Toshio Kinoshita

Enhancement of the fluorescence intensity of dansyl amino acids in aqueous media and its application to assay of amino acids

Abstract The fluorescence of dansyl amino acids in aqueous media was found to be greatly enhanced by addition of cycloheptaamylose. This finding was successfully applied for improvement of the assay procedure of amino acids. Interaction of cycloheptaamylose with dansyl amino acids was thermodynamically studied. The entropies and the enthalpies were found to vary with polarities of the dansyl amino acids.

Fluorescent labeling of the surface proteins of erythrocyte membranes using cycloheptaamylose-fluorescamine complex

Abstract Fluorescamine was incorporated in cycloheptaamylose and the resulting complex was utilized as a new probe for fluorescent labeling of the surface proteins of erythrocytes. The complex reacted with erythrocyte membrane at 37°, pH 7 to 8 without using organic solvents. The major membrane proteins of ghosts were labeled, whereas the complex did not penetrate in the intact erythrocytes and labeled only surface proteins. Particular advantages are that the complex itself and its hydrolysis product were nonfluorescent when electrophoresed in sodium dodecyl sulfate polyacrylamide gels.

Microassay of proteins on membrane filter in the nanogram range using cycloheptaamylose-dansyl chloride complex☆

Abstract A protein assay is described in which the sample is spotted on a Sartorius membrane filter and dansylated with cycloheptaamylose-dansyl chloride complex dissolved in aqueous urea solution. The fluorescence intensity of the spot on the membrane was directly measured by a scanning fluorometer. This assay is rapid, simple, highly reproducible, and linear from 0.05 to 4 μg of protein. Very dilute solutions of protein can also be determined by filtering off the sample on the membrane filter in the presence of 0.1 m MgCl 2 and dansylating the protein collected on the membrane. There is no interference either by commonly used reagents such as Tris, citrate, guanidine, and glycine, or by naturally occurring amines and amino acids.

A fluorophotometric method for the determination of amino acids by using pyridoxal and zinc(II) ion

Abstract A fluorophotometric method for the determination of α-oxo acids has been developed based on their interaction with pyridoxamine and Zn(II) ion in pyridine-methanol solution to yield highly fluorescent chelates. This fluorophotometric procedure is simple and highly sensitive to α-oxo acids. No detectable interference for the determination of pyruvate occurred with any of the amino acids, amines, sugars, organic acids, and inorganic salts. Recoveries for pyruvate added to bloods and serum ranged from 90.1 to 103.2%. As little as 5 × 10−11 mol of α-oxo acid may be measured.

A novel fluorophotometric method for determination of hexosamines using pyridoxal and zinc(II) ion.

Abstract A novel fluorophotometric method for the detection and determination of amino sugars was established utilizing pyridoxal-HCl and Zn(II) ion. This method was highly sensitive; its limit of determination was 1 × 10 −11 mole of amino sugars. The fluorogen of this fluorescence reaction was Zn(II) chelate of N -pyridoxylidene amino sugar containing a 1:1 ratio of amino sugar to Zn(II) ion. The hexosamine content of chondroitin sulfate A and C, hyaluronic acid, and heparin estimated by this method was higher than that obtained by the Elson-Morgan method. Only 2–3 hr of hydrolysis with 2 N HCl was sufficient for the assay of chondroitin sulfate and heparin.