Year: 1961
Type: article
Abstract: A method capable of measuring the activity of single enzyme molecules could initiate studies of a number of biological problems not yet amenable to experimen- tation.A technique for measuring f3-D-galactosidase in droplets of microscopic size using a new fluorogenic substrate,-6-hydroxyfluoran-13-D-galactopyranoside (6HFG) is described here.Results obtained by this technique indicate that it is capable of detecting individual molecules of the enzyme.As practical applications, the molecular weight and turnover number of f3-D- galactosidase were determined by this method.The values obtained are compar- able with those found by conventional techniques.Another application of this method is in determining whether or not an enzyme is composed of a homogeneous molecular species with respect to activity.This is exemplified here by experiments with a genetically altered f3-D-galactosidase.We concluded that the altered enzyme is a different species than normal f3-galactosidase.In contrast, thermal inactivation leads to a mixture of fully-active and inactive enzyme molecules.Materials and Methods.-Thesubstrate 6HFG5 was synthesized by coupling 6-hydroxyfluoran (6HF)1 and tetraacetyl-fD-galactopyranosylbromide2 in anhydrous benzene with silver oxide present.3The tetraacetyl galactoside was deacetylated with sodium methylate.4The final product, purified by ascendent paper chromatography in a 1-pentanol, 1-propanol, water system (40:11:15), moved with an Rr of 0.17 on Whatman #1 paper at room temperature (220C).6HF was located by the strong greenish fluorescence of 6HF liberated after spraying a portion of the paper with 6-D-galactosidase.A long wavelength "Mineralight" was used for excitation.UV light of shorter wavelength decomposes 6HFG on paper, forming a compound with bluish fluo- rescence.This can serve to locate 6HFG on chromatograms.The purity of 6HFG was established by paper chromatography in three different solvent systems at neutral, acid, and basic pH.5 Complete hydrolysis of 6HFG by a purified, crystalline preparation of O-D-galactosidase from Escherichia coli (kindly supplied by Dr. D. Perrin) with stoichiometric recovery of 6HF and D-galactose indicates that 6HFG has the formula: CH20H +
Author: Boris Rotman
Institution: Stanford University
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W2043127660