A method of resuspending small vesicles separated from suspension by protamine aggregation and centrifugation

• Published in: Analytical biochemistry Vol. 120; no. 1; pp. 113 - 124
• Main Authors: Gunter, K.K., Gunter, T.E., Jarkowski, A., Rosier, R.N.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.1982
• Subjects: Animals; Centrifugation; Erythrocyte Membrane; Heparin; Liposomes - isolation & purification; Methods; Phospholipids; Protamines; Proteolipids; Rats; Sarcoplasmic Reticulum; Submitochondrial Particles
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/0003-2697(82)90326-8

The technique of separating small vesicles from suspension by aggregation with protamine or polylysine, followed by either filtration or centrifugation has proved a very useful one. This is because the minimum times necessary to separate vesicles from the suspending medium are quite short (15 to 30 s) and the separation is almost complete (over 98% in the cases tested). The primary limitation of this technique, until now, has been that it could only be used for a terminal assay, because the aggregates produced could not be easily disaggregated. It has been found that after aggregation of vesicles by protamine and their separation from suspension by low-speed centrifugation, the addition of an excess of heparin over protamine, followed by vortexing or homogenizing the pellet in fresh medium, satisfactorily disaggregates the vesicles. This process has been tested with phospholipid vesicles and proteoliposomes and with vesicles from inner mitochondrial membrane, sarcoplasmic reticulum, and red blood cell membrane, producing satisfactory resuspensions in all cases tested. Leakage of [ 14C]sucrose or 86Rb + from phospholipid vesicles was not found to be significantly more rapid from protamine-heparin-treated vesicles, than from similar vesicles treated with protamine alone or from untreated control vesicles. Good separation of the resuspended vesicles from the remaining protamine and heparin was obtained with phospholipid vesicles and proteoliposomes using a gel filtration technique. Most of the protamine and heparin could be removed from the vesicles made of natural biological membrane using a combination of ion exchange chromatography and gel filtration. It was found possible to use this aggregation-centrifugation-disaggregation technique to increase the concentration of cytochrome oxidase vesicles without significantly decreasing the respiratory control ratio for these vesicles below that of a control which was only vortexed.