Adsorption of proteins from solution at the solid-liquid interface.

Globular proteins at solid-liquid interfaces.

Cytochrome P450: structure, function, and generation of reactive oxygen species.

The rapid adsorption of plasma proteins is the initial event in a complex series of reactions that occurs upon introduction of a synthetic material into the cardiovascular system. This protein surface layer has been shown to modify the interaction of various blood components at interfaces. The activity of soluble enzymatic factors involved in coagulation can be established, as in the case of factor XII,] or greatly enhanced as occurs following prothrombin adsorption to lipid vesicles.' Thrombosis may be induced through the rapid adsorption and desorption of denatured t h r ~ m b i n , ~ by alteration of the adhesion of platelets .l--B and red blood cells,' or the hemolysis of red blood cells.s Although the relative importance of coagulation and thrombosis remains at issue, protein adsorption has a central role in each case. The parallel mechanisms are illustrated schematically in FIGURE 1. In view of the ever-increasing use of artificial materials in the body, better techniques and information are required to characterize the major events occurring at the blood-implant interface. If the properties of proteins adsorbed at the solid/solution interface were known, the compatibility of synthetic materials in vivo might be predictable and subject to greater control. The ability to minimize thrombosis by an informed selection or design of materials might prevent such clinical problems as the formation of deposits impairing the mechanical function of heart valves, the shedding of emboli, or, in the case of large surface area dialyzers and oxygenators, the removal of blood factors from the circulation. This article summarizes recent quantitative studies concerning the surface chemical properties of plasma proteins. The review is not exhaustive, but it is hoped that most of the current concepts and approaches are indicated. Although the adsorption of plasma proteins and its role in surface-induced thrombosis are emphasized, many of the techniques described would be equally applicable to investigations of matrix insolubilized enzymes, clinical analysis by antibody flocculation, and cellular adhesion and aggregation.

The Adsorption and Conformation of Plasma Proteins: a Physical Approach

The cytochrome P-450-catalyzed insertion of an oxygen into a substrate culminates a process that reduces molecular oxygen to a species equivalent, in terms of formal electron count and reactivity, to an oxygen atom. The catalytic sequence for microsomal cytochrome P-450 enzymes, which has been reviewed previously,1–4 involves the following steps (Fig. 1): (1) binding of a substrate, (2) reduction of the two flavin prosthetic groups of cytochrome P-450 reductase by NADPH, (3) transfer of one of the two electrons thus made available to cytochrome P-450, (4) binding of molecular oxygen to give a ferrous cytochrome P-450—dioxygen complex, (5) transfer of a second electron from cytochrome P-450 reductase, or of an electron from cytochrome b 5, to the complex, (6) cleavage of the oxygen-oxygen bond with concurrent incorporation of the distal oxygen atom into a molecule of water, (7) transfer of the second oxygen atom to the substrate, and (8) dissociation of the product. The catalytic cycle for mitochondrial cytochrome P-450 enzymes differs in that the transfer of electrons from cytochrome P-450 reductase to the hemeprotein is mediated by adrenodoxin, an iron—sulfur protein (see Chapter 12). The steps in the catalytic sequence in which bonds to oxygen are made or broken are addressed in this chapter. The substrate-binding step is discussed in Chapter 3, however, and the electron transfer steps in Chapters 4, 5, 11, and 12.

Oxygen Activation and Transfer

Haem accessibility in cytochrome P-450 from rabbit liver. A proton magnetic relaxation study by stereochemical probes

The influence of the binding of methaemoproteins to latex particles with respect to protein conformation was examined using the longitudinal magnetic relaxation rates of solvent protons. The temperature dependences of the paramagnetically induced molar relaxation rates in bound metmyoglobin samples as well as their absolute values suggest an opening of the haem-pocket. The decrease of the same parameter for the immobilized methaemoglobin points to a smaller haem-pocket accessibility with respect to that in the solution. The changes of the haem-pocket structure found for both methaemoproteins support the idea that proteins change their conformation by their binding to the solid surface, although in opposite ways.

A proton magnetic relaxation study of methaemoproteins bound to monodisperse polystyrene latex particles

Using the solvent-protons' longitudinal magnetic relaxation rates (p.m.r.) for Lupinus luteus leghaemoglobin derivatives the accessibility of the haem has been evaluated by our "stereo-chemical p.m.r. titration" method with nonexchangeable protons of aliphatic lower alcohols in otherwise deuterated solutions. The haem in leghaemoglobin is more accessible and its protein environment more flexible compared with vertebrate haemoglobins. The correlation time in aquometleghaemglobin aqueous solution has been determined by measuring the frequency dispersion of the p.m.r. rates between 6.1 and 93 MHZ. Taking into account the measured value of tauc = (7.7 +/- 0.5 x 10(-10) s the iron-to-proton inter-spin distances have been calculated. The significance of these distances as well as the electronic g-factor anisotrophy for elucidation of fine structural details of the haem-environment are discussed.

The haem‐accessibility in leghaemoglobin of lupinus luteus as observed by proton magnetic relaxation

The molecular mechanism of the temperature enhancement of proton magnetic relaxation rates in methaemoprotein solutions.

Inositol hexaphosphate is the strongest allosteric effector even for the metform of haemoglobin. Its effects upon the quaternary structure of the tetramer have been studied in relation to the overall conformational state(s) of the haem-pockets in aqueous solutions of human haemoglobins. The method useci, proton magnetic relaxation, yields information about the accessibility of solvent pl.'otons towards the haem-iron. No differences in the relaxation rates were detected by this method between the unstripped carbonmonoxyhaemoglobin and the phosphate-stripped sample in the presence and absence of IHP. There are considerable changes in those relaxation rate·s due to the paramagnetic haem-iron of aquomethaemoglobin when IHP is added to the stripped adult haemoglobin, but none is observed for the foetal haemoglobin, although a similar shift in the spin-state equilibrium ts expected for both haemoglobins on addition of ,!HP. Neither was there any change with IHP in solutions of adult fluoromethaemoglobin. It is concluded thart there is no tightening of the haem-pockets upon addition of IHP to solutions of any of the three haemoglobin samples. An increase in the accessibility of the haem-pockets is probable only for the aquometfom1 of the adult haemoglobin. It is suggested that the structural aspect of ligand affinity, i.e. the haem-pocket conformation, is not as decisive in altering the affinity by IHP as is possibly the change in the haem-iron spin-state induced by !HP-binding.

S. Vuk-Pavlović

Papers

Haem accessibility in cytochrome P-450 from rabbit liver. A proton magnetic relaxation study by stereochemical probes

A proton magnetic relaxation study of methaemoproteins bound to monodisperse polystyrene latex particles

The haem‐accessibility in leghaemoglobin of lupinus luteus as observed by proton magnetic relaxation

The molecular mechanism of the temperature enhancement of proton magnetic relaxation rates in methaemoprotein solutions.

A Proton Magnetic Relaxation Study of tpe Interaction between Methaemoglobin and Inositol Hexaphosphate