The kinetic parameters of the reaction of lactate to pyruvate can be measured by following the formation of NADH from NAD+. It can also be measured in the reverse direction, however, at high concentrations of NADH the reaction cannot be monitored at 340 nm? Why is this?
I'm a bit confused at why high NADH concentrations would make it impossible to monitor a reaction at 340 nm. Any explanation of this concept would be greatly appreciated.
The measurement of the reaction at 340 nm involves using a spectrophotometer, which specifically detects changes in absorbance of light at that wavelength. In the case of monitoring the reaction of lactate to pyruvate, the formation of NADH is measured since it absorbs light at 340 nm.
However, at high concentrations of NADH, the absorption of light at 340 nm is saturated or maximal. This means that the maximum amount of light that can be absorbed by NADH has already been reached, and any additional NADH formed or present will not lead to a further increase in absorbance at 340 nm.
As a result, the reaction cannot be effectively monitored at 340 nm when NADH concentrations are high because there is no further increase in absorbance to measure. This limitation prevents accurate measurement of the reaction kinetics in the reverse direction when NADH concentrations become too high.
One way to overcome this limitation is to dilute the reaction mixture or use other methods to monitor the reaction at a different wavelength that is not affected by the high concentrations of NADH.
To understand why high concentrations of NADH can make it difficult to monitor the reaction at 340 nm, we need to first understand the principles behind monitoring reactions using spectrophotometry.
Spectrophotometry is a technique that measures the absorbance of light by a substance at different wavelengths. In this case, the reaction of lactate to pyruvate involves the formation of NADH from NAD+. NADH absorbs light at a specific wavelength of 340 nm.
When monitoring a reaction using spectrophotometry, we measure the rate of change in absorbance at a specific wavelength. The more NADH that is present, the higher the absorbance at 340 nm. This allows us to determine the rate of the lactate-to-pyruvate reaction by measuring the increase in absorbance at this specific wavelength.
However, at high concentrations of NADH, the absorbance can become so high that it reaches the upper limit of the spectrophotometer's sensitivity. Beyond this limit, the instrument becomes saturated, meaning it cannot accurately measure any further increase in absorbance.
In practical terms, this saturation means that when the concentration of NADH is too high, the spectrophotometer cannot detect any additional increase in absorbance at 340 nm. As a result, it becomes impossible to accurately monitor the reaction at this wavelength.
To overcome this limitation, researchers may have to dilute the sample or use other techniques, such as enzymatic methods, to indirectly measure the concentration of NADH in the reverse direction of the reaction.
In summary, high concentrations of NADH can make it impossible to monitor a reaction at 340 nm because the spectrophotometer becomes saturated and cannot accurately measure any further increase in absorbance. Dilution or alternative measurement methods may be necessary to overcome this limitation.