Albumin fluorescence

When LOOM species prepared by SLO oxidation of either linoleic or linolenic acid were incubated with bovine serum albumin, fluorescent products having excitation and emission spectra similar to those of oxidized LDL were observed (Fig. 6). From this, it has been proposed that after homolytic cleavage of LOOH to the more reactive LOO-, a concerted reaction occurs between LOO- and polypeptide amino groups to yield fluorescent adducts without prior LOOH fragmentation to aldehydes or other, more stable, products (Fruebis et al., 1992). During LDL or cell membrane oxidation... 

Utilizing time resolved internal reflection spectroscopic technique (Fig. 6), we were able to isolate the tryptophan intrinsic fluorescence and observe its = 20 ns fluorescence lifetime for albumin in bulk and in the surface microenvironment of a hydrophilic quartz material. The pH dependence of bulk albumin fluorescence lifetime served to "calibrate albumin in terms of native ( 7 ns time constant) protein at pH 7.2 and unfolded (c 4 ns) protein at the isoelectric pH 3.8. The fluorescence lifetime data (Tables I/II) supported the hypothesis that the adsorbed albumin exists in two forms on a hydrophilic quartz surface, each with a possibly different structure (] ). A loosely held "layer," consisting of microaggregates, native and partially unfolded albumin molecules with... 

BF breast-fed IF infant formula-fed MRP-F/Albumin fluorescence of advanced Maillard products/fluorescence of soluble tryptophan in plasma expressed in arbitrary units (AU) per gram albumin MRP-F/Creatinine fluorescence of advanced Maillard products per mmol creatinine in spot urines p<0.01, p<0.001 (vs. IF)... 

Sinn, H. Muelbaier, M. Preparation of albumin-fluorescent dye conjugate for diagnostics during surgery. Ger. Offen. DE 102005019104, 2006 Chem. Abstr. 2006, 145, 426066. 

Shaw, C.F. Ill, Schaeffer, N.A., Elder, R.C., Eidsness, M.K., Trooster, J.M. and Calls, G.H.M. (1984) Bovine serum albumin-gold thiomalate complex gold-197 Moessbauer, EXAFS and XANES, electrophoresis, sulfur-35 radiotracer, and fluorescent probe competition studies. Journal of the American Chemical Society, 106, 3511-3521. 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

The observed fluorescence blue shift following BLG adsorption was, however, small compared with the one observed for bovine serum albumin [28]. Univariate analysis... 

Figure 5. Fluorescence anisotropy of F-D labelled heparin-antithrombin interaction. F-D-heparin (0.02 fluoresceins per uronic acid) at 0.1 mg/ml was incubated with different concentrations of antithrombin (open circles) or bovine serum albumin (solid diamonds) in 20 mM sodium phosphate buffer, pH 7.4.

Recently, novel polymethine carbonyl-dyes based on coumarin moiety and their boron difluoride complexes 9a-d and lOa-d [34—36] were evaluated as fluorescent dyes for the detection of native proteins using bovine serum albumin (BSA) as a model protein, and as probes for the nonspecific detection of proteins using a BSA/ sodium dodecyl sulfate (SDS) mixture [37]. Optical properties of these compounds in the absence and presence of BSA, as well as in SDS and BSA/SDS mixture, were measured in Tris-HCl buffer (pH 8.0) (Table 1). 

Weber G (1952) Polarization of the fluorescence of macromolecules II. Fluorescent conjugates of ovalbumin and bovine serum albumin. Biochem J 51 155-167... 

Squaraine dyes 10b, 39a, 39b, 41a, 41c, 41d, and 41e were used to measure different proteins such as BSA, HSA, ovalbumin, avidin from hen egg white, lysozyme, and trypsin (Fig. 12) [58]. It is difficult to predict correlations between the dyes structures and the affinity or sensitivity of the dyes for different proteins. All squaraine probes exhibit considerable fluorescence increases in the presence of BSA. Dicyanomethylene-squaraine 41c is the brightest fluorescent probe and demonstrates the most pronounced intensity increase (up to 190 times) in presence of BSA. At the same time, the fluorescent response of the dyes 10b, 39a, 39b, 41a, 41c, 41d, and 41e in presence of other albumins (HSA and ovalbumin) is, in general, significantly lower (intensity increases up to 24 times). Dicyanomethylene-squaraine 41a and amino-squaraines 39a and 39b are the most sensitive probes for ovalbumin. Dyes 41d, 10b, and 41e containing an A-carboxyalky I -group demonstrate sufficient enhancement (up to 16 times) in the presence of avidin. Nevertheless, the presence of hydrolases like lysozyme or trypsin has only minor effects on the fluorescence intensity of squaraine dyes. 

Sytnik A, Del Valle JC (1995) Steady-state and time-resolved study of the proton-transfer fluorescence of 4-hydroxy-5-azaphenanthrene in model solvents and in complexes with human serum albumin. J Phys Chem 99 13028-13032... 

The F/P ratio of the purified, labeled protein may be determined by measuring the absorbance at 345 and 280nm. Ratios between 0.3 and 0.8 usually produce labeled molecules having acceptable levels of fluorescent intensity and good retention of protein activity. AMCA-labeled proteins may be lyophilized without significant loss of fluorescence. The addition of bovine serum albumin (15mg/ml) or another such stabilizer is often necessary to retain solubility of the freeze-dried, labeled protein after reconstitution. 

The interaction between 4-(4-hydroxybut-2-ynyloxy)-3-(phenylsulfonyl)-l,2,5-oxadiazole-2-oxide 16 and bovine serum albumin (BSA) was studied by spectroscopic methods including fluorescence and UV-Vis absorption spectroscopy. The results indicate that molecules 16 bind with BSA forming 1 1 complex. Thermodynamic parameters, such as AH, AG, and A.Y, were calculated. The results indicate that the binding reaction is mainly entropy driven and hydrophobic forces play a major role in this reaction <2006CHJ1050>. 

Several methods for measuring drug binding to human serum albumin involving the determination of retention times on HPLC columns with bound albumin have been reported [77,78]. Solid-phase microextraction [79,80], capillary electrophoresis [81], and displacement of near-infrared fluorescent labels [82] have all been studied. 

When staining with any of the fluorescent phalloidins, dilute 10 pL methanolic stock solution into 400 pi PBS. To reduce nonspecific background staining with these conjugates, add 1% bovine serum albumin (BSA) to the staining solution. It may also be useful to preincubate fixed cells with PBS containing 1 % BSA. 

Among the series of dyes, in which the effect of binding to human serum albumin (HSA) was studied [41], the dye 8 (Fig. 10) exhibited J-aggregate absorption and fluorescence bands, while these bands were not observed for the free dye. This led to the conclusion about the J-aggregation of the dye 8 in the HSA binding site. 

---