Denaturant, critical concentration

It is common knowledge that amphiphilic molecules, such as sodium dodecyl sulfate, above a certain critical concentration in water form assembled suiictures in which the hydrophobic units are clustered together. The notice of a hydrophobic effect was brought to light by Walter Kauzmann, whilst studying forces that influenced protein denaturation [27]. An excellent critical review on interfaces and the driving forces of hydrophobic assembly was written by Chandler in 2005 [28]. 

Sodium dodecyl sulphate is bound co-operatively to most protein, the critical concentration for co-operative binding being about 25 % of the CMC. Non-ionic surfactants and the bile salts do not usually induce co-operative binding and do not usually, therefore, denature proteins, although dissociation into inactive or... 

The existence of a critical concentration of denaturant was reported for the first time in the study by Goldberg (1972) of urea denatured j8-galactosidase refolding. This enzyme is completely renatured after denaturation by 8 M... 

When exposed for at least 1 hr to the critical concentration of denaturant (3 M urea), native tryptophanase also recovers the native structure after removal of the denaturant (London et u/., 1974). In this case the presence of aggregates was reported and this apparent irreversibility was attributed to aggregation. 

The occurrence of a critical concentration of denaturant, for which the irreversibility was observed, was interpreted to be caused by the formation of aggregates (London et a/., 1974). It was even proposed that aggregation arises from specific interactions between complementary association areas of two different molecules rather than within the same molecule. The dependency of the phenomenon on concentration and direct observation of aggregates was accepted as supporting this hypothesis. 

Mancini et aL, 1965). The reversibility of the process, when protein was incubated in intermediate concentrations of GuHCl, was analyzed by this method and compared with the return of enzymatic activity (see Chapter 5). Both methods indicate the same decrease in reversibility when elastase was incubated at critical concentration of denaturant. By contrast, for hen egg white and phage T4 lysozyme, a perfect reversibility was observed (Desmadril et aU 1982b). 

A preferential pathway of folding was shown for elastase. The same folded but inactive intermediates being formed and trapped in critical concentration of denaturant,in the denaturation as well as in the renaturation process (Ghelis and Zilber, 1982 Ghelis, 1980). 

Another aspect of polysorbates is that they are inherently susceptible to oxidative degradation. Often, as raw materials, they contain sufficient quantities of peroxides to cause oxidation of protein residue side chains, especially methionine (59). The potential for oxidative damage arising from the addition of stabilizer emphasizes the point that the lowest effective concentrations of excipients should be used in formulations. For surfactants, the effective concentration for a given protein will depend on the mechanism of stabilization. It has been postulated that if the mechanism of surfactant stabilization is related to preventing surface-denaturation, the effective concentration will be around the detergent s critical micellar concentration. Conversely, if the mechanism of stabilization is associated with specific protein-detergent interactions, the effective surfactant concentration will be related to the protein concentration and the stoichiometry of the interaction (39). 

One of the critical factors in excipient selection and concentration is the effect on preferential hydration of the biopharmaceutical product [53, 54], Preferential hydration refers to the hydration layers on the outer surface of the protein and can be utilized to thermodynamically explain both stability enhancement and denatur-ation. Typical excipients used in protein formulations include albumin, amino acids, carbohydrates, chelating and reducing agents, cyclodextrins, polyhydric alcohols, polyethylene glycol, salts, and surfactants. Several of these excipients increase the preferential hydration of the protein and thus enhance its stability. Cosolvents need to be added in a concentration that will ensure their exclusion from the protein surface and enhance stability [54], A more comprehensive review of excipients utilized for biopharmaceutical drug products is available elsewhere [48],... 

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