Use of isosbestic point wavelength shifts to estimate the fraction of a precursor that is converted to a given product.
An isosbestic point is observed in overlaid spectra when a chromophoric precursor is converted to a product with a different spectrum, so that it is often assumed that an isosbestic point occurs only when the precursor is quantitatively converted to a single product. We show experimentally and by computer simulations that more complex reactions also exhibit isosbestic points and that the wavelength of the isosbestic point may change. Such wavelength changes will occur if either (i) the molar absorbtivity of the precursor changes or (ii) the fraction of the precursor that is converted to multiple products changes. In the latter case, the isosbestic wavelength and molar absorbtivities of the precursor and product can be used to calculate the fraction of the precursor that is converted to products from the relationship, f = epsilon(Precursor)(M)/epsilon(Product)(M), where f is the fractional conversion, epsilon(Precursor)(M) is the molar absorbtvity of the precursor, and epsilon(Product)(M) is the molar absorbtivity of the product.
Berlett
B S
BS
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, MSC-0342, Bethesda, Maryland 20892-0342, USA.
Levine
R L
RL
Stadtman
E R
ER
D012015:Reference Standards; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*