Denatu ration

Friedlander, J., and Hastings, J. W. (1967). The reversibility of the denatu-ration of bacterial luciferase. Biochemistry 6 2893-2900. 

Epirubicin, a successor to adriamycin, was studied in ovalbumin microspheres (115). The microspheres, prepared by a heat denatu-ration process, were 20 ym in diameter and contained 12.5% drug. 

Synthetic and natural polymers have been investigated which are biodegradable and biocompatible. The nanospheres are formed by precipitation of synthetic polymers or by denatu-ration/solidification of polymers of natural origin. Four techniques have been reported for preparing nanoparticles from synthetic preformed polymers. These include ... 

The temperature dependency of enzymatic activity is usually asymmetric. With increasing temperature, the increased thermal movement of the molecules initially leads to a rate acceleration (see p.22). At a certain temperature, the enzyme then becomes unstable, and its activity is lost within a narrow temperature difference as a result of denatu-ration (see p. 74). The optimal temperatures of the enzymes in higher organisms rarely exceed 50 °C, while enzymes from thermophilic bacteria found in hot springs, for instance, may still be active at 100 °C. 

The equations so far apply to a simple two-state equilibrium of D N. That is, protein denaturation is a cooperative (or all-or-none) transition in which denatu-ration occurs in a single step without any intermediates accumulating. How can we tell that this is so ... 

J,2/5/W3) < 0. This serves to lower the free energy difference between the two states, and when the native state becomes the higher energy state, protein denatu-ration should result. 

Bell et al. [20] found, via differential scanning calorimetry, that the denatu-ration temperature of lyophilized bovine somatotropin and lysozyme decreased, and therefore stability decreased, with increasing moisture content. The denatura-tion temperature decreased with increasing moisture irrespective of the excipient. The magnitude of the decrease in denaturation temperature was, however, dependent on the type of excipient. 

Law, A. J., and Ijeaver, J. (1997). Effect of protein concentration on rates of thermal denatu-ration of whey proteins in milk. /. A ric. Fooii Chem. 45, 425.S-4261. 

Carbonic anhydrase provides a good example of a protein with many prolines. It contains 15 I,ram and 2 cis prolines. After long-term denatu-ration virtually all molecules have nonnative prolines and refold slowly (Fransson etal., 1992). More than half of the slow-folding molecules form rapidly after conformational unfolding in a reaction that is about tenfold faster than expected for a single prolyl isomerization (Kern et al., 1995). This increase in rate is probably caused by the additive and independent isomerizations of the many trans prolines of carbonic anhydrase. The remainder of the slow-folding molecules are then created slowly in a reaction that probably reflects the cis trans isomerizations of one or both cis prolines. 

Native Ure2p isolated from yeast is a dimer (Taylor et al., 1999), as is that produced in Escherichia coli (Perrett et al., 1999). Chemical denatu-ration studies of a series of deletion mutants showed that the absence of the prion domain did not affect the overall stability of the Ure2p molecule (Perrett et al, 1999). This indicates that any energy of interaction of the C-terminal and N-terminal domains is equivalent to that of alternative C-terminal - C-terminal interations. 

It is useful at this time to review briefly some basic aspects of enzyme inhibition and enzyme inhibitors. For a broader review of enzymatic mechanisms and kinetics consult any of several excellent biochemistry textbooks. Enzyme inhibitors are chemical agents capable of modifying an enzyme s capacity to catalyze the reactions of its normal substrates. Effecting such changes of enzyme function by altering the pH, changing the temperature, or subjecting the system to radiation such as UV should properly be considered a denatu-ration process. 

Before the use of recombinant DNA technology to obtain large amounts of ferrochelatase, the main biochemical studies focused on the characterization of the kinetic mechanism of the enzyme. Apart from Fe +, these studies established that Zn and Co could also be used as substrates [33-35]. Curiously, Fe + cannot be utilized by the enzyme [3], while Mn " and Hg + are inhibitors of ferrochelatase activity [9, 13]. Cd + was proposed as an inhibitor which is competitive versus Fe + [36]. However, in a study where cadmium chloride was added in stoichiometric amounts to a 500-pM solution of purified mouse ferrochelatase, irreversible denatu-ration of the enzyme was observed [37]. Reanalysis of the original inhibition data [36], indicated that the inhibition of ferrochelatase by Cd + is likely to be due to protein precipitation rather than to competition between Fe " " and Cd + for the active site [36]. 

Proteins Fibrous proteins Globular proteins Alpha-amino acids Side chains Dipeptide Peptide linkage Polypeptide Primary structure Secondary structure Alpha-helix Pleated sheet Tertiary structure Disulfide linkage Denatu ration Enzymes... 

It is now more readily accepted that this denatu-ration results from the adsorption of tannins without dehydration (Kawamoto and Nakatsubo, 1997) (Figure 9.5). The new complex is an electronegative hydrophobic colloid. It remains stable in a clear solution if no salts are present, otherwise it flocculates. In the same way, natural wine proteins are denatured by heating and flocculate as the liquid cools. 

Third and most important, theorists were able to look at the denatu-ration transition of a random sequence heteropolymer, and to show that it, too, does not have a significant latent heat. This was achieved first in 1987 by J. Bryngelson and P. Wolynes at the University of Illinois at Urbana-Champaign, who suggested a phenomenological description, and soon thereafter (and independently) by E. Shakhnovich and A. Gutin in Pouschino near Moscow, who developed a real microscopic model. Let s discuss briefly the main idea of their arguments. 

Step 1 Denatu ration of double-stranded DNA to single strands. 

Denatu ration process + Detergent-supplemented solution... 

As to the nano-dimension of the material, the enzyme adapts its 3D configuration to the morphology of the underlying nanostructure without inducing protein denatu-ration, resulting in intimate contact between the nano-objects fixed to the electrode surface and the redox active sites of the enzyme, which are often located deep inside... 

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