Poly(o-aminophenol)-modified bienzyme carbon paste electrode for the detection of uric acid.

A reagentless uric acid selective biosensor constructed by immobilising uricase and horseradish peroxidase (HRP) in carbon paste without the addition of an electron transfer mediator is described. The response of the electrode is based on the enzymatic reduction of hydrogen peroxide in the presence of uric acid. Uricase and HRP were dispersed in the carbon paste and the optimum paste mixture was determined. Poly(o-aminophenol) was electropolymerised at the working surface area of the electrode acting as a conducting polymer layer. Cyclic voltammetry was used to characterise the permselective characteristics of the polymer layer. At an applied potential of 50 mV vs. Ag/AgCl, a linear response was obtained up to 1 x 10(-4) M, with a limit of detection of 3 x 10(-6) M. The sensor had a response time of 37 s. a calibration precision of 2.2% (n = 4) and an estimated sample frequency of 20 h(-1). Responses to the analyte of interest were pH dependent. The sensor was incorporated into a flow injection system for the qualification of uric acid in human serum. Results compared favourably with a standard spectrophotometric method.

Effects of chemical modifiers on recombinant factor VIII activity.

Factor VIII performs a critical role in the blood coagulation cascade but is extremely labile. We have carried out chemical studies on a fully functional recombinant Factor VIII (rFVIII) using a variety of protein modifying agents, some of which were bifunctional. Thiol-specific maleimides cause no activity loss despite a substantial decrease in rFVIII's free thiol content. Mild oxidation procedures had either neutral or adverse effects on activity. Amino-specific reagents led to significant losses of rFVIII procoagulant activity. It appears that free amino groups are essential for Factor VIII's function while some at least of its many free thiol groups are not. None of these derivatives was any less labile than unmodified rFVIII. Systematic chemical modification of important proteins in this way can give useful insights into appropriate protein engineering strategies.

Understanding and increasing protein stability.

This review surveys the processes leading to loss of protein function and the types of molecular interaction that help stabilize proteins. It considers the effects of organic solvents on stability and the special features of thermophilic proteins. The deliberate manipulation of stability by protein engineering is discussed using the enzyme subtilisin as example. Both random and rational mutations of this protein have led to variants with greatly improved tolerances of high temperatures and organic solvents. One can also use chemical modification to modify protein stability and some of the main approaches are reviewed. The chemical and genetic strategies are complementary and have been combined to stabilize cytochrome c by metal-mediated cross-linking following site-specific mutagenesis. The article concludes by summarizing the beneficial effects of certain additives on protein stability.

Thermostabilized chemical derivatives of horseradish peroxidase.

Horseradish peroxidase finds a variety of uses in analysis, immunology, organic synthesis, and biosensors. Although moderately stable, its applicability to biosensors and other fields would be greatly enhanced if it could be made yet more stable. Appropriate chemical modification can substantially stabilize enzymes. Here we describe the use of bis-imidates and of bis-succinimides to modify free amino groups of commercial horseradish peroxidase under mild conditions of pH and temperature. Imidates yielded a marginal stabilization. Some of the succinimide derivatives, however, are much more thermostable than the native enzyme. Apparent half-lives indicate stabilizations of 6- to 23-fold, depending on the bis-succinimide used. These modifications preserve the carbohydrate side chains for subsequent reaction or immobilization.

Horseradish peroxidase: the analyst's friend.

HRP is a calcium-containing, extensively glycosylated, stable haemoprotein possessing four disulphide bridges and weighing 44 kDa. It is an oxidoreductase which can use a wide variety of hydrogen donors to reduce hydrogen peroxide. This gives rise to a range of colorimetric, fluorimetric, chemiluminescent and electrochemical assays for HRP activity. HRP is very widely used as an indicator in immunoassays, non-isotopic DNA probes, cytochemistry, bi-enzyme systems and biosensors. This is owing to its stability, high catalytic rates, ease of conjugation to other molecules and wide choice of assays of activity.

Functionally-stabilized proteins--a review.

The maintenance or stabilization of protein or enzyme function is of vital importance in Biotechnology. Investigations of thermophilic organisms, studies of denaturation and the use of enzymes in organic solvents have each contributed to an understanding of protein stability. Enzymes can reliably and reproducibly be stabilized by variety of means including immobilization, use of additives, chemical modification in solution and protein engineering. Examples of each of these are discussed. With these recent advances it appears that a rational strategy for achieving a particular stabilized enzyme or protein may be within reach.