Engineering cytochrome P-450s

Cytochrome P-450 isozymes represent a critical component of nature’s spectrum of detoxification catalysts that could be exploited for bioremediation. The ethanol-inducible human cytochrome P-450 2E1 serves as a model eukaryotic P-450 that complements the bacterial P-450 cam in dehalogenation and detoxification of environmental pollutants. We explored the construction of novel chimeric P-450s using cytochrome P-450 camC and 2E1 genes. For construction of chimera 1 (478 amino acids, 55.14 kDa), 145 amino acids from the N-terminus of P-450 2E1 protein (493 amino acids, 56.84 kDa) were replaced with 130 amino acids from the N-terminus of P-450 camC protein (415 amino acids, 46.66 kDa). In chimera 2 (525 amino acids, 60.24 kDa) the strategy involves replacement of 28 amino acids in the C-terminus of chimera 1 with 75 amino acids from the C-terminus of P-450 camC gene. Homology models of both the chimeric proteins were developed using SWISS-MODEL based on the known crystal structure of cytochrome P-450 camC, BM-3, 1DT6A, and 2C17A. The models indicated that the proposed heme-binding site was intact, which is inevitable for catalytic activity of cytochrome P-450s. The expression of chimera 1 and 2 genes in Escherichia coli DH5α was evident from light-pink cell pellets, protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis, and diagnostic carbon monoxide-difference spectra. Our studies show that strategies can be developed to exploit the natural diversity of the P-450 superfamily to generate chimeric biocatalysts that would provide new templates amenable to directed evolution.     

Erratum

A technique is reported for measuring triplet spin-lattice relaxation times in fluid solution. It is based on the observation of chemically induced electron spin polarization, in the presence of a triplet quencher. Spin-lattice relaxation times of the order of 10 ns are reported for the duroquinone triplet in various solvents.     

Biaxial bulge testing of polymeric sheets

The through thickness total strains of hydrostatically bulged domes of materials are usually determined indirectly. The in-plane circumferential strains are readily measured, and then constancy of volume is invoked to determine the “missing” or unknown strain. Some of our recent work with polymers has shown that volume constancy in large-scale deformation does not hold so that the usual computations of stress and strain during bulging of sheet may be in error. We describe an electrical device that reduces such errors by directly measuring the through thickness (and, thus, the thickness “strain”) for nonmetallic materials.     

Magnetic field effects on chemiluminescent fluid solutions

The effect of a magnetic field on the intensity of fluorescence from chemiluminescent reactions occurring in fluid solutions is calculated. The calculation first considers pairs of triplet molecules rotating in each other's vicinity and calculates the effect of a magnetic field on the rate at which the overall singlet state is populated. The field diminishes this rate, as in the solid-state situation. The triplets are then allowed to diffuse apart, and the process of populating the overall singlet state of the pair is treated as a relaxation process occurring during the diffusive trajectory. In this case too at high fields the intensity diminishes. The calculations are repeated for triplet-doublet quenching, and, in accord with the solid-state results, the intensity is enhanced at high fields because the quenching rate for doublet-triplet collisions is diminished, and the resultant fluorescence arises via triplets which escape abortive D-T encounters. Explicit expressions involving the dynamical parameters of the fluid (e.g. the translational and rotational correlation times of the species in the solutions) are given for the fluorescence intensity.     

A problem structuring method for ecosystem-based management: The DPSIR modelling process

The purpose of this paper is to learn from Complex Adaptive Systems (CAS) theory to inform the development of Problem Structuring Methods (PSMs) both in general and in the specific context of marine management. The focus on marine management is important because it is concerned with a CAS (formed through the interconnection between natural systems, designed systems and social systems) which exemplifies their particularly ‘wicked’ nature. Recognition of this compels us to take seriously the need to develop tools for knowledge elicitation and structuring which meet the demands of CAS. In marine management, chief among those tools is the DPSIR (Drivers – Pressures – State Changes – Impacts – Responses) model and, although widely applied, the extent to which it is appropriate for dealing with the demands of a CAS is questionable. Such questioning is particularly pertinent in the context of the marine environment where there is a need to not only recognise a broad range of stakeholders (a question of boundary critique) but also to manage competing knowledge (economic, local and scientific) and value claims. Hence this paper emphasises how a CAS perspective might add impetus to the development of a critical perspective on DPSIR and PSM theory and practice to promote a more systemic view of decision-making and policy development.     

Phase calibration of sonar systems using standard targets and dual-frequency transmission pulses

The phase angle component of the complex frequency response of a sonar system operating near transducer resonance is usually distorted. Interpretation and classification of the received sonar signal benefits from the preservation of waveform fidelity over the full bandwidth. A calibration process that measures the phase response in addition to the amplitude response is thus required. This paper describes an extension to the standard-target calibration method to include phase angle, without affecting the experimental apparatus, by using dual-frequency transmission pulses and frequency-domain data processing. This approach reduces the impact of unknown range and sound speed parameters upon phase calibration accuracy, as target phase is determined from the relationship of the two frequency components instead of relying on a local phase reference. Tungsten carbide spheres of various sizes were used to simultaneously calibrate the amplitude and phase response of an active sonar system in a laboratory tank. Experimental measurements of target phase spectra are in good agreement with values predicted from a theoretical model based upon full-wave analysis, over an operating frequency of 50-125 kHz.