[The use of fluorescence spectroscopy for identification of A- and B-thrombin chains during their chromatographic...

The separation of A- and B-chains of human thrombin has been performed by gel filtration on Sephadex G-100 under the reduction of disulphide bonds with dithiothreitol. Identification of A- and B-chains has been provided by measurements of the fluorescence intensity of fractions at 310 nm and 350 nm which are near the maximum positions of tyrosine and tryptophan fluorescence, respectively. The appearance of A-chain was monitored by an increase of the ratio of Ifl310/Ifl350 greater than 2. The fluorescence spectrum of A-chain has maximum position at 304 nm, which is characteristic of tyrosine fluorescence. The fluorescence spectrum of B-chain has maximum position at 347.5 nm which corresponds to fluorescence of tryptophan residues. The identification of A- and B-chains has been confirmed by the gel electrophoresis data.