3.

3 Determining Km and Vmax of lactate dehydrogenase

Results:

Tub e No. 1 2 3 4 5 Time (sec) 0 30 60 90 120 150 180 210 240

10mM NAD+ (ml) 0.1 0.1 0.1 0.1 0.1

200mM lactate (ml) 0.03 0.06 0.12 0.15 0.3

0.05M buffer pH 9.0 (ml) 2.67 2.94 2.58 2.55 2.4

LDH (ml) 0.2 0.2 0.2 0.2 0.2

mM lactate (final []) 2 4 6 8 10

Table 3: Volumes used for dilution of 200mM lactate (ml) for Tubes 1-5

Tube 1 0 0.031 0.049 0.064 0.078 0.09 0.102 0.111 0.119

Tube 2 0 0.052 0.081 0.103 0.122 0.139 0.154 0.168 0.181

A340 Tube 3 0 0.074 0.111 0.141 0.166 0.188 0.206 0.224 0.24

Tube 4 0 0.086 0.132 0.165 0.195 0.22 0.242 0.263 0.28

Tube 5 0 0.123 0.182 0.232 0.271 0.304 0.339 0.365 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 1 2 3 4 Initial Velocity, Vo (Absorbance units/min)

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 (Absorbance/min) 0.040 0.080 0.093 0.106 0.167 Vl (M/min) Vll (moles/min) 1/[S] (mM-1) 0.500 0.250 0.175 0.100 0.050 1/Vll (moles/min)-1

6.99 10-6 1.40 10-5 1.62 10-5 1.85 10-5 2.92 10-5

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 Michaelis-Menten 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 x-intercept of this straight line is the affinity, 1/KM, of the enzyme for the substrate. The y-intercept 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.