studied by equilibrium dialysis at 5°. When the 

 fraction of the occupied poly U binding sites is 

 plotted as a function of free adenosines (Fig. 3), 

 the resulting adsorption isotherm shows a very 

 steep transition. No binding was detectable 

 until a critical threshold concentration of adeno- 

 sine was reached. This steep curve of adsorp- 

 tion isotherm is analyzed by the following 

 equation derived from lattice statistics based 

 on the nearest neighbor interaction (8). 



ee 



din Y, 



e- 



_ exp (-W/2kT) 

 4 



where d is the fraction of sites occupied, W is 

 the interaction energy of the nearest neighbor 

 and Y is the function of absolute activity of the 

 adsorbate (A= eu/kT)and the partitian function 

 for a molecule of bound adsorbate, q; Aq at 

 dilute solution is equivalent to KqM, where Kq 

 is intrinsic association constant for 1 molecule 

 of adsorbate with a single site and M is the 

 molar concentration of free adsorbate. There- 

 fore: 



39 



ain M 



exp (-W/2kT) 



9=% 



Estimation from the slope of the curve (Fig. 3, 

 open circle) yields a value of 30-60. In equation 



—I 1 — I 1 M 



Fig. 3. 



Adenosine bound per UMP of the poly U (1.5 x 10-2 W) 

 versus adenosine input concentration at 5°C, 0.4 ,W NaCl, 



O.OU/ phosphate (HMP) ( — • ). The fraction of poly 



U sites occupied versus free adenosine concentration 

 under the same condition is given by — o — . (From 

 Huang and Ts'o, J. Mvt. Biol. 16. 523, 1966; reproduced 

 with permission of Academic Press.) 



2 the W is calculated to be -5 to 6 Kcal/mole 

 which is the stacking energy of adenosine upon 

 pairing with 2 U of poly U. This is comparable 

 to the value of -4.8 Kcal/mole or -7.5 Kcal/mole 

 calculated for the stacking energy of poly dAT 

 and poly dI:dBC respectively by Crothers and 

 Zimm (9). 



Similar experiments were also performed 

 using cytidine or inosine as the dialysable 

 components. No detectable binding was found 

 even at input nucleoside concentration as high 

 as 2 X 10"2 M. Therefore, this interaction has 

 the same specificity as the system of long 

 chain polymers, i.e., the base pairing scheme 

 of Watson-Crick. The stoichiometry of this 

 binding reaction was studied by the solubility 

 measurements, and it was found at low tem- 

 perature that the stoichiometry is 2 U to 1 A, 

 while at 20° the stoichiometry becomes 1 A to 

 1 U. The physical properties of this poly U- 

 adenosine complex were further analyzed by 

 sedimentation, viscosity, and by optical rotation 

 measurement. 



The formation of poly U- adenosine (AR) 

 complex can be demonstrated by analytical 

 ultracentrifugation. Sedimentation coefficients 

 (S) of poly U in the absence (control) and pres- 

 ence of nucleosides are given in Table VII and 

 the patterns are shown in Fig. 4. When N-6- 

 methyladenosine, cytidine or inosine was mixed 

 with poly U in equal amounts (1.5 x 10"^ M each) 

 at 5° and 0.4 M NaCl, no change in either the 

 pattern or the S value was found. As adenosine 

 was mixed with poly U under identical condi- 

 tions, a 33% increase in S value and a sharpen- 

 ing of the boundary was observed as compared 

 with the control (Fig. 4). Similar results were 

 obtained in 0.02 M MgCl2 with the same mixture. 

 In 0.4 M NaCl, as the temperature was raised, 

 the percentage change in S value also increased 

 to 43% at 10° C and 53% at 19° C, but it was 

 accompanied by decrease in sharpness of the 

 boundary (Fig. 4). The specific viscosities of 

 poly U (1.5 X 10-2M) and poly U-AR complex 

 (1.5 X 10-2 Af of each) in 0.4 M NaCl at 5° were 

 respectively 0.602 and 1.05. As previously 

 stated a parallel increase in S value (33%) 

 has also been observed. The concurrent in- 

 crease in both specific viscosity and the sedi- 

 mentation coefficient of the poly U-AR complex 

 as compared with those of poly U, unambigu- 

 ously showed that there is a molecular weight 

 increase in the polymer resulting from the 

 complex formation. 



Optical rotation measurement at 350 mn 

 was used to determine the conformation and 



190 



