.1

200mM lactate (ml) 0.03

0.05M buffer pH 9.0 (ml) 2.67

LDH (ml) 0.2

mM lactate (final []) 2

2 0.1 0.06 2.94 0.2 3 0.1 0.12 2.58 0.2 4 0.1 0.15 2.55 0.2 5 0.1 0.3 2.4 0.2 Table 3: Volumes used for dilution of 200mM lactate (ml) for Tubes 1-5

4 6 8 10

Time A340 (sec) Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 0 0 0 0 0 0 30 0.031 0.052 0.074 0.086 0.123 60 0.049 0.081 0.111 0.132 0.182 90 0.064 0.103 0.141 0.165 0.232 120 0.078 0.122 0.166 0.195 0.271 150 0.09 0.139 0.188 0.22 0.304 180 0.102 0.154 0.206 0.242 0.339 210 0.111 0.168 0.224 0.263 0.365 240 0.119 0.181 0.24 0.28 0.396 Table 4: Absorbance of Tubes 1-5 at wavelength 340nm over 240 seconds at 30 second intervals

Graph of A340 against time

Discussion of results
Initial velocity = Gradient of the tangent of the curve = yx Hence, a tangent has to be drawn at t=0 for each curve. Tube Sample Initial Velocity, Vo (Absorbance units/min)

1 2 3 4 5

0.061.50= 0.040 0.081.00= 0.080 0.131.40= 0.093 0.171.60= 0.106 0.100.60= 0.167

Table 5: Initial velocity, Vo (Absorbance units/min) of Tubes 1-5 In order to get the initial velocity in the form of moles/min, we must apply the Beer-Lambert equation, which states that:
Abs = c l where A = absorbance = molar absorption coefficient c = molar concentration l = path length

c (mol.) = Abs/ l V0 (mol./min) = Abs/min / l

From 3.2.3, = 5.85 mM-1cm-1 The initial velocity from the graph of absorbance against reaction time is similar to the value of y in the equation of the linear graph of absorbance against concentration for NADH, which is y=5.7269. Hence: Initial velocity in , mM min-1 =V 5.7269 10-3 = V1M min-1 Initial velocity in, mM min-1= MV1000 (where M=V1 and V=3ml) = 3V'1000 = V11moles min-1 Tube No. 1 2 3 4 5 Initial velocity, Vo Vl (M/min) (Absorbance/min) 0.040 0.080 0.093 0.106 0.167
6.99 10-6 1.40 10-5 1.62 10-5 1.85 10-5 2.92 10-5

Vll (moles/min)

1/[S] (mM1 ) 0.500 0.250 0.175 0.100 0.050

1/Vll (moles/min)1

2.097 10-8 4.20 10-8 4.86 10-8 5.55 10-8 8.76 10-8

4.77 107 2.38 107 2.06 107 1.80 107 1.14 107

Table 6: Calculation of Initial velocity to moles per min.

From Michaelis Menten Curve, Vmax 0.5 Vmax KM = 8.00 10-7 = 4.00 10-7 = 7.80 mM

From Lineweaver Burk Plot,

1Vo =KmVmax1[S]+ 1Vmax

- 1Km= -0.15 mM
1Vmax= 1.20 107

Thus,

KM

= 10.15 = 6.67 mM

Vmax

= 1 / 1.20 107 = 8.33 10-8 molesmin-1

Michaelis-Menton plot Vmax KM 8.00 10-7 7.80 mM

Lineweaver-Burk plot 8.33 10-8 molesmin-1 6.67 mM

The KM and Vmax values obtained from Michaelis Menten curve and the Lineweaver Burk plot are different. As the concentrations of substrate used in the experiment are not high enough, the MichaelisMenten curve obtained from the experimental results does not level off. Hence, the values of Km and Vmax are estimated by extrapolation. Since Km is determined at half Vmax, an inaccurate Vmax would cause an inaccurate Km obtained. In the Lineweaver Burk plot, the Michaelis Menten curve is transformed to a straight line from a hyperbolic curve and their inverses are plotted. Hence, the absolute value of the xintercept of this straight line is the affinity, 1/KM, of the enzyme for the substrate. The yintercept is 1/Vmax. It is from these intercepts that we are then able to calculate the Vmax and Km which are independent of each other.

Hence, the Lineweaver-Burk plot is the more accurate measure of both Vmax and Km. However, the disadvantage of the Lineweaver- Burk plot is that it places undue weight on the points obtained at low concentrations of substrate (the highest values of 1/[S] and 1/Vo). These are the points at which the precision of determining the rate of reaction is lowest, because the smallest amount of product has been formed.