Analysis of the Domain Structure of Membranes by Fragmentation and Separation
Our aim with separation is often preparative. We want to obtain a purification of a desired substance. Separation can also be used as an analytical tool if we want to learn about the composition of a mixture or the heterogeneity of a cell population. I would like to point out the possibility of using separation in combination with fragmentation for gaining information on the structure of a cell organelle or a biological membrane. Selective fragmentation with specific enzymes followed by separation and puzzle analysis is used for the determination of the sequence of proteins and nucleic acids. A similar approach should be possible in the case of larger structures such as membranes. Since we do not yet have specific enzymes available which can cleave the membranes selectively, we have to resort to random fragmentation. Consider for example an object (Fig. 1) composed of three domains A, B and C. If we break this to pieces by a mechanical random fragmentation we will get fragments varying in size and composition. If we have in our hands two separation methods, one of which can separate according to size and the other according to chemical composition, we can get the diagrams of Fig. 1. The large fragments obtained by centrifugation will not give distinct peaks (Fig. 1a) while the smallest fragments will give three well separated peaks when separated by a method, such as aqueous phase partition, which separates according to surface chemical composition. Between peak A and B we will find fragments composed of A and B, and between peaks B and C we will find fragments composed of B and C. However, since we will not find any fragments composed of only A and C, we can conclude that A and C cannot be neighbours in the original object. Thus, fragmentation and separation has given us information of the structure of the original object.
KeywordsDistinct Peak Specific Enzyme Large Fragment Fraction Number Cell Organelle
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