A sensitive method for determining preferential binding of substances to enzymes and biological membranes

A systematic study of the factors affecting the channel ratio of a mixture of ( 3H) and ( 14C) as measured by liquid scintillation counting has been carried out. The variation in the estimation of the channel ratio by repeated counting of the same vial is shown not to exceed that expected from statistical fluctuations of the counting rate. However, variations between the channel ratio of different counting vials containing the same solution of radioactivity and scintillator fluid is larger than can be accounted for by statistical fluctuations. A three-channel procedure is devised to correct for the different counting properties of the vials. Furthermore, the effect of the resolving time of the scintillation counter on the channel ratio at different counting rates is noted. The calculations involved in the use of the method are demonstrated by an examination of the binding of d-glucose to hexokinase. Finally, the applicability of the method for determining low levels of specific binding of substances to enzymes and cellular membranes is discussed.