so i had to find the Km and Vmax for Lactate dehydrogenase from given absorbance
ID: 85049 • Letter: S
Question
so i had to find the Km and Vmax for Lactate dehydrogenase from given absorbance values of NAD+ and i got the below values. i dont really know what i was doing and now its asking for what assumptions were made. i have no idea what to answer for the bottom question about assumptions below is the excel data for the calculations. Also i am unsure how to calculate the specific activity of lactate dehydrogenase if the protein concentration is 1.05mg.ml-1
Table 3: Calculations for Lineweaver-Burk plot Reaction no. [NAD+] 1/[NAD+] DA v v 1/v ie [S] ie 1/[S] (min-1 per 20 µl of enzyme) (µmol. min-1 per 20 µl of enzyme) (20 µl of enzyme. min. µmol-1) (mM) (mM-1) for 120 s per minute from sample calculation reciprocal 1 1 mM 1 0.0952 0.0476 0.007677419 130.2521008 2 0.1 mM 10 0.0323 0.01615 0.002604839 383.9009288 3 0.2 mM 5 0.0479 0.02395 0.003862903 258.8726514 4 0.4 mM 2.5 0.0686 0.0343 0.005532258 180.7580175 From the graph, y intercept: 110 x intercept: -3.9 Vmax: 1/y intercept Km: - 1/x intercept Vmax: 0.009091 µmol.min-1 Km: 0.256410256 mM or mmol.L-1 If your line does not meet the negative x-axis, calculate Km from the slope of the line: a. Slope = Km/Vmax = rise/run = (y2 - y1) / (x2-x1) b. Using y intercept = 1/Vmax , Km = (Slope / y intercept) mM Note: Km has the same units as [S] i.e. [NAD+] Q: What assumption have you made with respect to the other substrate in determining the Km for NAD+ and the Vmax for the enzyme?Explanation / Answer
A)
slope = Km / Vmax. or (Y2-Y1) / (X2-X1)
slope = 0.256 / 0.009091 or (383.9 - 258.87) / (10 -5)
slope = 28.1. or 125.03 / 5
25.006
B)
Y intercept = 1/ Vmax = 1 /0.009091 = 110.
Km = slope / y intercept
Km = 28.1 / 110
Km = 0.255 mM.
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