Rate theories and puzzles of hemeprotein kinetics.

The binding of dioxygen and carbon monoxide to heme proteins such as myoglobin and hemoglobin has been studied with flash photolysis. At temperatures below 200 K, binding occurs from within the heme pocket and, contrary to expectation, with nearly equal rates for both ligands. This observation has led to a reexamination of the theory of the association reaction taking into account friction, protein structure, and the nature of electronic transitions. The rate coefficients for the limiting cases of large and small friction are found with simple arguments that use characteristic lengths and times. The arguments indicate how transition state theory as well as calculations based on nonadiabatic perturbation theory, which is called the Golden Rule, may fail. For ligand-binding reactions the data suggest the existence of intermediate states not directly observed so far. The general considerations may also apply to other biomolecular processes such as electron transport.

The energy landscapes and motions of proteins.

Recent experiments, advances in theory, and analogies to other complex systems such as glasses and spin glasses yield insight into protein dynamics. The basis of the understanding is the observation that the energy landscape is complex: Proteins can assume a large number of nearly isoenergetic conformations (conformational substates). The concepts that emerge from studies of the conformational substates and the motions between them permit a quantitative discussion of one simple reaction, the binding of small ligands such as carbon monoxide to myoglobin.

Tunneling in ligand binding to heme proteins.

Rebinding of carbon monoxide to the beta chain of hemoglobin after photodissociation by a laser flash is intramolecular below about 200 K. Above 25 K, rebinding occurs via classical over-the-barrier motion; below, quantum-mechanical tunneling dominates. Both are described by an energy spectrum peaked at Epeak=4.0 kilojoules per mole. The barrier width d(E), determined from the energy dependence of the tunneling rate, depends on barrier height, d(E) approximately 0.05 nanometer X (E/Epeak) 1.5.

Dynamics of carbon monoxide binding by heme proteins.

Rebinding of carbon monoxide to myoglobin and to cytochrome P-450 after removal by a light flash occurs down to 50 degrees K for myoglobin and 25 degrees K for cytochrome P-450 in glycerol-water solution. Above 240 degrees K the reaction is second order; between 240 degrees and 200 degrees K the rebinding becomes exponential and independent of the carbon monoxide concentration. Below 150 degrees K the reaction follows a power law and is approximately 10(3) times faster for cytochrome P-450 than for myoglobin.

Recombination of carbon monoxide to ferrous horseradish peroxidase types A and C.

The recombination of carbon monoxide to isoenzymes A2 and C of horseradish peroxidase (HRP) was studied as a function of temperature (2 to 320 K) and pH (5 to 8.3) with flash photolysis and infrared difference absorption. At low temperatures three geminate recombination processes are observed. One of these internal processes, denoted by I*, is exponential in time with a rate coefficient that deviates strongly from an Arrhenius behavior below 100 K, implying phonon-assisted tunneling. The two other processes, denoted by I, are non-exponential in time and related to different carbonyl isomers, as shown by the infrared difference spectra. The existence of three internal processes indicates that HRP differs considerably from myoglobin where only one internal process, I, is seen. Moreover, the internal processes in HRP are faster than process I in myoglobin. At 300 K, only one recombination process from the solvent is observed and it is very slow (lambda s approximately 1 s-1 at 1 atm CO (1 atm = 101,325 Pa)), much slower than the corresponding association process in myoglobin. Since process I is fast, but binding from the solvent is slow, the barrier at the heme cannot be responsible for the small association rate. The infrared absorption difference spectra of the amide I/II bands indicate that photolysis and recombination trigger a two-step structural change. The slow recombination rate at 300 K can thus be explained by the large Gibbs energy of the conformational transition that is necessary to let CO move into the heme pocket. The partition coefficient for the CO in the heme pocket and the solvent is extremely small, while bond formation with the heme iron occurs in less than 100 nanoseconds.

Rebinding and relaxation in the myoglobin pocket.

The infrared stretching bands of carboxymyoglobin (MbCO) and the rebinding of CO to Mb after photodissociation have been studied in the temperature range 10-300 K in a variety of solvents. Four stretching bands imply that MbCO can exist in four substates, A0-A3. The temperature dependences of the intensities of the four bands yield the relative binding enthalpies and and entropies. The integrated absorbances and pH dependences of the bands permit identification of the substates with the conformations observed in the X-ray data (Kuriyan et al., J. Mol. Biol. 192 (1986) 133). At low pH, A0 is hydrogen-bonded to His E7. The substates A0-A3 interconvert above about 180 K in a 75% glycerol/water solvent and above 270 K in buffered water. No major interconversion is seen at any temperature if MbCO is embedded in a solid polyvinyl alcohol matrix. The dependence of the transition on solvent characteristics is explained as a slaved glass transition. After photodissociation at low temperature the CO is in the heme pocket B. The resulting CO stretching bands which are identified as B substates are blue-shifted from those of the A substates. At 40 K, rebinding after flash photolysis has been studied in the Soret, the near-infrared, and the integrated A and B substates. All data lie on the same rebinding curve and demonstrate that rebinding is nonexponential in time from at least 100 ns to 100 ks. No evidence for discrete exponentials is found. Flash photolysis with monitoring in the infrared region shows four different pathways within the pocket B to the bound substates Ai. Rebinding in each of the four pathways B----A is nonexponential in time to at least 10 ks and the four pathways have different kinetics below 180 K. From the time and temperature dependence of the rebinding, activation enthalpy distributions g(HBA) and preexponentials ABA are extracted. No pumping from one A substate to another, or one B substate to another, is observed below the transition temperature of about 180 K. If MbCO is exposed to intense white light for 10-10(3) s before being fully photolyzed by a laser flash, the amplitude of the long-lived states increases. The effect is explained in terms of a hierarchy of substates and substate symmetry breaking. The characteristics of the CO stretching bands and of the rebinding processes in the heme pocket depend strongly on the external parameters of solvent, pH and pressure. This sensitivity suggests possible control mechanisms for protein reactions.

Relaxations and fluctuations in myoglobin.

One major goal of biological physics is the discovery and understanding of the concepts and laws that govern biomolecules, in particular proteins. Since there exist at least 10(5) different proteins, the choice of a suitable prototype is necessary. Myoglobin (Mb) has for many years played the role of such a prototype. It appears to be simple enough so that many of its properties can be understood, yet it is complex enough to display many of the fascinating characteristics of biomolecules. One major achievement in the study of any protein would be the establishment of convincing connections among structure, kinetics, energy landscape, dynamics, and function. We believe that this goal has not yet been reached in any protein, but the present knowledge of Mb gives some hope that the end is near in this case. Here, we sketch some of the results that have been obtained in the past 50 or more years in the research on Mb, obtained by an army of investigators.

Changes in serum muscle enzyme levels associated with training schedules and stage of the oestrous cycle in Thoroughbred racehorses.

Blood samples were collected twice weekly over a nine month period from 24 Thoroughbred racehorses in training at Newmarket to study the effects of daily training schedules and stage of oestrous cycle on serum enzyme levels and clinical signs of equine exertional myopathy. The sampling period extended from November to July and was performed between 16:00 h and 18:00 h so as to be at least 6 h after exercise. Serum creatine kinase, aspartate aminotransferase and plasma progestogen concentrations were measured. All horses were subjected to a similar traditional training programme. The study demonstrated marked fluctuations in serum muscle enzyme concentrations which were more pronounced in two-year-old fillies than three-year-old fillies and colts. There was no correlation, however, with the stage of oestrous cycle. It was concluded that some degree of muscle cell damage, which results in a variable increase in serum creatine kinase levels, occurs in all horses following light exercise after a rest day. Traditional flat race training may not be ideal in this respect and it is suggested that, for horses with an observed tendency towards exertional myopathy, more consistent and longer periods of less strenuous exercise may be advantageous.

Effects of dexamethasone on endotoxin shock in the anesthetized pony: hematologic, blood gas, and coagulation changes.

The effects of dexamethasone (1 mg/kg of body weight) on hematologic, blood gas, and blood coagulation values in anesthetized ponies during endotoxin-induced shock were evaluated. Fifteen ponies were assigned to 3 groups of 5 ponies each: group 1, anesthetized nontreated and dexamethasone-treated controls; group 2, endotoxin, nontreated; group 3, endotoxin, dexamethasone treated. The hematologic changes in this endotoxin shock model included leukopenia and hemoconcentration. Significant hematologic effects were not seen in ponies after administration of dexamethasone. However, dexamethasone treatment resulted in an increased trend in total WBC counts and neutrophils. The blood gas changes reflected a respiratory component resulting from anesthesia and a greater metabolic component from the endotoxemia. The plasma lactate increase was significantly (P less than 0.05) less in ponies treated with dexamethasone, compared with plasma lactate in non-treated ponies. During endotoxin shock, the changes observed in the blood coagulation values included a significant (P less than 0.05) prolongation of the activated partial thromboplastin time and thrombin time and an insignificant prolongation of the prothrombin time. Dexamethasone treatment prevented prolongation of thrombin time and permitted only a mild prolongation of activated partial thromboplastin time. Seemingly, corticosteroids are useful in the treatment of clinical endotoxin shock in horses as indicated by their desirable effects on total WBC, neutrophils, cellular metabolism, and blood coagulation.

Endotoxin-induced hemodynamic changes in ponies: effects of flunixin meglumine.

A study was made of flunixin meglumine, an analgesic agent with antiinflammatory and antiprostaglandin activity, for the management of endotoxin-induced cardiovascular derangements. Three groups of 5 ponies each were used: controls--group 1; given endotoxin but not treated--group 2; and given endotoxin and treated with flunixin meglumine--group 3. Shock was induced in anesthetized ponies with IV injection of Escherichia coli endotoxin. Hemodynamic changes were monitored, and regional blood flow was determined at 4 different times, using microspheres labeled with 1 of 4 nuclides. There were extensive vasodilation and decreased blood return to the heart of group 2 ponies, as indicated by decreased mean arterial blood pressure and central venous pressure and by increased heart rate and cardiac output. Blood flow, as determined by radioactive microspheres, to gastrointestinal regions, skeletal muscle, and skin was increased and that to the CNS was decreased. Treatment with flunixin meglumine (group 3 ponies) exerted selective microvascular actions which helped to reverse endotoxin-induced changes. This included the maintenance of mean arterial blood pressure and the enhanced perfusion of vital organs (eg, brain and heart) by preventing extensive vasodilation in the gastrointestinal tract.

Endotoxin-induced change in hemograms, plasma enzymes, and blood chemical values in anesthetized ponies: effects of flunixin meglumine.

A study was made of flunixin meglumine (FM), an analgesic agent with antiprostaglandin activity, in the management of endotoxin-induced changes in ponies. Three groups of 5 ponies each were used: A--controls, B--nontreated ponies with endotoxin-induced shock, and C--ponies with endotoxin-induced shock treated with FM. Shock was induced in anesthetized ponies with IV injections of Escherichia coli endotoxin. Disruption of glucose homeostasis, insulin levels, hemograms, aerobic metabolism, and cell damage as indicated by plasma enzymes were observed. Treatment with FM (5 minutes) after shock was induced did not prevent general tissue damage as indicated by plasma enzymes, but separation of creatine phosphokinase into its 3 isoenzymes revealed a significant increase in the amount of the creatine phosphokinase isoenzyme bb in group B ponies, but not in FM-treated ponies (group C). The source of this isoenzyme is believed to be brain tissue. Acidosis as indicated by lactic acid and venous pH was less in FM-treated ponies than in nontreated (group B) ponies. Blood glucose and insulin concentrations changed in both groups B and C (endotoxin-induced shock), but the patterns of change were different. The only effect of FM on hematologic values was a significant decrease in blood platelet counts. The results of these experiments indicate that FM improved cellular metabolism and reduced brain damage. These effects were believed to be the result of the maintenance of mean arterial blood pressure and enhanced perfusion of vital organs by preventing extensive vasodilation in the gastrointestinal tract.

Rn for treatment of periocular fibrous connective tissue sarcomas in the horse.

Twelve periocular fibrous connective tissue sarcomas in 11 horses were treated with 222Rn. Follow-up periods ranged from 1 to 6 years; the overall nonrecurrence rate at 12 months after therapy was 92%. Two lesions recurred 2 years after treatment, and 1 after 3 years. One of the former lesions has not recurred after a 2nd 222Rn treatment.

External cardiovascular resuscitation of the anesthetized pony.

External cardiac massage and concomitant respiratory support were used successfully 6 of 8 anesthetized ponies sustaining unexpected cardiac arrest while being used in a study of shock. Approximately 20 thoracic compressions/min maintained systolic and diastolic aortic blood pressures in excess of 50% of the corresponding base-line values in 5 ponies. The high success rate was attributed to early recognition of the problem, the small size of the patient, and the relatively short duration of cardiopulmonary resuscitation (average, 2.9 minutes). It was concluded that external cardiac message can be effective for cardiopulmonary resuscitation in selected equine patients that have sustained cardiac arrest.