lucifer-yellow and 5-((4-6-dichloro-1-3-5-triazin-2-yl)amino)fluorescein

Fluorescent dye conjugates of arylsulfatase A (ASA) from rabbit liver were prepared at pH 9.0 in 0.1 M sodium bicarbonate buffer. The modification of amino or sulphadryl groups by dichlorotriazinylamino-fluorescein or Lucifer yellow fluorescent dyes did not alter the characteristic features of the enzyme molecule such as enzyme activity, dimerization of the protein molecule at pH 4.5 and anomalous kinetics of the native enzyme. The fluorescence intensity of the Lucifer yellow enzyme conjugates were quenched when the pH of the protein solution was changed from pH 7.5 to 4.5. Therefore, the Lucifer yellow enzyme conjugate can be used to study the kinetics of pH-dependent association and dissociation of the ASA. Availability of such fluorescent dyes conjugates of ASA or other lysosomal enzyme may be used as a biological tracer to study the receptor dependent endocytosis of enzyme molecules.

Topics: Animals; Carrier Proteins; Cerebroside-Sulfatase; Endocytosis; Fibroblasts; Fluoresceins; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Isoquinolines; Liver; Lysosomes; Molecular Imaging; Rabbits; Skin