Development and validity of the Burns-Child Adult Medical Procedure Interaction Scale (B-CAMPIS) for young children.

BACKGROUND: Young children are at increased risk of burn injury and of procedural distress during the subsequent wound care. There are currently few observational measures validated for use with young children during medical procedures. The aim of this research was to adapt the Child-Adult Medical Procedure Interaction Scale-Revised (CAMPIS-R) to assess parent-young child interactions during burn wound care by including nonverbal behavioral coding. METHOD: Eighty-seven families of children (1-6years old) were recruited at their first burn dressing change. Potential behaviors were identified through a literature review, consulting health professionals, and direct observation of parents and children during burn wound care. Nonverbal behaviors were coded live, and verbal behaviors were audio recorded for later assessment. RESULTS: Inter-coder reliability was good to excellent for the Burns-CAMPIS (B-CAMPIS). The additional behaviors were correlated with the hypothesized coping, distress, coping-promoting and distress-promoting categories of the CAMPIS-R. Some behaviors differed in frequency across child age groups, with older children demonstrating more verbal behaviors. Convergent validity was demonstrated through correlations with previously validated observational parent-child behavior measures, and parent- and nurse- reported measures of child pain and anxiety. Univariate regression analyses demonstrated the child categories of the B-CAMPIS accounted for equal or more of the variance of parent- and nurse- reported child pain and anxiety, compared to the CAMPIS-R. CONCLUSIONS: The B-CAMPIS is a reliable and valid measure, for assessing coping and distress relationships in young children and their families. Pending further validation, the B-CAMPIS assists researchers and clinicians to recognize and target important behaviors to improve young child coping during pediatric burn wound care.

Thermodynamic studies on the equilibrium properties of a series of recombinant betaW37 hemoglobin mutants.

In human hemoglobin (Hb) the beta37 tryptophan residue (betaW37), located at the hinge region of the alpha1beta2 interface, forms many contacts with alpha subunit residues of the opposite dimer, in both the T and R quaternary structures. We have carried out equilibrium O2 binding studies on a series of recombinant Hbs that have mutations at this residue site: betaW37Y, betaW37A, betaW37G, and betaW37E. Binding isotherms measured at high concentrations of these mutants were found to be shifted toward increased affinity and decreased cooperativity from that of the normal HbA0 tetramer. Analysis of these binding isotherms indicated that amino acid substitutions at the beta37 position could both destabilize the tetrameric form of the mutants relative to their constituent dimers and also alter cooperativity of the intact tetrameric species. These alterations from wild-type function are dependent on the particular side chain substituted, with the magnitude of change increasing as Trp is substituted by Tyr, Ala, Gly, and Glu. The dimer to tetramer assembly free energy of deoxy-betaW37E, the most perturbed mutant in the series, was measured using analytical gel chromatography to be 9 kcal/tetramer less favorable than that of deoxy HbA0. Stabilizing the betaW37E tetramer by addition of IHP, or by cross-linking at the alphaK99 positions, does not restore normal O2 binding behavior. Thermodynamic parameters of all the mutants were found to correlate with their CO binding rates and with their high-resolution X-ray crystal structures (see accompanying papers: Kwiatkowski et al. (1998) Biochemistry 37, 4325-4335; Peterson & Friedman (1998) Biochemistry 37, 4346-4357; Kavanaugh et al. (1998) Biochemistry 37, 4358-4373].

Isolation and stability of partially oxidized intermediates of carp hemoglobin: kinetics of CO binding to the mono- and triferric species.

The monoliganded and triliganded forms of the asymmetric valency hybrids of carp hemoglobin were isolated using high-performance liquid chromatography. These partially oxidized hybrids were shown to be sufficiently stable to permit the measurement of the kinetics of CO binding. The effects of protons and inositol hexaphosphate on the rates of these reactions were examined. The kinetics of CO recombination with these partially oxidized derivatives were compared to the kinetics of CO binding to the fully ferrous molecule. To a first approximation, the kinetic behavior of the monoferric derivative was consistent with a small shift in the T<==>R equilibrium in favor of the R state. The presence of three ferric ligands resulted in a still greater shift in the conformational equilibrium in favor of the R state. The kinetic behavior of the triferric molecule was similar, but not identical, to that of a fully ferrous molecule which is triliganded with CO. The properties of both asymmetric valency hybrids were responsive to the nature of the ligand; i.e., the rate of CO binding was increased more by the presence of cyanide on the ferric hemes than by water. Not all of the data could be accommodated within the two-state model. For example, there was evidence of an altered T state in the case of the tricyanomet derivative at low pH in the presence of inositol hexaphosphate.

Functional properties of human hemoglobins synthesized from recombinant mutant beta-globins.

The previous and following articles in this issue describe the recombinant synthesis of three mutant beta-globins (beta 1 Val----Ala, beta 1 Val----Met, and the addition mutation beta 1 + Met), their assembly with heme and natural alpha chains into alpha 2 beta 2 tetramers, and their X-ray crystallographic structures. Here we have measured the equilibrium and kinetic allosteric properties of these hemoglobins. Our objective has been to evaluate their utility as surrogates of normal hemoglobin from which further mutants can be made for structure-function studies. The thermodynamic linkages between cooperative oxygenation and dimer-tetramer assembly were determined from global regression analysis of multiple oxygenation isotherms measured over a range of hemoglobin concentration. Oxygen binding to the tetramers was found to be highly cooperative (maximum Hill slopes from 3.1 to 3.2), and similar patterns of O2-linked subunit assembly free energies indicated a common mode of cooperative switching at the alpha 1 beta 2 interface. The dimers were found to exhibit the same noncooperative O2 equilibrium binding properties as normal hemoglobin. The most obvious difference in oxygen equilibria between the mutant recombinant and normal hemoglobins was a slightly lowered O2 affinity. The kinetics of CO binding and O2 dissociation were measured by stopped-flow and flash photolysis techniques. Parallel studies were carried out with the mutant and normal hemoglobins in the presence and absence of organic phosphates to assess their allosteric response to phosphates. In the absence of organic phosphates, the CO-binding and O2 dissociation kinetic properties of the mutant dimers and tetramers were found to be nearly identical to those of normal hemoglobin. However, the effects of organic phosphates on CO-binding kinetic properties of the mutants were not uniform: the beta 1 + Met mutant was found to deviate somewhat from normalcy, while the beta 1 Val----Met mutant reproduced the native allosteric response. Further characterization of the allosteric properties of the beta 1 Val----Met mutant was made by measuring the pH dependence of its overall oxygen affinity by tonometry. Regulation of oxygen affinity by protons was found to be nearly identical to normal hemoglobin from pH 5.8 to 9.3 (0.52 +/- 0.07 protons released per oxygen bound at pH 7.4). The present study demonstrates that the equilibrium and kinetic functional properties of the recombinant beta 1 Val----Met mutant mimic reasonably well those of normal hemoglobin. We conclude that this mutant is well-suited to serve as a surrogate system of normal hemoglobin in the production of mutants for structure-function studies.

Studies on the linkage between spin equilibria and protein structure in carp ferric hemoglobin.

The effects of protein conformation on the spin-state equilibria of several derivatives of carp hemoglobin have been examined. This has been done by measuring the pH dependence of the paramagnetic susceptibilities of these derivatives in the presence and absence of inositol hexakisphosphate, P6-inositol. In all cases the addition of P6-inositol at low pH and the lowering of the pH in the presence of P6-inositol shift the spin-state equilibrium in favor of the high-spin electronic configuration. The P6-inositol and pH dependence of these magnetic properties parallels the pH and P6-inositol dependence of the conformational state of the hemoglobin as determined in earlier studies and further supports a thermodynamic linkage between the electronic state of the iron atoms and the quaternary structure of the hemoglobin molecule.

Distal residues in the oxygen binding site of haemoglobin studied by protein engineering.

The geometries of the Fe-O2 and Fe-CO bonds in myoglobin and haemoglobin differ significantly from those in free porphyrin model compounds. It has been suggested that steric hindrance by Val-E11 and His-E7 and a hydrogen bond between His-E7 and oxygen affect the geometry and electronic state of the Fe-ligand bond, and that these interactions may be important in controlling oxygen affinity. We have produced mutant haemoglobins in E. coli having Val(67 beta)E11 replaced by Ala, Met, Leu or Ile and His(58 beta)E7 by Gln, Val or Gly. We have studied the effect of these mutations on the equilibrium and kinetics of ligand binding. The conformation of the new side chains and their effect on the protein structure have been examined by X-ray crystallography, and the vibrational properties of the Fe-CO bond observed by resonance Raman spectroscopy. We found that the steric hindrance of ligand binding by the E11 residue and the polarity of the E7 residue in the beta subunit are critical for fine-tuning ligand affinity.

Functional properties of hemoglobins from deep-sea fish: correlations with depth distribution and presence of a swimbladder.

The ligand binding properties of the hemoglobins of several deep-sea, bottom-living fish have been examined. These include five species of rattails (Macrouridae) and Antimora rostrata, all of which possess swimbladders, and two unrelated species without swimbladders, Bathysaurus mollis and Alepocephalus sp. All of the hemolysates of these fish exhibited the Root effect with a minimum ligand affinity at about pH 6 in the presence of organic phosphate. Under these conditions the hemolysates from fish which possess swimbladders exhibit two roughly equal populations of heme groups with markedly different ligand affinities. For the deeper-dwelling species the affinities for carbon monoxide differ by some 500-fold, the low-affinity population having a p50(CO) of 100 mmHg at 15 degrees C. This very low affinity is associated with a second-order rate constant for CO combination of the order of 10(3) M-1 X s-1. Those species without swimbladders have hemoglobins which do not have such heterogeneous binding sites, suggesting a relationship between these very-low-affinity heme groups and the pumping of oxygen into a swimbladder at high hydrostatic pressures.

A high-resolution proton nuclear-magnetic-resonance investigation of carp hemoglobin. Conformational differences between carp and human normal adult hemoglobins in solution.

The high-resolution proton nuclear magnetic resonance spectra of carp hemoglobin have been compared to those of human normal adult hemoglobin. Carp deoxy and carbonmonoxy hemoglobins in the deoxy-type quaternary state exhibit two downfield exchangeable proton resonances as compared to four seen in human normal adult deoxyhemoglobin. This suggests that two of the hydrogen bonds present in human normal adult deoxyhemoglobin are absent or occur in very different environments in carp hemoglobin. One of the exchangeable proton resonances of carp hemoglobin, while present in the deoxy-type quaternary state of the carbonmonoxy and deoxy derivatives, is absent in the oxy-type quaternary state of both, in agreement with the assignments of these quaternary structures by other methods. The ring-current-shifted proton resonances (sensitive tertiary structural markers) of carp carbonmonoxyhemoglobin are substantially different from those of human normal adult hemoglobin. The aromatic proton resonance region of carp hemoglobin has fewer resonances than that of human normal adult hemoglobin, consistent with its much reduced histidine content. The hyperfine-shifted proximal histidyl NH-exchangeable proton resonances of carp hemoglobin suggest that during the transition from the oxy to the deoxy quaternary structure, there is a greater alteration in the heme pocket of one type of subunits (presumably the beta chain) than that in the other subunit. The present results suggest that there are differences in both tertiary and quaternary structures between carp and human normal adult hemoglobins which could contribute to the great differences in the functional properties between these two proteins.

Quaternary structure and spin equilibria in ferric hemoglobins. A room temperature study.

The effective magnetic moments for a number of human and carp methemoglobin derivatives were determined in solution at room temperature. The data permit us to confirm the dependence of the spin-state equilibrium of azide methemoglobin on the quarternary state of the hemoglobin and to demonstrate a similar dependence for both human and carp aquomethemoglobin. In addition, the pH dependence of the effective magnetic moment and the Soret spectrum of carp azidemethemoglobin are compared.

Magnetic and spectral properties of carp carbonmonoxyhemoglobin. Competitive effects of chloride ions and inositol hexakisphosphate.

We have extended our studies on the magnetic properties of carp carbonmonoxyhemoglobin and the dependence of these properties upon solution variables. Using an improved version of the superconducting magnetometer, we have found that the magnetic susceptibility of carp carbonmonoxyhemoglobin is sensitive to both inositol hexakisphosphate and chloride ion. The dependence upon chloride ion concentration is complex. At relatively low concentrations this anion reverses the effect of inositol hexakisphosphate, restoring paramagnetism. At higher chloride concentrations the protein is converted to a roughly diamagnetic state in the absence of inositol hexakisphosphate. Along with these susceptibility studies, we have examined the effects of these anions on other properties of carp carbonmonoxyhemoglobin. The positions of the Soret bands of human and carp methemoglobin derivatives are correlated with spin state; changes in the magnetic susceptibility of carbonmonoxyhemoglobin are similarly associated with alterations in this spectral band. We have also examined the effects of these anions on the proton nuclear magnetic resonance spectrum of carp carbonmonoxyhemoglobin. Both chloride and inositol hexakisphosphate alter the position of the proton resonances in the ring-current-shifted region of the spectrum.

Variability of the magnetic moment of carbon monoxide hemoglobin from carp.

Deionized carp carbon monoxide hemoglobin in distilled water or in bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane or Tris buffer exhibits a slight but significant paramagnetism. This is most clearly demonstrated by the decrease in this paramagnetism that is caused by the addition of inositol hexaphosphate to this protein in the former buffer at pH 6.3-6.4. No such effect is seen when inositol hexaphosphate is added to carp cyanomethemoglobin, demonstrating that the change observed with carbon monoxide derivative is not due to a modification in the diamagnetic properties of the protein.