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Fluoresceinisothiocyanate was applied to fluorescent labeling of protein for the determination of molecular weight by SDS Polyacrylamide gel electrophoresis. Proteins having molecular weight ranging from 15,000 to 70,000 were tested. The resolved FITC-labeled protein was eluted from the gel and was submitted to amino acid sequence analysis.

We investigated the subunit structures of the three agglutinins (BRA-1, BRA-2, and BRA-3) isolated from the coelomic fluid of the acorn barnacle, Megabalanus rosa, by high-speed gel filtration, gel electrophoresis, CD spectra, and peptide mapping. BRA-2 (Mr 140,000) and BRA-3 (Mr 64,000) were glycoproteins composed of subunits having two identical basic units cross-linked by disulfide bonds. The basic units of BRA-2 and BRA-3 had molecular weights of 22,000 and 18,000, respectively. They did not have a common structure. BRA-1 (Mr 330,000) was composed of subunits homologous to that of BRA-2. The binding properties of these agglutinins against rabbit erythrocyte ghosts were also investigated after labeling the agglutinins with fluorescein isothio-cyanate.

The addition of phenol (about 1%) to 6 m HCl largely prevented destruction of tryptophan during hydrolysis of peptides at 110°C for 22 hr. Tryptophan recovery depended on the volume of 6 m HCl containing phenol and the concentration of phenol. The maximum tryptophan recovery was 85% for a standard amino acid mixture. The recovery was slightly lower for proteins. This hydrolytic procedure was advantageous for micro amino acid analysis using a conventional high-performance liquid chromatography with a precolumn labeling technique. The method was used in the amino acid sequence analysis of a minor component of sea anemone toxins isolated from Anthopleura fuscoviridis. The toxin consisted of 48 amino acid residues with three tryptophan residues.

A cleavable photoactivable heterobifunctional reagent, the N-hydroxysuccinimide ester of 1-azido-5-naphthalene sulfonyl S-carboxymethylthiocysteamine (NHS-ANS-CTC), was synthesized and characterized. The reagent was applicable to the group-directed modification of protein ligands, such as an invertebrate lectin and a trypsin inhibitor. The modified ligands bound to rabbit erythrocyte ghosts and trypsin, respectively. Upon exposure to ultraviolet light, the modified ligands reacted with their binding proteins to form cross-linked fluorescent products. The crosslinked fluorescent complexes were readily cleaved by reducing the disulfide bond of the reagent, leaving the fluorescent probe on the binding proteins. The photolabeled binding proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The fluorescence intensity of the fluorescent probe was enhanced by 4 - 8 times to improve sensitivity when the SDS-gel was dehydrated with methanol. This phenomenon was also observed with the proteins labeled with l-dimethylamino-5-naphthalene sulfonyl chloride.

A fluorometric manual technique has been demonstrated for determining the amino acid sequence of a polypeptide, by the combined use of fluoresceinisothiocyanate (FITC) and phenylisothiocyanate (PITC). This method permits continuous assignments of 10 residues with 5.6 nmoles of myoglobin or 23 residues with 45 nmoles.

Two new photoactivable heterobifunctional reagents, 1-azido-5-naphthalene sulfonyl chloride (ANS-C1) and the N-hydroxysuccinimide ester of 1-azido-5-naphthalene sulfonyl aminopropionate (NHS-ANS-AP), were synthesized and characterized. The reagents were applicable to the group-directed modification of ligands. Upon exposure to ultraviolet light, the non-fluorescent modified ligands generated reactive nitrene intermediates which could react with neighboring molecules to form fluorescent products. This was demonstrated by the covalent modification of bovine serum albumin with ANS-AP by photolysis. NHS-ANS-AP was used for the preparation of a photore-active invertebrate lectin.

(1976). Analysis of Fluorescein-thiohydantoin Amino Acids by High Speed Liquid Chromatography. Agricultural and Biological Chemistry: Vol. 40, No. 4, pp. 815-817.