Fluorescent spin label

As alkylating agents haloidalkylnitrones 64, 65, and 72a and iodo-enaminoketone 122 can be used. Alkylation with iodoenaminoketone 122 of 2,6-diaminoacridine yielded a convenient fluorescent spin label for nucleic acids 123 (Reznikov and Volodarsky, 1979). 

A. Chattopadhyay and E. London, Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipids, Biochemistry 26, 39-45 (1987). 

E. London and G. W. Feigenson, Fluorescence quenching in model membranes. 1. Characterization of quenching caused by a spin-labeled phospholipid, Biochemistry 20, 1932-1938 (1981). 

Lateral diffusion of phospholipids in model membranes at ambient pressure has been studied over the years by a variety of techniques including fluorescence recovery after photobleaching (FRAP), spin-label ESR, pulse field gradient NMR (PFG-NMR), quasielastic neutron scattering (QENS), excimer fluorescence and others.In general, the values reported for the lateral diffusion coefficient (D) range from 10 to 10 cm /s in the... 

Figure 26 also depicts the distances calculated from fluorescence quenching experiments by enzyme-bound Co2+ on the s-adenosine moiety of s-ATP adenylylated glutamine synthetase (e-AMP-GS). Additional data were gathered by an EPR method that measured the decrease in EPR amplitude of the nitroxide spin-labeled adenylylated enzyme (TEMPO-AMP-GS) owing to enzyme-bound Mn2+ (122). Distances between Mn2+ and the N—O of the spin label are also shown in Fig. 26. 

Thus, we have determined the distances between the adenylyl moiety and the two divalent metal ion binding sites on glutamine synthetase by 13C and 3 P NMR, spin-labeled EPR, and fluorescence energy transfer methods. The results obtained from each method are in good agreement. The data show that the adenylyl regulatory site is close to the catalytic site (12-20 A). Additional data on the rotational correlation time of the adenyl derivatives reveal that the adenylyl site is located on the surface of the enzyme. 

Size exclusion/molecular sieve chromatography Ultra Itration/dia lysis Ultracentrifugation Fluorescent probes Spin label EPR NMR probes Calorimetry Microelectrophoresis Zeta potential... 

The fluorescence probe l-anilinonaphthalene-8-sulfonate was used to examine the binding of spin-labeled local anesthetics to membranes of human blood cells and rabbit sarcoplasmic reticulum. Two distinct fluorescence lifetimes are exhibited by this probe the shorter life time represents the probe associated with pure lipid, the... 

As an example, we will consider the molecular dynamical behavior of egg white lysozyme. The temperature dependence of mobility of fluorescence, spin and Mossbauer labels attached to lysozyme was found to be similar to other investigated proteins the monotonic increase typical for rigid polymers in dry states and in samples with water content (wt) was less than the critical value (wtcr) and drastically burst when wt > wtcr at T > 200 K took place (Frolov et al., 1978 Likhtenshtein, 1979). At similar conditions, experiments on the temperature dependence of heat capacity indicated only a monotonic steady increase for rigid organic material. Recently, in the fully dried lysozyme crystal, similar monotonic behavior of heat capacity was observed in temperatures between 8 and 30°C. At D20 content more than 24 wt %, a slight deviation from the monotony was observed at temperatures above approximately 185 K, which most probably is due to the eutectic melting of NaCl/2H20 present in the samples to prevent water crystallization (Miyazaki et al., 2000). 

Figure 4.1. Arrhenius dependence of different characteristics of lysozyme (1) enzyme activity (2) relative fluorescence intensity (3) water proton relaxation in the presence of the sample spin labelled by His-lS (4) partial spin capacity (5) H-D exchange, (6,7) spin labels rotation and (8) the lysozyme globule rotation. Likhtenshtein et al., 2000. Reproduced with permission.

Very fast electron transfers from P+ to bacteriochlorophyl (Bchl) and from (Bchl)- to QA do not depend on media dynamics and occur via conformationally non-equilibrium states (Fig.3.18). The dual fluorophore-nitroxide molecules (D-A) are also convenient objects for analysing the activity-dynamics relationship. The marked irreversible photoreduction of the nitroxide fragment of the dual probe incorporated into the binding site of HSA only took place when the nanosecond dynamical processes around the probe traced by ESR and fluorescence methods were detected (Rubtsova et al., 1993, Fogel et al, 1994 Likhtenshtein, 1986 Lozinsky et al., 2002). Similar results were reported for another model protein system, i.e. a-chymotrypsin with spin labeled methionin-92 groups (Belonogova et al., 1997). In the latter enzyme, the excited tryptophan group serves as an electron donor. 

Besides radionuclides (detected on the basis of beta and gamma radiation), labels detected by the measurement of magnetic resonance have been proposed (spin labels - spin immunoanalysis, abr. SIA). Other labels are fluorescent dyes already... 

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