Question

The enzyme lactate dehydrogenase catalyzes the reaction \\[\text { Pyruvate }+\mathrm{NADH}+\mathrm{H}^{+} \rightarrow \text { lactate }+\mathrm{NAD}^{+}\\].NADH absorbs light at \(340 \mathrm{nm}\) in the near- ultraviolet region of the electromagnetic spectrum, but \(\mathrm{NAD}^{+}\) does not. Suggest an experimental method for following the rate of this reaction, assuming that you have available a spectrophotometer capable of measuring light at this wavelength.

Short answer

Monitor the decrease in NADH absorbance at 340 nm using a spectrophotometer to follow the reaction rate.

Step-by-step solution

Understand the Reaction

The reaction involves pyruvate and NADH converting to lactate and NAD+. The concentration change of NADH can be monitored because NADH absorbs light at 340 nm, while NAD+ does not.

Preparing the Spectrophotometer

Set up the spectrophotometer to measure absorbance at a wavelength of 340 nm.

Create a Calibration Curve

Prepare a series of NADH standards of known concentrations and measure their absorbance at 340 nm. Plot the absorbance against the concentration to create a calibration curve.

Prepare the Reaction Mixture

Mix pyruvate, NADH, and any additional required buffer or co-factors. This mixture is the reaction sample for the experiment.

Measure Initial Absorbance

Measure the initial absorbance of the reaction mixture at 340 nm to determine the starting concentration of NADH.

Monitor the Reaction Over Time

At regular time intervals, measure the absorbance of the reaction mixture at 340 nm to track the decrease in NADH concentration.

Calculate the Reaction Rate

Using the calibration curve, convert the absorbance values to NADH concentrations. Plot these concentrations against time, and determine the slope (change in concentration over time) which is the reaction rate.

Key concepts

headline of the respective core concept

Spectrophotometry is a technique used to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through the sample solution. In this exercise, we use spectrophotometry to monitor the concentration of NADH since it absorbs light at a specific wavelength of 340 nm. By setting up the spectrophotometer to this wavelength, we can selectively measure the absorbance of NADH and ignore other substances that do not absorb at this wavelength.
By preparing a series of known NADH concentrations and measuring their absorbances, we can create a calibration curve. This curve helps us determine the concentration of NADH in our reaction mixture based on their absorbance measurements. Spectrophotometry is ideal for this experiment because it provides a rapid and non-destructive way to determine concentration changes in real-time.

headline of the respective core concept

NADH is a crucial molecule in metabolic reactions and absorbs light at 340 nm. This property allows us to track its concentration in a solution by measuring the absorbance at this specific wavelength using a spectrophotometer. The unique absorbance of NADH, and not NAD+, is key to monitoring the reaction.
During the reaction catalyzed by lactate dehydrogenase, NADH is converted to NAD+, resulting in a decrease in absorbance at 340 nm. By measuring this decrease over time, we can determine how much NADH is being consumed and consequently how much pyruvate is being converted to lactate.

headline of the respective core concept

Measuring the reaction rate involves tracking how quickly reactants are converted to products. In this context, we observe the decrease in NADH concentration as the reaction proceeds. The spectrophotometer readings at 340 nm give us real-time data.
By plotting the NADH concentration against time, we generate a graph where the slope represents the reaction rate. A steeper slope indicates a faster reaction. Using the calibration curve, we convert absorbance values to NADH concentrations to accurately determine this rate.
This method is efficient for studying enzyme kinetics and understanding how different conditions (e.g., pH, temperature) affect the enzyme's activity, providing valuable insights into the enzyme's functionality in biological systems.