Studies on enzymatic histochemistry. 29: Dilatometric micro-kestimation of peptidase activity.

Compt. rend. Laborat. Carlsberg, Sér. chim., 21/24. - Copenhagen , H. Hagerup, 8°, pp.315-337, 11 Abbildungen, 13 Tab., orig. Broschur; Stemp. a.Tit. This is the paper that first described the gradient tube. The method essentially solved the single-cell problem for reactions that were associated with an appreciable change in volume such as the proteases and fat-splitting enzymes that were under study at the Carlsberg Laboratory at the time. The idea was simple and ingenious.Two fluids, immiscible in water and of slightly different density, are prepared. The lighter of the fluids is carefully overlaid above the heavier in a tube with a long cylindrical section in the middle. The center section is gently stirred and after a day or so it is found that there is a linear density gradient in the long central section. 'Qpically, the density changes only by 0.001 g.mL" per cm. Small drops (about 0.1 pL) of a solution to be analyzed are added to the column and fall until their density matches that of their surroundings. This converts the measurement of density to the measurement of position in the tube, which can be done with great accuracy with a cathetometer. A series of KC1 solutions of known density are run alongside the unknowns for calibration. We omit details concerning saturation, calibration, rejuvenation of the column, etc., which are covered in the original paper.With this procedure complicated chemical or titrimetric techniques are replaced by a simple, non-invasive physical measurement As illustrated in the figure it also permitted the study of kinetics, provided that the reaction was slow compared to the equilibration time in the column. This technique increased the sensitivity by a factor of 50 relative to the 1931 paper described above, and could measure the peptidase activity of single cells. It was also used in isotope experiments to determi) One of us used the method to measure the partial specific volume of proteins many years ago with very satisfactory results on extremely small samples of protein. The procedure was also the basis of Lang's method for the hydrogen exchange of proteins, ." J. A. SCHELLMAN and C. G. SCHELLMAN: Kaj Ulrik Linderstrom-Lang (1896-1959), Protein Sci. 1997 6: 1092-1100. Buchnummer des Verkäufers 17016

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Titel: Studies on enzymatic histochemistry. 29: ...

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