Studies on Fluorescence Associated with Proteins

Abstract

The relative fluorescence intensities of the proteins and their hydrolysates were determined by measuring the dilution required to reduce the fluorescence of a given amount of protein or protein hydrolysate to the same intensity as the fluorescence of a diluted standard solution of quinine hisulfate.

The proteins prepared and studied in this investigation were: casein, wheat gluten, gliadin, glutenin, blood fibrin, gelatin, ovalbumin and zein. Hair and wool were also compared to the above proteins.

When examined in ultraviolet light of wavelengths 3100-4100 Å the proteins give a uniform bluish-white fluorescence in the solid state and a somewhat more green fluorescence in solutions. The fluorescence of these proteins is more green in basic solution than in acid, but the color change is not sharp.

Fluorescence of proteins is destroyed by oxidation with strong nitric acid or by ashing. The small amount of protein ash is not fluorescent in the solid state nor in acid, basic or neutral solution.

Organic solvents do not extract the fluorescent material from the solid protein nor from the protein hydrolysates in acid or basic solution. Likewise, dialysis experiments failed to remove the fluorescent material from protein solutions but after hydrolysis with strong acid the fluorescent material is readily removed from proteins by dialysis.

Hydrolysis of proteins by proteolytic enzymes or alkali produced only a slight increase in the amount of fluorescence. However, hydrolysis with hydrochloric acid, sulfuric acid, perchloric acid or phosphoric acid produced large increases in the fluorescence of those proteins containing tryptophane and only slightly increased fluorescence in those proteins which are deficient in this amino acid. The color of the fluorescence produced during acid hydrolysis is blue-green.

Crude commercial proteins produce the same amount of fluorescence, during acid hydrolysis, as these same proteins prepared in a purified state.