7] DIALYSIS STUDIES 113 



These two fractions showed no significant difference in enzymatic activity, 

 optical activity, or in their chromatographic behavior on an ion exchange 

 column.^'* When both fractions were separately re-run in the dialysis pro- 

 cedure there was a noticeable difference in the slope and position of the 

 slower component, but quantitatively much less than would have been 

 expected from the initial run. 



20 r 



Effect of salt in 0.01M acetic acid 



.00001 



.0001 .001 .01 .1 

 Ionic atpength 



Fig. 6. Effect of ionic strength on 50 % escape time. 



That the effect noted was due to the solute rather than to some property 

 of the membrane was shown by an experiment with a larger amount of 

 the protein initially. At a time just prior to the break in the curve the 

 inside solution was transferred directly to a new, freshly prepared membrane 

 and cell. The effect from this point on was much more pronounced with a 

 retarded escape rate and with the break much nearer the origin. This de- 

 monstrated that the effect was not due to a tendency of the membrane to 

 become 'plugged'. The most likely explanation of the break in the curve 

 would appear to be based on the possibility of more than one configuration, 

 state of aggregation, degree of folding or shape in a single protein and with 

 a sufficiently slow rate of interconversion so that two escape rates are regi- 

 stered in the curves. 



Thus far, different membranes from the same roll have shown remarkable 

 uniformity. Even from roll to roll in the same size there has been good 

 uniformity. However, it would appear advisable to standardize each roll 

 since two 20/32 rolls obtained recently have shown a slower escape rate 

 than the earlier ones used to accumulate most of the data presented in this 

 paper. 



The escape rate can be reproducibly increased by stretching a wet mem- 

 brane and decreased by acetylating it. A slower membrane when acetylated 

 gives a slower escape rate for a solute (e.g., with 18/32 bacitracin barely 

 passes) than a faster one (e.g., 20/32 gives a 50% escape time of 3 hours)« 



Hps 



