Site Exploration - Direct Methods

There are no given rules regarding the location of boreholes or drillholes, or the depth to which they should be sunk. This depends upon the geological conditions and the type of project concerned. The information provided by the desk study, the preliminary reconnaissance and from any trial trenches should provide a basis for the initial planning and layout of the borehole or drillhole programme. Holes should be located so as to detect the geological sequence and structure. Obviously, the more complex this is, the greater the number of holes needed. In some instances, it may be as well to start with a widely spaced network of holes. As information is obtained, further holes can be put down if and where necessary. 

Determining main soil type boundaries, relative permeabilities and periglacial studies 

Possible diversion routes. Ground needing clearing. Suitable areas for irrigation 

Location of sand and gravel, clay, rip-rap, borrow and quarry sites 

Avoidance of major obstacles and expensive land. Best graded alternatives and ground conditions. Sites for bridges. Pipe and power line reconnaissance. Best routes through urban areas Detection of shafts and shallow abandoned workings, subsidence features 

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Chemical modification of surface residues of HRP is one method which may offer some improvement in thermal or long-term stability of the enzyme. The -amino groups of the six surface Lys residues can be modified by reaction with carboxylic anhydrides and picryl sulfonic acid (296). In this example the number of sites modified was found to be more significant than the chemical nature of the modification, at least as a criterion for improved stability. Other methods explored include the use of bifunctional crosslinking reagents to couple surface sites on the enzyme (297). Future developments are likely to be concerned with the selection of site-directed mutants of HRP C that show enhanced thermal stability. Dramatic increases in thermal stability of up to 190-fold have been reported recently for mutants of Coprinus cinereus peroxidase (CIP) generated using a directed evolution approach (298). 

The enzyme mechanism, however, remains elusive. Quantum mechanical models generally disfavor C6-protonation, but 02, 04, and C5-protonation mechanisms remain possibilities. Free energy computations also appear to indicate that C5-protonation is a feasible mechanism, as is direct decarboxylation without preprotonation O-protonation mechanisms have yet to be explored with these methods. Controversy remains, however, as to the roles of ground state destabilization, transition state stabilization, and dynamic effects. Because free energy models do take into account the entire enzyme active site, a comprehensive study of the relative energetics of pre-protonation and concerted protonation-decarboxylation at 02, 04, and C5 should be undertaken with such methods. In addition, quantum mechanical isotope effects are also likely to figure prominently in the ultimate identification of the operative ODCase mechanism. 

A. Overview of Methods for Exploring the Solution Structure of Rhodopsin Site-Directed Spin Labeling, Sulfhydryl Reactivity, and... 

The effect of pH variation and isotope (or elemental) substitution on reaction kinetics has been used in the steady state to explore the roles of active site acid/base catalysts and to attempt to define the nature of the transition state (8a, 8b, 58). Each of these methods also depends on the extent to which the rate of the chemical reaction is rate limiting in the steady state. If some other step limits the rate of steady-state turnover, then changes in the rate of the chemical reaction will be obscured. Use of pH variation or isotope effects in transient kinetic experiments has been useful in a number of cases (27), especially where it has been possible to examine directly the rate of the chemical reaction at the enzyme active site. In these cases, the effect of pH or isotope substitution can be interpreted directly in terms of the effect on a single reaction. 

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