You perform the LDH enzyme assay and measure absorption at 340 nm 30 seconds after addition...

You perform the LDH enzyme assay and measure absorption at 340 nm 30 seconds after addition of the enzyme. Your absorbance reading is 0.97. What is the enzyme activity (in units of µmol NADH/min)? (show your work)

Expert Solution

You need to know the the extinction coefficient (epsilon: e) of your product then you apply the Beer Lambert Abs= e c l (l is the pathlength if you use cuvette of 1 cm then you can calculate c (concentration of product that appeared or substrate that disappeared) by Abs/el . Be careful with the units of e, to determine the C (usually in mM). If you have c in mM for instance and you are working in 1 mL you will know that you have let say if c = 0.2 mM 0.2 µMol in 1 mL . If now you know that you have a delta Abs in 1 min then means you have 0.2 µmol (200 nmol) per 1 min and you have to know how much enzyme you put in your cuvette (let say 2 nM) then your kcat (catalytic constant) will be 100 min-1. You can also work out activity as nmol/min/mg (then you need to know how much you put in the cuvette let say 1 µg in the 1 mL then meaning that you got 200 nmol/min for 100 µg so you mutliply by 10 to get 2000 nmol/min/mg or 2 µmol/min/mg that is also the enzyme activity.

Value of extinction coefficient must be given to calculate the concentration?

queen_honey_blossom answered 2 years ago

A colorimetric LDH assay provides a method to measure the enzyme activity of LDH. NADH (MW...

A colorimetric LDH assay provides a method to measure the enzyme activity of LDH. NADH (MW 633.4 g/mol) absorbs light at 340 nm while NAD+ does not show any absorption at that wavelength. The Beer-Lambert Law relates the absorbance of a solute to its concentration: A = ε*c*L A is absorbance, ε is the molar extinction coefficient, c is concentration, and L is the path length of the solution in the spectrophotometer (usually in cm). In the lab you have...

We will assay enzyme activity at 340 nm. The molar absorbance is higher at 260, meaning...

We will assay enzyme activity at 340 nm. The molar absorbance is higher at 260, meaning we can detect three times less cofactor. Why don’t we use that wavelength? If you had to pick the worst possible wavelength to monitor, what would it be more context: doing assays after purifying proteins from animal tissue. Testing for enzymatic activity

Using 1.0 nM enzyme X for the enzyme kinetics experiments, you obtained the reaction rates at...

Using 1.0 nM enzyme X for the enzyme kinetics experiments, you obtained the reaction rates at different concentrations of substrate (shown in the following table). Assume that the enzyme X follows a typical Michaelis-Menten kinetics. [substrate] (μM) Reaction rate (μmol/min) 2 7 5 17 10 29 20 44 50 67 100 80 200 89 500 95 1000 98 a) Estimate the Vmax and Km values of the enzyme in this reaction using double reciprocal plot. b) If 5.0 nM of...

Using 1.0 nM enzyme X for the enzyme kinetics experiments, you obtained the reaction rates at...

Using 1.0 nM enzyme X for the enzyme kinetics experiments, you obtained the reaction rates at different concentrations of substrate (shown in the following table). Assume that the enzyme X follows a typical Michaelis-Menten kinetics. [substrate] (μM) Reaction rate (μmol/s) 2 7 5 17 10 29 20 44 50 67 100 80 200 89 500 95 1000 98 a) Estimate the Vmax and Km values of the enzyme X in this reaction using the double reciprocal plot. b) If 5.0...

Exercise : Enzyme-Linked Immunosorbent Assay (ELISA) At the conclusion of this exercise you should be able...

Exercise : Enzyme-Linked Immunosorbent Assay (ELISA) At the conclusion of this exercise you should be able to: explain the principles of the enzyme-linked immunosorbent assay (ELISA). determine the qualitative results of a clinical scenario using ELISA. understand the purpose of the reagents used in ELISA. Background Immunology is the study of the immune response. This involves the interaction between antigens, antibodies, and cells. Immune responses are generated by the body to recognize substances considered to be “foreign”. Microorganisms can generate...

If you excite a fluorescent sample with light of 310 nm, in addition to the peaks...

If you excite a fluorescent sample with light of 310 nm, in addition to the peaks from the light emitted by the molecule of interest, at what other wavelengths (in nm) would you expect to see peaks in the spectrum? Why?

Suppose you were using a direct hemagglutination assay as a way to measure the amount of...

Suppose you were using a direct hemagglutination assay as a way to measure the amount of an antigen that causes red blood cell clumping. Describe how you would report the results as a titer, and also describe how you could go from this semi-quantitative measure to an absolute quantitation using a standard curve.

In the lab, you measure the initial rate of an enzyme reactionas a function of...

In the lab, you measure the initial rate of an enzyme reaction as a function of substrate concentration in the presence and absence of an unknown inhibitor (0.05 mM). The following data are obtained when the total enzyme concentration is 1 x 10-6mM:[S] mM0.00010.00020.00050.0010.0020.0050.010.020.050.10.2NoInhibitorV0 (mM/min)3350718391969899100100100WithInhibitorV0 (mM/min)172950678091959899100100A) What are Vmax and Km in the absence of inhibitor? (2 marks)B) What kind of inhibitor is it likely to be and why? (1 mark)C) Calculate the inhibition constant for this inhibitor with the...

You are looking at an illustration depicting the process of telomere addition. Which enzyme is most...

You are looking at an illustration depicting the process of telomere addition. Which enzyme is most likely to be found on the illustration: DNA dependent RNA polymerase RNA dependent DNA polymerase DNA dependent DNA polymerase Reverse transcriptase

1. You observe an AGN where you measure the MgII line (λ0 = 280 nm) at...

1. You observe an AGN where you measure the MgII line (λ0 = 280 nm) at an observed wavelength of λobs = 336 nm. Assuming a Hubble constant of 70 km s−1 Mpc−1 , what is the distance of this AGN in Mpc? 2. Jupiter has a mass of 2 × 1027 kg. If the Jupiter would collapse into a black hole, what would be its Schwarzschild radius? 3. You measured the redshift of a galaxy to be z= 0.35....

Question

You perform the LDH enzyme assay and measure absorption at 340 nm 30 seconds after addition...

Solutions

Expert Solution

Related Solutions

A colorimetric LDH assay provides a method to measure the enzyme activity of LDH. NADH (MW...

We will assay enzyme activity at 340 nm. The molar absorbance is higher at 260, meaning...

Using 1.0 nM enzyme X for the enzyme kinetics experiments, you obtained the reaction rates at...

Using 1.0 nM enzyme X for the enzyme kinetics experiments, you obtained the reaction rates at...

Exercise : Enzyme-Linked Immunosorbent Assay (ELISA) At the conclusion of this exercise you should be able...

If you excite a fluorescent sample with light of 310 nm, in addition to the peaks...

Suppose you were using a direct hemagglutination assay as a way to measure the amount of...

In the lab, you measure the initial rate of an enzyme reactionas a function of...

You are looking at an illustration depicting the process of telomere addition. Which enzyme is most...

1. You observe an AGN where you measure the MgII line (λ0 = 280 nm) at...