Enzymes critical parameters

A two-variable model taking into account the allosteric (i.e. cooperative) nature of the enzyme and the autocatalytic regulation exerted by the product shows the occurrence of sustained oscillations. Beyond a critical parameter value, the steady state admitted by the system becomes unstable and the system evolves toward a stable limit cycle corresponding to periodic behavior. The model accounts for most experimental data, particularly the existence of a domain of substrate injection rates producing sustained oscillations, bounded by two critical values of this control parameter, and the decrease in period observed when the substrate input rate increases [31, 45, 46]. 

Pro-drugs must not be cleaved by digestive enzymes of the upper GI tract and should not be susceptible to chemical hydrolysis. Moreover, pro-drug absorption in the small intestine should be negligible. Because of these requirements, the hydrophUicity, the molecular weight and the charge of the carrier molecules have to be regarded as critical parameters. 

In the described systems the length is not a critical point by itself. The critical parameter is the Thiele modulus (including length, enzyme density, and diffusion properties). This parameter is a dimensionless one. 

A critical parameter for the development and application of this approach is the use of enzyme-specific assays for the assessment of the catalytic activities of individual enzymes in human liver microsomes. Cytochrome P450 form-specific assays have been developed for in vitro measurement of most human cytochrome P450 forms. Many of these specific assays will be discussed in Section 6. 

As previously discussed, the protection of pDNA against degrading enzymes is a critical parameter for a non-viral carrier. Such ability is needed for the polyplex to protect the nucleic acid for an extended period of time in the blood while the polyplex circulates and distributes. Research conducted in 1999 by Richardson and coworkers [101] to study the ability of chitosan to protect against DNase degradation revealed that incubation of polyplexes prepared at NIP ratio of 3/1 in the presence of DNase I (8 U, 1 h incubation) protected pDNA from degradation. Other studies of chitosans as gene delivery vehicles confirm that the NIP ratio has to be at least 3/1 to 5/1 in order to provide a sufficient protective effect against DNases. 

These studies of protein structure, like those of urea and methylamine effects on enzyme kinetic parameters, reveal that physiological mixtures of counteracting solutes achieve essentially the same end result as use of compatible solutes regulating cell volume through adjusting concentrations of compatible solutes or counteracting solutes (at the appropriate ratio of concentrations) conserves critical aspects of protein structure and function as well as cell volume per se. 

One of the critical factors in the design of experiments to determine the values of steady-state enzyme kinetic parameters is the optimal choice of a range of substrate concentrations. On the basis of the data in Fig. 5-28, suggest a possible optimal range of substrate concentrations. 

Stability of enzymes immobilized in electrodes of EFC power sources remains a critical parameter of the technology. Enzymes dispersed in buffer solutions typically have lower stability compared with firozen or lyophilized (freeze-dried) enzyme preparations. Therefore, it is expected that EFC power systems will be delivered and stored in dry or frozen form. For both cases, recommended and maximum allowed storage temperature ranges must be defined. Despite enzyme immobilization protocols (see Chapter 11) that improve enzyme stability, long storage periods at temperatures above 60 °C remain a challenge for most EFC systems. 

Enzyme amount and incubation time are critical parameters for successful digestions. For example, too much enzyme will only contribute, on gel analysis, to the smearing of DNA after only 5 to 10 min incubation. For best results, a given batch of Bal31 enzyme should be calibrated for its digestion rate with a standard substrate such as linearized plasmid DNA or, preferably, with the target DNA. 

The solvent is also a critical parameter as it must be compatible with both catalysts avoiding their deactivation and must be inert with the acyl donor. The requirement of an initial activation of metal catalyst for the racemization step has led to the common selection of THF and toluene as ideal solvents, the latter having the advantage of its higher boiling point where a high temperature is required in the system although temperatures over 100 °C are not recommendable due to enzyme denaturation. 

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