Recovery of biological activity

Protein tertiary structure is also influenced by the environment In water a globu lar protein usually adopts a shape that places its hydrophobic groups toward the interior with Its polar groups on the surface where they are solvated by water molecules About 65% of the mass of most cells is water and the proteins present m cells are said to be m their native state—the tertiary structure m which they express their biological activ ity When the tertiary structure of a protein is disrupted by adding substances that cause the protein chain to unfold the protein becomes denatured and loses most if not all of Its activity Evidence that supports the view that the tertiary structure is dictated by the primary structure includes experiments m which proteins are denatured and allowed to stand whereupon they are observed to spontaneously readopt their native state confer matron with full recovery of biological activity... 

Poor recovery of biological activity with good recovery of protein... 

Recovery of biological activity too high (compared to the amount loaded)... 

Denaturation is accompanied by changes in both physical and biological properties. Solubility is drastically decreased, as occurs when egg white is cooked and the albumins unfold and coagulate. Most enzymes also lose all catalytic activity when denatured, since a precisely defined tertiary structure is required for their action. Although most denaturation is irreversible, some cases are known where spontaneous renaturation of an unfolded protein to its stable tertiary structure occurs. Renaturation is accompanied by a full recovery of biological activity. 

In addition to high resolution and short analysis times, perfusion chromatography has the advantage of improved recovery of biological activity because active biomolecules spend less time on the column, where denaturing conditions may exist. 

The volatile components from the urine of normal and adrenalectomized female mice were compared and chemical differences identified through gas chromatographic analysis.131 Six simple compounds accounted for the majority of the difference, at least from the perspective of concentration. These were 2-heptanone (41), ( )-hept-5-en-2-one (42), ( )-hept-4-en-2-one (43), -pentyl acetate (44), (Z)-pent-2-enyl acetate (45), and 2,5-dimethylpyrazine (46). When authentic samples of 41—46 were added to urine from adrenalectomized animals and their concentrations restored to the levels of normal urine, full recovery of biological activity (delay of first estrus) was observed.131 Subsequent studies have confirmed the biological responses to pyrazine 46.132... 

Many of the unfolding processes are irreversible however, some are reversible with the return of full biological activity. For example, proteins dissolved in DMSO refold upon dilution in water (Chang et ah, 1991). Supercritical anti-solvent precipitation of insulin, lysozyme and trypsin from DMSO yielded partially unfolded intermediates, as characterized by spectroscopy (Yeo et al., 1994 Winters et ah, 1996). However, these structures were reversible upon reconstitution in aqueous media, with recovery of biological activity (Yeo et ah, 1994 Winters et ah, 1996). Chymotrypsin also completely unfolded in DMSO, but regained activity upon rehydration (Zaks and Klibanov, 1988a Yeo et ah, 1994). 

An additional factor in selecting a resin is recovery of biological activity. The choice of elution conditions is critical to effect a good recovery. A proper pH-stability study must have been carried out and the biologically active compound must have been found to be stable over the range of pH values utilized. 

Biotechnology, chemistry, physics provide tire tools for target identification, for the creation of new molecular stnrctures, and for the recovery of biologically active molecules provided by the biosphere and efficiency-honed during continuous evolutionar y processes. 

Nykiforuk, C.L. et al.. Transgenic expression and recovery of biologically active recombinant human insulin from Arabidopsis thaliana seeds. Plant BiotechnoL, 4,77, 2006. 

The compounds listed in Table 1 were added, individually or in groups, to the urine of adrenalectomized female mice in order to determine their biological activity (Novotny et al., 1985). Our bioassay procedure and urine collection were similar to those described by Drickamer and coworkers (1978). As seen from Table 2, the mice treated with pyrazine alone and the acetates, or both types of compounds together, spiked into the urine from adrenalectomized, grouped females reached puberty significantly later than young females treated with ketones plus urine from adrenalectomized females, or water (control). The urine of adrenalectomized animals with all six compounds resulted in a full recovery of biological activity. 

Sarney DB, Hale C, Frankel G, VuHson EN. A novel approach to the recovery of biologically active ohgosaccharides fiom milk using a combination of enzymatic treatment and nanofiltration. Biotechnol Bioeng 2000 69(4) 461-7. 

If the antigen is to be used for immunization it may be sufficient to test that the required epitopes are present, but often it will be necessary to assay for recovery of biological activity. The antigen may be denatured or misfolded in some way that would not show up by these techniques. It is useful therefore to have some form of bioassay by means of which the purified ligand and the original starting material can be compared, this will of course depend on the nature of the antigen itself. 

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