RAPID METHOD FOR SEPARATING SMALL VESICLES FROM SUSPENSION

Techniques for rapidly aggregating suspensions of small vesicles made either of natural biological membrane or of phospholipid or phospholipid-protein mixtures by addition of one of the basic polypeptides, polylysine or protamine, have been investigated. Either filtration or centrifugation may be used to rapidly (15 to 30 s) and completely (over 98%) separate the vesicular aggregate from the suspending medium. At low values of the polylysine-to-vesicle weight ratio, aggregation is observed to increase with increasing polylysine concentration. At high values of this same ratio, aggregation decreases with increasing polylysine concentration. With protamine, like polylysine, aggregation increases with protamine concentration at low values of the weight ratio. At high values of the ratio, aggregation induced by protamine does not rapidly decrease with increasing protamine concentration as it does with polylysine. Explanations are given for these observations. While leakage induced by polylysine may be troublesome under some conditions with phospholipid vesicles or reconstituted systems, protamine aggregation has been found to induce much less leakage than polylysine aggregation. The leakage rate induced by protamine was not found to be significantly different from the control leakage rate for the first few minutes after addition of protamine under any of the conditions tested. Since this provides ample time for filtration, the protamine aggregation-filtration technique seems to be the method of choice for separation of many types of small vesicles from the suspending medium. It combines the advantages of rapid separation, complete separation, low rate of vesicle leakage, and versatility in being able to separate the vesicles from uncharged molecular components such as sugars where ion-exchange techniques will not work (1), as well as from ionic components. Low cost and simplicity are further advantages of the technique. © 1979.