Optical Density of Compound X: Calculate Molar Extinction Coeff.

[bio_monkey]

Homework Statement

In an experiment it is found that the optical density of a solution of compound X was 0.7 when measured at 400nm. in a 1cm glass cuvette. The solution had been prepared by dissolving 35mg of X in 50 ml of water. What is the molar extinction coefficient of X at 400nm? (mol. wt. of X is 450)
(20% of the marks)


(ii) Measurements of lactate dehydrogenase (LDH) activity were carried out on plasma from an adult patient admitted to hospital with a suspected heart attack. The assay was carried out in the direction:

Pyruvate + NADH  lactate + NAD+

The oxidation of NADH was observed spectrophotometrically at 340nm in a 1cm cuvette.
Patient plasma samples were prepared by adding 0.3ml of plasma to 2ml of a suitable buffer and then adding a further 1ml of a solution to the diluted plasma to stabilise it.
Assay mixtures were prepared in a final volume of 3ml and contained 0.5ml of the stabilised diluted plasma, 0.15mM NADH and 1mM pyruvate. The reaction was carried out at 25oC and started by adding the pyruvate last. The following readings were obtained:

Time after pyruvate addition/min Absorbance at 340nm
0 0.98
1 0.82
2 0.66
3 0.50
4 0.40
5 0.35
6 0.35

Given that the extinction coefficient of NADH is 6.22 x 103 l.mol.-1 cm.-1at 340nm, calculate the activity of LDH in the patient plasma in terms of mol. pyruvate reduced. min.-1. (ml plasma) –1
(80% of the marks)

The Attempt at a Solution

Im not too sure where to start with this Q..Im guessing that for the first part, I use A=ecl, where l is 1, A is 0.7 and I work out c to then find e??

 

[nate808]

For the first part of the question, you are correct in using the Beer-Lambert law, A=ecl. However, you also need to know the concentration (c) of the solution in order to calculate the molar extinction coefficient (e). In this case, the concentration can be calculated using the formula c = (mass of X / molecular weight of X) / volume of solution. This would give you a concentration of approximately 0.0044 mol/L. Then, you can rearrange the Beer-Lambert law to solve for e, which would give you a molar extinction coefficient of 159.1 l.mol-1 cm-1.

For the second part of the question, you can use the formula for enzyme activity, which is activity = change in absorbance / (extinction coefficient x path length x reaction time). In this case, the change in absorbance is 0.63 (0.98-0.35), the extinction coefficient is given as 6.22 x 10^3 l.mol-1 cm-1, the path length is 1 cm, and the reaction time is 6 minutes. This would give you an enzyme activity of approximately 0.0018 μmol pyruvate reduced/min (ml plasma).

 

[Blueshift5]

For the second part, I can use Beer-Lambert law again, but I dont know the concentration of NADH, so I cant calculate the activity of LDH.

I would first start by understanding the experiment and the data provided. From the given information, we can see that the experiment is measuring the optical density of a solution of compound X at a specific wavelength (400nm). The solution is prepared by dissolving a known amount of X in a known volume of water. We are also given the molecular weight of X.

To calculate the molar extinction coefficient, we can use the Beer-Lambert law, A=εcl, where A is the measured optical density, ε is the molar extinction coefficient, c is the concentration, and l is the path length (in this case, 1cm). We have A and l, and we can calculate c by converting the given mass of X (35mg) to moles and then dividing by the volume (50ml) to get the concentration. Once we have c, we can solve for ε.

For the second part, we are given a series of readings for the absorbance at 340nm over time. The experiment is measuring the activity of LDH in a patient's plasma sample. We are given the reaction that takes place and the concentrations of the reactants. To calculate the activity of LDH, we can use the Beer-Lambert law again, but this time we need to calculate the concentration of NADH. This can be done by using the extinction coefficient of NADH and the absorbance readings at different time points. Once we have the concentration of NADH, we can use the reaction equation to calculate the activity of LDH. It is important to note that the activity of LDH is expressed in terms of μmol of pyruvate reduced per minute per milliliter of plasma.