MadSci Network: Biochemistry

Re: in spectrofluorimetry, what is the purpose of having an emission wavelength

Date: Tue Apr 4 10:41:47 2006
Posted By: Karl A. Wilson, Faculty (Professor), Biological Sciences, S.U.N.Y. at Binghamton (Binghamton University)
Area of science: Biochemistry
ID: 1143325192.Bc

I presume you are assaying for a Caspase using a substrate such as acetyl- Asp-Glu-Val-Asp-AMC, where AMC is the 7-amino-4-methylcoumarin group. The appropriate protease, such as a Caspase, can cleave the amide bond linking the AMC group to the rest of the peptide substrate, releasing free AMC. While the substrate is only very weakly fluorescent, the free AMC is highly fluorescent. The increase in fluorescence is then a measure of the enzyme activity.

To answer your question about fluorescence intensity vs emission wavelength, letís first consider what is happening in the fluorimeter you are using. You irradiate the sample in a suitable cuvette with light of a particular wavelength. The wavelength is usually at or near the wavelength of maximum absorbance of the substance to be measured. In this case, thatís AMC, with light at about 342 nm wavelength. This is the excitation wavelength. Some of this absorbed light is then emitted by the excited AMC as more light, but at a longer wavelength (less energy/photon). This occurs at a wavelength specific to the AMC molecule in a given solvent. This wavelength is the emission wavelength, about 441 nm for AMC. The amount of light (how many photons) emitted (the emission intensity) will depend upon how many AMC molecules are in the solution (i.e. its concentration). When you set up the fluorimeter, you set the excitation and emission wavelengths (in nm), either with filters or monochrometers. You then measure the intensity of the emitted light Ė this is the measure of the product (AMC) produced. You can calibrate this process with an AMC solution of know concentration, and thus are able to calculate how many units of enzyme activity you have.

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