Biological macromolecules, purification

Macromolecules such as proteins, polysaccharides, nucleic acids differ only in their physicochemical properties within the individual groups and their isolation on the basis of these differences is therefore difficult and time consuming. Considerable decreases may occur during their isolation procedure due to denaturation, cleavage, enz3rmatic hydrolysis, etc. The ability to bind other molecules reversibly is one of the most important properties of these molecules. The formation of specific and reversible complexes of biological macromolecules can serve as basis of their separation, purification and analysis by the affinity chromatography [6]. 

The interaction between biological macromolecules can be of various degrees of complexity. These interactions may be of high specificity, as in antigen-antibody complexes, or of low specificity, as in protein-lipid associations. The complexes that are formed may be very stable, or highly unstable. These properties of a complex must be considered in selecting an affinity adsorbent to be used for the purification of a specific substance. 

Affinity chromatography can be applied to the isolation and purification of virtually all biological macromolecules. It has been used to purify nucleic acids, enzymes, transport proteins, antibodies, hormone receptor proteins, drug-binding proteins, neurotransmitter proteins, and many others. 

The development of methods and techniques for the separation and purification of biological macromolecules such as proteins has been a prerequisite for advances in bioscience and biotechnology during the last five decades. 

The economic feasibility of a bioreaction process clearly depends on the characteristics of the associated bioseparation process, especially in the usual case when the product is present at low concentration in a complex mixture. For example, the existence of an extremely efficient and low-cost separation process for a particular compound could significantly lower the final concentration of that compound required in the bioreactor to achieve a satisfactory overall process. After noting that special approaches and processes are needed for efficient recovery of small molecules (ethanol, amino acids, antibiotics, etc.) from the dilute aqueous product streams of current bioreactors, I shall discuss further only separations of proteins. These are the primary products of the new biotechnology industry, and their purification hinges on the special properties of these biological macromolecules. 

I continue with biological macromolecules but in moving to a medical center I realized that the photosynthetic reaction center should not be my main interest. I also knew that studying more photosynthetic reaction centers was an entirely new issue. It required different approaches and sources of protein, purification, and it was a good point in time to switch. There were many scientists here that needed structural information on medically relevant proteins. Although nobody told me what to do, I sensed that the expectation was to focus on mammalian and human proteins and that s what I did. 

Hydrophobic interaction chromatography (HIC) is a mode of separation in which molecules in a high-salt environment interact hydrophobically with a nonpolar bonded phase. HIC has been predominantly used to analyze proteins, nucleic acids, and other biological macromolecules by a hydrophobic mechanism when maintenanee of the three-dimensional structure is a primary eoneern [1-4]. The main applications of HIC have been in the area of protein purification because the reeovery is frequently quantitative in terms of both mass and biological activity. 

The separation of nucleic acids, particularly t-RNA, has been another useful application of HIC for biological macromolecules. The tertiary structure of t-RNA has made analysis under the gentle conditions of HIC very feasible [12]. Figure 2 shows an example of the purification of t-RNA molecules specific for different amino acids on a lOO-nm polyol HIC column. Separation of t-RNA molecules has also been accomplished successfully by using HIC conditions on supports with alkylamino ligands, which are functionally similar to those traditionally used to separate nucleic acids [I]. 

Beis, K., Whitfield, C., Booth, I., and Naismith, J.H. 2006. Two-step purification of outer membrane proteins. International Journal of Biological Macromolecules 39 10-14. 

Gel filtration[91 is a key method in the purification of enzymes as well as biological macromolecules. It is reliable and simple as a separation technique without adsorption and interaction on gel filtration media. In gel filtration, the principle of separation is very simple, and macromolecules in solution are separated based on differences in their size as they pass through a column (Fig. 2-12). Large molecules pass through the stationary phase first while smaller molecules move about the gel filtration medium slowly. Gel filtration is also called molecular-sieve chromatog-... 

Crystallisation. The ultimate in purification of proteins or nucleic acids is crystallisation. This involves very specialised procedures and techniques and is best left to the experts in the field of X-ray crystallography who can provide a complete picture of the structure of these large molecules. [A. Ducruix and R. Giege Eds, Crystallisation of Nucleic Acids and Proteins A Practical Approach, 2nd Edition, 2000, Oxford University Press, ISBN 0199636788 (paperback) T.L. Blundell and L.N. Johnson Protein Crystallisation, Academic Press, NY, 197, A. McPherson Preparation and Analysis of Protein Crystals, J.Wiley Sons, NY, 1982, A. McPherson, Crystallisation of Biological Macromolecules, Cold Spring Harbour Laboratory Press, 2001 ISBN 0879696176, see also Bibliography in Chapter 1.]... 

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