Proflavine absorbance

Acriflavin Mixture (Euflavin, 3,6-diamino-lO-metbylacridiniuni chloride) [8063-24-9] M 259.7, m 179-181 . Purified by dissolving in 50 parts of H2O, shake with a small excess of freshly ppted and washed Ag20. The mixture is set aside overnight at 0 and filtered. The cake is not washed. The pH of the filtrate is adjusted to 7.0 with HCl and evaporated to dryness. The residue is then crystd twice from MeOH, twice from H2O and dried at 120 X ,ax at 452nm has a loge value of 4.67. It is a red powder which readily absorbs H2O. The solubility is increased in the presence of proflavin. The dihydrochloride is a deep red crystn powder. It is available as a mixture of 3,6-diaminoacridinium chloride (35%) and its 10-metho-chloride (65%). [see Albert, The Acridines Arnold Press p. 346 1966 Chem Ber 45 1787 1912]. 

Fig. 26. Sensitized anti-Stokes delayed fluorescence from anthracene.60 Normal fluorescence (curve 1) and delayed fluorescence (curve 2) from a solution containing 10- Af proflavine hydrochloride and 5 X 10-4M anthracene in ethanol excited by 436 m/ , 3 X 10-4 einstein liter-1 sec.-1 absorbed. Curve 1 at a sensitivity 3000 times less than curve 2. Temperature was — 66°C. 3°C.

Chromophoric inhibitor displacement.6 1 The spectrum of the dye proflavin changes significantly with solvent polarity. It is a competitive inhibitor of chymotrypsin, trypsin, and thrombin, and it undergoes a large increase in absorbance at 465 nm (Ae 2 X 104 M l cm 1) on binding (Figure 7.1). 

The rapid exponential decrease in absorbance at 465 nm is caused by the displacement of proflavin from the enzyme on formation of the acylenzyme. The slow increase in absorbance is due to the depletion of the substrate and the consequent decrease in the steady state concentration of the acylenzyme. [From A. Himoe, K. G. Brandt, R. J. Desa, and G. P. Hess, J. Biol. Chem. 244, 3483 (1969).]... 

When an ester such as acetyl-L-phenylalanine ethyl ester is mixed with a solution of chymotrypsin and proflavin, the following events occur. There is a rapid displacement of some of the proflavin from the active site as the substrate combines with the enzyme, leading to a decrease in A465. (This is complete in the dead time of the apparatus.) Then, as the acylenzyme is formed, the binding equilibrium between the ester and the dye is displaced, leading to the displacement of all the proflavin. The absorbance remains constant until the ester is depleted and the acylenzyme disappears. The dissociation constant of the enzyme-substrate complex may be calculated from the magnitude of the initial rapid displacement, whereas the rate constant for acylation may be obtained from the exponential second phase. 

Quantum Yields. The quantum yield is the ratio of the fluorescence intensity to the amount of light absorbed. The ratios of the quantum yield of a 2.5 10 6 M solution, containing only PFH+, and the quantum yields of proflavine on the clays, 0/ with 0 0.34 (1), are shown in figures 5-8 as a function of the loading, expressed in % of the CEC. An increase of 0/ means a decrease of the fluorescence intensity for adsorbed PFH+, as illustrated in the spectra of figure 4. It is due to increasing amounts of dimers on the surface (5). On the basis of these plots the clay suspensions can be divided in three categories. 

Figure 23. A plot of the change in the 444-nm absorbance of 0.02mM proflavine on addition of dC-dC-dG-dG (O) and dG-dG-dC-dC (0) in 0.1 M phosphate...

Macromolecules may or may not fluoresce. Those that do are considered to contain intrinsic fluors. The common intrinsic fluors for proteins are tryptophan, tyrosine, and phenylalanine (the same three groups that absorb UV radiation). Macromolecules that have no intrinsic fluors can be made fluorescent by adding an extrinsic fluor to them. This is done by the process of chemical coupling or sample binding. The most common extrinsic fluors for proteins are l-aniline-8-naphthalene sulfonate, l-dimethylaminonaphthalene-5-sulfonate, dansyl chloride, 2-p-toluidyl-naphthalene-6-sulfonate, rhodamine, and fluorescein. The most common extrinsic fluor for nucleic acids are various acridienes (acridine orange, proflavin, acriflavin) and ethidium bromide. 

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