Mechanism of Denaturation

Protein Denaturation Part C. Theoretical Models for the Mechanism of Denaturation Charles Tanford... 

Gurtoo HL, Marinello AJ, Struck RF, et al. 1981. Studies on the mechanism of denaturation of cytochrome P-450 by cyclophosphamide and its metabolites. J Biol Chem 256 11691-11701. 

The mechanisms of denaturation during frozen storage and of the cryoprotective effects have been discussed and a hypothetical model has been presented. 

An alternative approach to the patterning of enzymatic activity is to start with a substrate uniformly coated with enzyme and to locally deactivate the enzyme in order to generate the pattern. This can be achieved by the generation of denaturing agents at the tip, e.g., Br2/HOBr (15). Although the mechanism of denaturation is not well understood, geometrically well-defined disks and lines of deactivated diaphorase could be produced and im-... 

Most of the early work on this subject was done by extracting actomyosin from fish fillets following frozen storage and thawing. On the other hand, many studies designed to determine the mechanisms of denaturation have involved frozen solutions or suspensions of isolated protein preparations. 

Tanford, C. Protein denaturation. Part C. Theoretical models for the mechanism of denaturation. Adv. Protein Chem. 1970,25, 1-95. 

The previous section has clarified the mechanism of denaturation induced by the addition of cosolvents. Here, we discuss how the cosolvents can enhance or protect proteins from denaturation, which can be caused by the change in the environment (temperature, pressure, etc.). Some organisms, living under extreme conditions, exploit various cosolvents to protect their precious proteins from denaturation. Here, I focus particularly upon cosolvent effects on thermal denaturation. 

C. Tanford, Adv. Protein Chem., 24, 1 (1970). Protein Denaturation. Part C. Theoretical Models for the Mechanism of Denaturation. 

The mechanism of antiperspinant action has not been fully estabHshed but probably is associated with blockage of ducts leading to the surface by protein denaturation by aluminum salts. The FDA has mandated that an antiperspinant product must reduce perspiration by at least 20% and has provided some guidelines for testing finished products. Some antiperspinant chemicals are Hsted in Table 14 (63). 

Another important protein of the Clp family is ClpB which possesses ATPase activity. In a clpB mutation, 45% of the denatured and aggregated protein arising transiently after the transfer of an E. coli culture from 30 to 45 °C is stabilized [14]. ClpB seems to play an important role in the renaturation or proteolysis of the aggregated proteins, but the mechanism of action of ClpB is not yet known. One can suppose that it might participate in the resolubilization of aggregated proteins. 

The shaking of protein solutions may lead to aggregation and precipitation as a result of several mechanisms, such as air oxidation, denaturation at the interface, adsorption to the vessel, or mechanical stress. These possibilities were systematically examined for solutions of human fibroblast interferon [50]. In this example, mechanical stress was identified as the causative factor in the inactivation. The proposed mechanism of inactivation by mechanical stress was through orientation of the asymmetrical protein in the... 

In general, the mechanism of heat and alkaline solution for DNA extraction may be based upon a hypothesis, previously proposed for the AR technique.32 Strong alkaline solution may denature and hydrolyze proteins, resulting in breaking cell and nuclear membranes as well as disrupting cross-linkages due to formalin fixation. It is no surprise to observe the similarity between retrieval of nucleic acid and retrieval of protein (antigen) based on a similar chemical reaction of formaldehyde with these two kinds of macromolecules (Fig. 3.1).15"19... 

These thermal analysis studies serve to establish a direct relationship between a heat-induced AR method and the reversal of formalin-induced intra- and intermolecular protein cross-links.10 2831 Further, while formalin-treatment provides thermal stability to RNase A, this stabilization is not sufficient to prevent thermally induced protein denaturation at temperatures (>100°C) typically used in heat-induced AR methods.32 34 The implications of this finding for the mechanism of AR will be discussed further in Section 15.6. 

The derivation of an intercalation association constant from kinetic studies of BPDE hydrolysis presumes that the reaction proceeds via an intercalated complex This mechanism is supported by the observations that the catalytic activity of denatured DNA is lower than that of native DNA (8), that catalysis is inhibited at high ionic strengths ( 3, 8, 17), and that mononucleotides such as GMP exhibit much greater catalytic activity than does free phosphate (80). 

Given that hydroxylamine reacts rapidly with heme proteins and other oxidants to produce NO [53], the hydrolysis of hydroxyurea to hydroxylamine also provides an alternative mechanism of NO formation from hydroxyurea, potentially compatible with the observed clinical increases in NO metabolites during hydroxyurea therapy. Incubation of hydroxyurea with human blood in the presence of urease results in the formation of HbNO [122]. This reaction also produces metHb and the NO metabolites nitrite and nitrate and time course studies show that the HbNO forms quickly and reaches a peak after 15 min [122]. Consistent with earlier reports, the incubation ofhy-droxyurea (10 mM) and blood in the absence of urease or with heat-denatured urease fails to produce HbNO over 2 h and suggests that HbNO formation occurs through the reactions of hemoglobin and hydroxylamine, formed by the urease-mediated hydrolysis of hydroxyurea [122]. Significantly, these results confirm that the kinetics of HbNO formation from the direct reactions of hydroxyurea with any blood component occur too slowly to account for the observed in vivo increase in HbNO and focus future work on the hydrolytic metabolism of hydroxyurea. 

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