Serum albumin labelling

Serum albumin labeled with an iodine radionuclide was firstly used as a substrate for determining protease activity by Absolon This method was later on modified several times and applied for assaying various proteolytic activities in different materials. Mego et al. injected denaturated I-human %rum albumin into the tail vein of rats and measured the rate of intralysosomal proteolysis on isolated lysosomes containing endocytosed substrate. This method was also used for the determining the intralysosomal pH on the basis of differences found in the rate of I-albumin breakdown in intact and lysed lysosomes C-bovine serum albumin, I-casein or I-albumin have been alternatively used as substrate for measuring the activity of trypsin, chymotrypsin and papain - ). 

Hill, R.D. and Laing, R.R., Specific reaction of dansyl chloride with one lysine residue in rennin, Biochim. Biophys. Acta 132, 188-190, 1967 Chen, R.F., Huorescent protein-dye conjugates. I. Heterogeneity of sites on serum albumin labeled by dansyl chloride. Arch. Biochem. Biophys. 128, 163-175, 1968 Chen, R.F., Dansyl-labeled protein modified with dansyl chloride activity effects and fluorescence properties. Anal Biochem. 25, 412M16, 1968 Brown, C.S. and Cunningham, L.W., Reaction of reactive sulfydryl groups of creatine kinase with dansyl chloride. Biochemistry 9, 3878-3885, 1970 Hsieh, W.T. and Matthews, K.S., Lactose repressor protein modified with dansyl chloride activity effects and fluorescence properties. 

Likhtenshtein and colleagues (Belonogova et al., 1978, 1979 Likhten-shtein, 1976) carried out a series of measurements on the hydration dependence of the mobility of spin labels covalently bound to several proteins. The results were correlated with Mossbauer spectroscopic data obtained in parallel experiments. Spin-labeled human serum albumin and a-chymotrypsin showed a critical hydration level for onset of motion at relative humidity 0.8, equivalent to 0.2 h. The temperature dependence of the spin label spectrum showed a critical temperature of 230 K, below which motion was frozen. Serum albumin labeled at surface sites... 

Fig. 46. Sum S(t) and difference D(t) curves for Bovine Serum Albumin labeled with DNS in aqueous solution at pH 7.2 S(t) - upper curve, Dlt) - hiver curve. Points are experimental data,...

An interesting study on the fate of injected placental alkaline phosphatase (N17) has been reported by Posen et al. (P18), who measured heat-stable alkaline phosphatase. Following injection, there was an initial rapid fall in enzyme activity lasting 3-5 hours and then a less rapid disappearance. After 4 hours, two thirds of the initial activity remained, but it took several weeks for complete disappearance. These kinetics resembled those known for serum albumin labeled with iodine-131. 

Cheung D, Bowen B, Rashid F, Rhem R, Dolovich M. Preparation of microaggre-gated human serum albumin labelled with Tc to enable in vivo evaluation of suspension aerosols. Am J Resp Crit Care Med 1998 157 A638. 

Scheme 8.20 Synthesis of human serum albumin labeled with a rhenium tricarbonyl fragment.

Some work has been completed on reaction of proteins with nitrite followed by hydrolysis and analysis for amino acids It has been shown that 3-nitrotyrosine and 3,4-dihydroxyphenylalanine are formed from bovine serum albumin when nitrosation occurs under conditions similar to those found in the human stomach (36), Direct demonstration that nitrite reacts with protein has been made by using NaN02 with bovine serum albumin (pH 5.5, 20 C and 200 ppm nitrite). A 60% loss of the originally added nitrite was observed in one week and nearly half of the nitrite (labelled %) could be recovered from the protein. Similar work with myosin revealed that 10-20% of the incorporated label was present as 3-nitrotyro-sine (J7). 

Catalase has also been used as an enzyme label in competitive heterogeneous enzyme immunoassays. Catalase generates oxygen from hydrogen peroxide with the oxygen determined amperometrically with an oxygen electrode. This approach has been demonstrated for a-fetoprotein theophylline and human serum albumin... 

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.

The syntheses, photophysical and electrochemical properties of [Ir(ppy)2(phen-NS-5)]PF6, (181), [Ir(ppy)2(phen-NHCOCH2I-5)]PF6, (182), and [Ir(ppy)2(phen-NH2-5)]PF6 are reported.342 Complexes (181) and (182) have been used to label amine- and sulfhydryl-modified oligonucleotides and human serum albumin to give luminescent bioconjugates. 

The flux of 3H-labeled PNU-78,517 across MDCK cell monolayers shows the characteristic disparity between the kinetics of disappearance from the donor solution and appearance in the receiver sink (Fig. 32). Drug uptake is rapid and exponential with time and approaches a quasi-equilibrium state in contrast, the concomitant efflux of drug into the receiver is slow and linear. While maintaining a 3% bovine serum albumin (BSA) concentration in the donor and varying the BSA concentration between 0.5 and 5% in the receiver, the results show that the... 

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. 

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