Glucose oxidase, horseradish peroxidase, xanthine oxidase, and carbonic anhydrase have been adsorbed to colloidal gold sols with good retention of enzymatic activity. Adsorption of xanthine oxidase on colloidal gold did not result in a change in enzymatic activity as determined by active site titration with the stoichiometric inhibitor pterin aldehyde and by measurement of the apparent Michaelis constant (K'(M)). Gold sols with adsorbed glucose oxidase, horseradish peroxidase, and xanthine oxidase have also been electrodeposited onto conducting matrices (platinum gauze and/or glassy carbon) to make enzyme electrodes. These electrodes retained enzymatic activity and, more importantly, gave an electrochemical response to the enzyme substrate in the presence of an appropriate electron transfer mediator. Our results demonstrate the utility of colloidal gold as a biocompatible enzyme immobilization matrix suitable for the fabrication of enzyme electrodes.
Particularly stable elements of noncovalent structure in bovine carbonic anhydrase have been detected and studied. These are present in a highly populated intermediate state formed during denaturation of the enzyme with guanidinium chloride. The intermediate has been detected by analysis of the denaturation profiles, and some of its structural properties have been characterized by CD and fluorescence spectroscopy, including fluorescence polarization and lifetime measurements. Measurements have been made on the Zn2+-enzyme, Co2+-enzyme, and apoenzyme to ascertain the structural effects of the active-site Zn2+. Kinetic measurements indicate that this intermediate is on the folding pathway from the random coil to the native state.
Significance: The broad classes of O 2 -binding proteins known as hemoglobins (Hbs) carry out oxygenation and redox functions that allow organisms with significantly different physiological demands to exist in a wide range of environments. This is aided by allosteric controls that modulate the protein's redox reactions as well as its O 2 -binding functions. Recent Advances: The controls of Hb's redox reactions can differ appreciably from the molecular controls for Hb oxygenation and come into play in elegant mechanisms for dealing with nitrosative stress, in the malarial resistance conferred by sickle cell Hb, and in the as-yet unsuccessful designs for safe and effective blood substitutes. Critical Issues: An important basic principle in consideration of Hb's redox reactions is the distinction between kinetic and thermodynamic reaction control. Clarification of these modes of control is critical to gaining an increased understanding of Hb-mediated oxidative processes and oxidative toxicity in vivo. Future Directions: This review addresses emerging concepts and some unresolved questions regarding the interplay between the oxygenation and oxidation reactions of structurally diverse Hbs, both within red blood cells and under acellular conditions. Developing methods that control Hbmediated oxidative toxicity will be critical to the future development of Hb-based blood substitutes. Antioxid. Redox Signal. 18, 2298-2313.
General PrinciplesEmergence of proteins capable of transporting O 2 I n this review, we examine the adaptive changes in the molecular controls of hemoglobin (Hb) oxygenation and oxidation that have evolved to meet the highly varied physiological and environmental demands of respiring organisms. Globins came into being during the planet's long early period of anoxia/hypoxia, and recent studies show that globins are either expressed or inducible in almost all cells (98). Studies have shown that one evolutionary pathway of Hb is that of a multipurpose domain attached to a variety of unrelated proteins, thus forming molecules with different functions (126). This pathway has allowed structurally distinct Hbs to evolve: (i) to protect against the high levels of nitrosative stress of the earth's early environment; (ii) to protect against O 2 -linked oxidation; (iii) to act as O 2 sensors that help regulate the expression of proteins during periods of hypoxia or anoxia; and (iv) to enable aerobic respiration by facilitating diffusion and/or acting as O 2 carriers (44,56,57,70,71,73,107). A common theme in the fascinating story of Hb evolution is the emergence of distinct mechanisms for controlling Hb's oxygenation and redox functions.Since increased amounts of O 2 were released in our planet's early history, O 2 toxicity brought about species extinction on a global scale. On the other hand, this ''oxygen pollution'' made possible a new biological process, that of aerobic respiration. A tremendous gain in the energy obtainable from oxidation of energy-rich metabolites was achieved when organisms evolve...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.