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The mechanism of water exchange between the haem-pocket and bulk solvent in aqueous methaemopiotein solutions was firmly substantiated by using the aliphatic protons of certain lower alcohols in an otherwise deuterated solution for measuring the incremental relaxation rates resulting from their magnetic interaction with the haem-iron. The fast-exchange condition was established for solutions of horse fluorometmyoglobin, human A fluoromethaemoglobin and Chironomus thummi aquomethaemoglobin. The distances between the exchangeable protons and the haem-iron obtained from these PMR measurements concur with the presence of the fluoride ion, while for Chironomus aquomethaemoglobin this distance is also much larger than that resulting from the location of the 6th site Water molecule. The latter finding is the first clear-cut evidence that the exchanging protons belong to the next neighbour water molecule, a previously advanced hypothesis. The exchanging water molecule may thus serve as a natural probe for comparing the haem-pocket conformational state(s) under different conditions or in various haemoproteins.

End-tidal steady state diffusing capacity (DLCOss2) was measured with different ventilation rates at rest in healthy subjects and patients with pulmonary diseases. The results in the patients were compared with the Filley method (DLCOss1) during exercise. The DLCOss2 values in the patients at rest during normoventilation correlated better to exercise DLCOss1 than the DLCOss2 values obtained during hyperventilation. The reproducibility of DLCOss2 at rest with ‘standardized’ ventilation was comparable to that reported for exercise DLCOss1. The healthy subjects had low DLCOss2 values at rest when breathing quietly, but if correction for ventilation was made, they could be separated from those with pulmonary diseases.

An investigation was undertaken to determine the influence of single doses of carbon monoxide plus single doses of ethanol, repeated simultaneous doses of carbon monoxide plus ethanol, and repeated dosage of carbon monoxide (ethanol) plus single doses of ethanol (carbon monoxide) on the activities of leucine amino-peptidase and glutamic-pyruvic transaminase in the plasma of rats. Combinations of carbon monoxide and ethanol were found to produce additive effects with respect to the increase of indicator enzymes if at least 50 % of the hemoglobin was saturated with carbon monoxide and if one of the noxious agents was administered in large doses.

Photodissociation of the CO molecule from carbon monoxide complexes of myoglobin and hemoglobin was examined between 4.2 and 80 °K. Experimental results have demonstrated that: 1. (I)|The photodissociated form was stable below 10 °K. 2. (II)|The ratio of the quantum yield of hemoglobin to myoglobin was roughly estimated to be 0.38. 3. (III)|The recombination of dissociated CO occurred by raising the temperature. 4. (IV)|The recombination reaction of photodissociated myoglobin and hemoglobin with CO was biphasic, whereas that of cobalt myoglobin with O2 was monophasic. 5. (V)|Activation energies for fast and slow reactions of myoglobin and hemoglobin, and for monophasic reaction of cobalt myoglobin are listed below: Fast Slow Mb(Fe 2+ ) + CO 124 cal/mole 124 cal/mole Hb(Fe 2+ ) + CO 124 cal/mole 230 cal/mole Mb(Co 2+ + O 2 570 cal/mole 6. (VI)|In the near infrared region the photodissociated forms of myoglobin and hemoglobin gave absorbance peaks at 770 nm, whereas deoxygenated forms of myoglobin and hemoglobin gave absorbance peaks at 758 and 750 nm, respectively, which suggests that the near infrared band is sensitive to the protein structure and may be called the “conformation band”. Several discussions have been held on the nature of the photodissociated form and the biphasic property of the reaction of myoglobin and hemoglobin, compared with those of cobalt myoglobin. It is now concluded that the new species, corresponding to the “rapidly reacting form” of hemoglobin by Gibson, Q. H. (1959) Biochem. J. 71, 293–303, and, more recently, of hemoglobin, single chain proteins and noncooperative aggregated forms by Alpert, B., Banerjee, R., and Lindqvist, L. (1974) Proc. Natl. Acad. Sci. U.S. 71, 558–562, is certainly trapped at 4.2 °K by photodissociating the carbon monoxide and oxygenated myoglobin and hemoglobin as well as the oxygenated cobalt myoglobin and hemoglobin.

The Evelyn and Malloy procedure for methemoglobin determination in blood has been modified to decrease changes which occur in shed blood and in blood hemolysates. Immediate hemolysis, acidification to pH 6.9, and cooling to 0°C serve to minimize effectively metabolic changes in the MetHb content of shed blood and autoxidation in the hemolysates. The MetHb content is evaluated from the change in absorbance at 630 nm caused by addition of KCN to the buffered hemolysate. A dilution of the hemolysate in which K3Fe(CN)6 is employed to convert all possible forms of hemoglobin to methemoglobin is used as a reference solution. Quantitative conversion of COHb to MetHb by K3Fe(CN)6 at room temperature and pH 6.9 requires 30 min. The procedure has a coefficient of variation of about 3.5% for samples of low MetHb content. Recovery of added MetHb is essentially quantitative over a wide range of concentration.

Twenty young men, smokers and non- smokers, were tested for maximal aerobic power in a 35 C environment under four ambient air conditions: (1) filtered air (FA), (2) 50 ppm carbon monoxide (CO), (3) 0.27 ppm peroxyacetyl nitrate (PAN), and (4) CO plus PAN. Carbon monoxide was effective in reducing the work time of the nonsmokers and in eliciting changes In the respiratory patterns of both smokers and nonsmokers. No significant physiological effects were noted during the PAN exposure. Maximal aerobic power was not affected by any pollutant condition. Since the concentrations of CO represented a first-stage alert level for the Los Angeles area, it appears that the air pollution standard for this pollutant is set at a realistic level for healthy young men doing strenuous work for a relatively short period of time.

Mice were pretreated with phenobarbital, chlorpromazine, or alcohol prior to exposure to 1900 ppm carbon monoxide (CO) or 7.5% oxygen (O 2 ) environments. Pretreatment for 1 hr with chlorpromazine or ethanol increased the lethality of mice exposed to both CO and 7.5% O 2 , while 1-hr phenobarbital pretreatment had no effect on CO lethality but increased 7.5% O 2 lethality. Changes in carboxyhemoglobin concentrations that were observed were inconsistent with the alteration in CO lethality. Studies of red blood cell 2,3-diphosphoglycerate concentrations and rate of carboxyhemoglobin formation in vitro showed that the apparent affinity of hemoglobin for oxygen and CO remained unchanged following drug or alcohol pretreatments. The different effects of the pretreatments on CO and 7.5% O 2 lethality and the lack of correlation of CO lethality with carboxyhemoglobin concentrations suggests that there are other factors besides extracellular events directly associated with oxygenation of tissues which are critical determinants of the lethal potential of CO or 7.5% O 2 .

The carbon monoxide (CO) sources of carboxyhemoglobin (COHb) in the St. Louis metropolitan population are primarily smoking (first) and industrial work exposure (second). The mean COHb saturations of the population studied were the following: total (16,649)–2.30%; smokers (6,492)–4.58%; nonsmokers (10,157)– 0.85%; smoking industrial workers (1, 738)–5.01%; nonsmoking industrial workers (1, 523)–1.38%; smoking other persons (4, 754)–4.42%; nonsmoking other persons (8, 634)–0.75%. Study samples were obtained from 16, 649 individual blood donors of the Red Cross blood program during a one-year period. The COHb saturation was correlated with smoking histories, travel, and socioeconomic data obtained during phlebotomy. Air-monitoring data were obtained from governmental agencies. Ambient air exposure produces very small increases in COHb levels in this regional population, as does increase in travel time or distance to donor site. These results indicate a need to reassess the environmental sources of CO in rel...

Sixteen rabbits were placed in exposure chambers: eight were continuously exposed to 180 ppm of carbon monoxide and eight were exposed to atmospheric air for 2 weeks. The myocardial ultrastructure of all the rabbits was examined. In rabbits exposed to carbon monoxide, local areas of partial or total necrosis of the myofibrils and degenerative changes of the mitochondria were found. Extra- and intracellular edema, increases in the number of ribosomes and lipofuscin granules, and reparative fibrotic changes also occurred. Varying degrees of injury were noted in the blood vessels. Capillary edema, but never total stenosis, was seen in some areas. Stasis and occasional small perivascular hemorrhages were typical on the venous side; on the arterial side the characteristic picture was one of endothelial swelling, formation of subendothelial edema, and degenerative changes of the myocytes. The present study supports the hypothesis that chronic exposure to low levels of carboxyhemoglobin can produce myocardial damage and account for the increased risk of myocardial infarction and sudden death seen in heavy cigarette smokers.

Summary Using a pH titration method we have observed the ligation Bohr effect in sulfhemoglobin and find that it is increased upon the addition of 2,3-diphosphoglycerate. These results indicate that sulfhemoglobin undergoes a quaternary structure change upon ligation.

Hemoglobin molecules after digestion with carboxypeptidase A or B show large differences in their ligand affinity, heme-heme interaction, Bohr effect and response to organic phosphates. Removal of β146 histidine and β145 tyrosine produces a molecule of high oxygen affinity, low Bohr effect and no heme-heme interaction. Removal of α141 arginine produces similar, but less drastic, changes. In both digestion products normal functional behavior may be partially restored by addition of inositol hexaphosphate. Doubly digested hemoglobins (carboxypeptidase A followed by B or vice versa) have properties similar to the initial digestion products but are no longer responsive to organic phosphates. The equilibrium and kinetic properties of these proteins are presented and are consistent with the idea that various conformations of the unliganded molecules may be in equilibrium.

Effects of carbon monoxide (CO) on various cardiopulmonary parameters were determined in adult, male. White Leghorn type chickens. Four groups of ten birds each were allowed to breathe spontaneously 0, 0.01, 0.1 and 0.5% CO, respectively, while cardiopulmonary and carboxy hemoglobin HbCO) measurements were obtained. No significant differences in respiratory frequency (fresp), tidal volume (V t ), minute volume (V), heart frequency (fresp), or systolic and diastolic blood pressure between birds receiving 0 or 0.01% CO for 60 min were detected (HbCO = 9%). Inhaling 0.1% CO for 60 min (HbCO = 39%) increased V T and V, and decreased blood pressure while fresp was unchanged. Inhaling 0.5% CO was lethal after an average of 20.5 min (range, 17.5–24.5 min). This concentration increased V t and V but decreased fresp, blood pressure and fresp and resulted in HbCO concentrations of 52% in 15 min. The effects of CO on cardiopulmonary control cannot be explained by an action on peripheral chemoreceptors (carotid body) because stimulation of those receptors increases fresp as well as V t . Carbon monoxide may, therefore, act directly on other peripheral receptors (intrapulmonary CO2-sensitive receptors) or on the CNS.

The effect of changes in effector ligand activity on the oxygen affinity of human whole blood was calculated for protons, carbon dioxide, and 2,3-diphosphoglycerate (DPG) at various concentrations of these ligands. The data were used to obtain, from experimentally obtained oxygen dissociation curves of blood from apparently healthy subjects, the standard oxygen dissociation curve (pH = 7.40, Pco2 = 40 mm Hg, DPG = 5.0 mmol/l, and HbCO = 1.0%) of normal human blood.

To decide whether the change in the quaternary state of unligated hemoglobin affects the electronic and structural properties of the heme group unligated hemoglobins modified with bis-(N-maleimidomethyl)ether and 2, 2′-dicarboxy-4, 4′-diiodoacetamidoazobenzol, which are in the high-affinity form, have been compared with unligated normal deoxyhemoglobin A by absorption spectroscopy and Raman scattering. The absorption spectra show no difference in the frequency of the bands in the visible and near ultraviolet region but differences were found in peak height and shape of the Soret band. The Raman resonance spectra recorded between 300 and 1700 cm−1 show no difference at all. These spectral observations indicate that the change of the quaternary structure of unligated hemoglobin slightly influences the excited electronic states of the heme but does not alter the electronic ground state. Along with other spectroscopic evidence from Mossbauer and nuclear magnetic resonance spectroscopy this suggests that only a minor fraction of the free energy difference of ligand binding in the two quaternary states may originate from differences in the electronic properties of the heme in unligated hemoglobin.

Photolysis of carbon monoxide and oxygen derivatives of hemoglobin by a short laser pulse produces a transient species that rapidly decays to normal deoxyhemoglobin. The effect, which is also observed on single chain proteins and on noncooperative aggregated forms, has been interpreted as corresponding to structural changes in the heme pocket on ligand dissociation. The decay of the transient species follows first-order kinetics with constants ranging from 0.8 to 1.8 × 10 7 sec -1 . In cooperative hemoglobins, the kinetic constants are pH-dependent, though remaining first- or pseudo first-order at all wavelengths. This shows the close linkage of tertiary and quaternary structure changes in normal hemoglobin.