Fluorescence fading

As an example of the potential usefulness of such fractions, we also report their use as a probe of the physical basis of the photochemical fading of acridine orange bound to heparin. Fluorescence fading under continuous irradiation is a second-order photochemical reaction of adjacently-bound acridine orange ions in which photooxidation leads to irreversible loss of fluorescence, with the role of heparin being catalytic... 

A quite unexpected finding was that anticoagulant activity and fluorescence fading both showed qualitatively the same peculiar dependence upon anionic density. This suggested... 

Fluorescence Fading. The fluorescence fading constant, r", was measured for each of the heparin fractions in 8 IM... 

Thus, a knowledge of the physical basis for fluorescence fading may yield information concerning the role of physical forces in governing anticoagulcint activity. 

Figure 3. Relationship between fluorescence fading constant (t") (9—left ordinate) and T3. The anticoagulant activity —right ordinate) shown in Figure 2 are repeated in order to show the similarity in shape.

Figure 5. Relationship between relative activation entropies of fluorescence fading, AS t (in eu) and the logarithm of the anticoagulant activity. Activation entropies are relative to the value observed with the 0./50M fraction.

The work with fluorescence fading permits some speculation about a mechanism by which anionic density governs anticoagulant activity. This work suggests that the mechanism may involve the dynamic conformational properties of the heparin, and not the static conformational properties. 

It must be emphasized that the relationships between anticoagulant activities and r" (Fig 4) and activation entropy (Fig 5) are strictly empirical. Nevertheless, the existence of an underlying physical basis is consistent with models of heparin action. As summarized by Laurent and coworkers (I.) heparin promotes the interaction between thrombin and antithrombin which are bound to the same heparin chain. In this respect the heparin can be considered as a catalyst, and, as discussed above, its role in this process and fluorescence fading may well be quite analogous. A number of authors have shown that heparin Induces conformational changes in antithrombin or, possibly, in the serine proteases of the coagulant cascade, and that this leads to an increase in the rate of inactivation of the serine protease by antithrombin. Our... 

J. M. Menter, R. E. Hurst, and S. S. West, Photochemistry of Heparin-Acridine Orange Complexes in Solution Photochemical Changes Occurring in the Dye and Polymer on Fluorescence Fading, Photochem. Photobiol 29, 473-478 (1979). 

Notes The slides may be stored in the dark at 4 C for 1-2 days before observation we have found that this treatment reduces fluorescence fading. 

Fluorescence fading depends on both the lamp and the set of filters used for epifluorescence. If the degree of fading is too high, the slides may be mounted in Vectashield (H-1000 Vector Laboratories) or in similar media containing glycerol and antifading compounds (see Appendix 3). 

Note This treatment improves the degree of differentiation and reduces fluorescence fading. Here again (see Protocol 1.3, step 6) if fluorescence fading is too high, mount the slides in Vectashield (H-1000 Vector Laboratories) or similar media. 

The major Chi a/b-protein 2 and minor Chi a/b-protein 1 were isolated from barley (Hordeum vulgare L. cv Bonus) free from contamination by other Chl-proteins using sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (Machold et al. 1979). FDMR at 4.2 K was performed as previously described (Searle et al. 1981) except that the 476 nm line of a Coherent Radiation CR3 Ar" " (CW) laser was used to excite Chi b specifically. The same set-up (detailed in Searle et al., to be published) was used to measure front-surface fluorescence excitation and emission spectra, and also to carry out the fluorescence fading measurements (Avarmaa 1977), all at 4.2 K. Chi fluorescence decay kinetics and fluorescence emission spectra at 293 K were measured using excitation from a mode-locked Coherent Radiation CR18 Ar+ laser (100 ps FWHM pulses with < 10 photons cm 2 pulse" ), the pulse train being modulated at 330 kHz. 

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