PTEN hamartoma of soft tissue: a distinctive lesion in PTEN syndromes.

PTEN hamartoma tumor syndrome (PHTS) presents in a spectrum that encompasses the eponymous disorders Cowden and Bannayan-Riley-Ruvalcaba. Herein, we delineate the distinctive histopathology of a predominantly intramuscular lesion in PHTS, often called "arteriovenous malformation," because of certain imaging and histopathologic features. Cases were identified by review of lesions resected from patients with PHTS registered at our Vascular Anomalies Center and of unusual intramuscular vascular anomalies in our pathology database from 1985 to 2008. Thirty-four patients with this lesion were identified: 20 had a clinical diagnosis of, or were suspected to have, PHTS (genetically confirmed in 16). In 4 patients without clinical manifestations of PHTS, 2 had PTEN mutations, 1 did not, and in 1 the mutation was intronic. In the remaining 10, there was insufficient clinical information to fully assess whether they had manifestations of PHTS. Lesions manifested by 15 years of age, normally with pain and swelling, and were most often located in the lower extremity. The major mass was usually intramuscular, but often there were fascial and subcutaneous components and not infrequently a cutaneous vascular stain. Magnetic resonance imaging generally showed an infiltrative soft tissue lesion involving the muscle, fascia, and subcutis with frequently enlarged, serpiginous vessels, small arteriovenous fistulae with disproportionately dilated draining veins, and a prominent adipocytic component. Some lesions involved contiguous muscles, and 20% were multifocal. Resected specimens ranged in size from 1.2 to 25 cm; in 1 patient, amputation was necessary. Histopathologically, these unencapsulated masses, often with a nodular appearance at scanning magnification, consisted of: (1) a variable admixture of mature adipocytic and dense and/or myxoid fibrous tissues (50% to 90% of surface area); (2) a vascular component (10% to 50% of surface area) with: (a) clusters of venous channels, some with excessively and irregularly muscularized complex walls and lumens, and others with thin walls resembling pulmonary alveoli, (b) tortuous, thick-walled arteries with concentric muscular hyperplasia and relatively small lumens, (c) numerous small vessels (arteries, veins, and indeterminate channels), and (d) occasional arteriovenous communications; (3) lymphoid follicles (50%); (4) foci of bone (20%); and (5) hypertrophic nerves with "onion bulb" proliferation of periaxonal spindled cells (9%). We designate this disorganized overgrowth of essentially mesenchymal elements as PTEN hamartoma of soft tissue. It differs from other vascular and connective tissue lesions that occur in patients with PHTS. PTEN hamartoma of soft tissue is histopathologically distinctive, and its identification should prompt a thorough investigation for PHTS.

Effects of strategy use on acquisition of a motor task during various stages of learning.

To specify the optimal point for introducing a learning strategy, 50 participants were randomly assigned into five groups based on the timing of strategy introduction while learning a badminton serve. Groups were instructed in the use of Singer's Five-step Strategy either prior to starting their acquisition trials (100% group) or following acquisition Trial Blocks 1 (83% group), 3 (50% group), 5 (17% group) or were assigned to a control (0% group) group). Participants were asked to complete six acquisition trial blocks of 10 serves each, followed by a break and then two retention trial blocks. Scores were obtained by hitting shuttles into a scoring grid, which served as the dependent measure. Data were analyzed using a mixed-model analysis of variance with a group x trial blocks design, which yielded significant main effects for both factors during acquisition. Introduction of a learning strategy may be more efficient once participants have become familiar with the task. No significant differences were observed between groups who received the strategy early and the control group. Thus, it appears that learning strategies should be introduced later in the learning process and may distract if provided too early.

Spinal cord modulation associated with isometric contractions.

OBJECTIVE: The dual-strategy hypothesis explains single-joint voluntary movement by dividing movements into two different strategies and suggesting that different excitation pulses modulate these movements. The existence of this excitation pulse was evaluated by quantifying magnitude and timing changes in the H-reflex (changes in spinal excitability) prior to a voluntary contraction. These changes in spinal excitability were assessed during a ballistic plantar flexion isometric contraction, where both the target size and force level was manipulated. METHODS AND MATERIALS: Subjects were seated in a modified chair with a force transducer placed under the metatarsal heads to measure ankle force output. Following a visual stimulus subjects were trained to produce a plantar flexion force of 25% and 50% of a maximum voluntary contraction, within target sizes of 5% and 15% of the selected force level. Soleus motor neuron reflex excitability was analyzed by measuring changes in the H/M ratio. The H-reflex was randomly elicited by tibial nerve stimulations at 15, 30, 45, 60, 75 and 90 ms prior to the recorded average soleus premotor time for each of the force and target size conditions. RESULTS: A two-way repeated-measures analysis of variance indicated a significant effect among target sizes for the time of change in spinal excitability, slope of facilitation (rate of rise of spinal excitability), and peak facilitation. A significant difference was also established between force levels for the slope and peak facilitation, but there was no difference with time of facilitation. CONCLUSIONS: These results indicate that changes in both target size and force level can influence slope and peak of facilitation. However, only target size appears to affect the time of facilitation. Results clearly support the existence of an excitation pulse that is regulated by the type of movement.

Chronic myelogenous leukemia: an overview.

Important advances have been made in the treatment of chronic myelogenous leukemia (CML). In the past two decades, treatment of this disease has changed and now includes the use of hydroxyurea, interferon, tyrosine kinase inhibitors, and high-dose chemotherapy followed by hematopoietic stem cell transplantation. Because several relatively effective forms of therapy now exist, the care and management of patients with CML has become more complex. Through continued research and drug development, additional therapies are expected to emerge offering new hope and expanded treatment options for patients with CML.

The Effect of Life Events on Incidence of Injury in High School Football Players.

OBJECTIVE: To investigate the potential relationship between life events and injury among high school football players. DESIGN AND SETTING: This was a prospective cohort study. We collected athlete-exposure and injury data through 2 consecutive seasons at 13 high schools. Injury data consisted of injury status, frequency, severity, practice versus game, time of season, and total time lost from football participation. A life event survey was administered at the end of each season to obtain total, negative, and positive life event scores. SUBJECTS: A total of 331 varsity high school football players. MEASUREMENTS: Data collected included athlete exposures, injury reports, and life event surveys. Statistical analysis was completed using the Wilcoxon rank sum W test, the Pearson chi(2) test using an iterative proportional fitting procedure, and contingency chi(2) test. RESULTS: Total and negative life change measures significantly affected injury status and frequency of injury (P <.05). However, they did not reflect differences in severity of injury or time lost due to injury, and the positive life change measure was not associated with significant differences for any injury factor (P >.05). Preseason injury rates were significantly higher than season and playoff injury rates (P <.05). CONCLUSIONS: High school football players who experience high degrees of total and negative change were at greater risk of becoming injured and of sustaining multiple injuries. Playing situation and time of season also affected risk of injury.

Cognitive learning strategies: their effectiveness in acquiring racquetball skill.

Racquetball players were compared to assess whether a Self-directed strategy (self-monitoring), a Task-oriented strategy (attentional focusing), or a Combined use of both strategies would be beneficial in acquisition of racquetball skills. According to skill (Beginning, Advanced), players (N=80) were assigned into treatment groups. After treatment, participants executed diagonal lob serves and rallies for Acquisition and Retention phases (Session 1). During Session 2, subjects competed in a modified play setting (Transfer phase). Analysis of variance with repeated measures showed differences by skill during the basic tests favored Advanced players. During modified play, the Task-oriented group won significantly more points and games compared to the Self-directed and Control groups, regardless of skill. Results are discussed relative to skill and the literature on learning strategies.

Minimizing error in measurement of error: a proposed method for calculation of error in a two-dimensional motor task.

Traditional one-dimensional error scores are still consistently used in research on motor learning to quantify two-dimensional error; however, the inherent differences in two-dimensional tasks render that application inappropriate and often misleading. Consequently, the purpose of this paper was to propose a novel method of presenting errors, which more precisely represents the accuracy, direction, and variability of error in two-dimensional settings. Although closely related to several alternatives for representing errors, the methodology used and the results obtained provide a more accurate procedure for pinpointing critical trends in what have been commonly referred to as AE (absolute error), VE (variable error), CE (constant error), and E (total variability). The proposed measurements of AVE (adjusted variable error), DE (directional error), TSE (total spread of error), and RE (radial error) provide composite error scores carrying a variety of information about performance on two-dimensional tasks. Formulas and examples are provided to facilitate computation and enhance understanding of the proposed scores.

Determination of Fe-ligand bond lengths and the Fe-N-O bond angles in soybean ferrous and ferric nitrosylleghemoglobin a using multiple-scattering XAFS analyses.

The NO adducts of leghemoglobin (Lb) are implicated in biological processes, but only the adduct with ferrous Lb (Lb(II)NO) has been characterized previously. We report the first characterization of ferric nitrosylleghemoglobin (Lb(III)NO) and XAS experiments performed on frozen aqueous solutions of Lb(II)NO and Lb(III)NO at 10 K. The XANES and electronic spectra of the NO adducts are similar in shape and energies to the myoglobin (Mb) analogues. The environment of the Fe atom has been refined using multiple-scattering (MS) analyses of the XAFS data. For Lb(II)NO, the MS analysis resulted in an averaged Fe-N(p)(pyrrole) distance of 2.02 A, an Fe-N(epsilon)(imidazole) distance of 1.98 A, an Fe-N(NO) distance of 1.77 A, and an Fe-N-O angle of 147 degrees. The Fe-N(NO) distance and Fe-N-O angle obtained from the analysis of Lb(II)NO are in good agreement with those determined crystallographically for [Fe(TPP)(NO)] (TPP, tetraphenylporphyrinato), with and without 1-methylimidazole (1-MeIm) as the sixth ligand, and the MS XAFS structures reported previously for the myoglobin (Mb(II)NO) analogue and [Fe(TPP)(NO)]. The MS analysis of Lb(III)NO yielded an average Fe-N(p) distance of 2.00 A, an Fe-N(epsilon) distance of 1.89 A, an Fe-N(NO) distance of 1.68 A, and an Fe-N-O angle of 173 degrees. These bond lengths and angles are consistent with those determined previously for the myoglobin analogue (Mb(III)NO) and the crystal structures of the model complexes, [Fe(III)(TPP)(NO)(OH(2))](+) and [Fe(OEP)(NO)](+) (OEP, octaethylporphyrinato). The final XAFS R values were 16.1 and 18.2% for Lb(II)NO and Lb(III)NO, respectively.

NMR characterization of a single-cysteine mutant of Escherichia coli thioredoxin and a covalent thioredoxin-peptide complex.

The mechanism of disulfide reduction by thioredoxin in the cell is thought to occur through the formation and subsequent destruction of a mixed-disulfide intermediate between thioredoxin and the substrate. In order to model the interaction, we have prepared a mutant of Escherichia coli thioredoxin where the second cysteine residue of the active site has been replaced by an alanine residue. A specific covalent complex has been prepared between the remaining cysteine residue and a short cysteine-containing peptide. This paper describes the preparation and characterization of the mutant protein both free and in the peptide complex.

Chemical shift as a probe of molecular interfaces: NMR studies of DNA binding by the three amino-terminal zinc finger domains from transcription factor IIIA.

We report the NMR resonance assignments for a macromolecular protein/DNA complex containing the three amino-terminal zinc fingers (92 amino acid residues) of Xenopus laevis TFIIIA (termed zf1-3) bound to the physiological DNA target (15 base pairs), and for the free DNA. Comparisons are made of the chemical shifts of protein backbone 1HN, 15N, 13C alpha and 13C beta and DNA base and sugar protons of the free and bound species. Chemical shift changes are analyzed in the context of the structures of the zf1-3/DNA complex to assess the utility of chemical shift change as a probe of molecular interfaces. Chemical shift perturbations that occur upon binding in the zf1-3/DNA complex do not correspond directly to the structural interface, but rather arise from a number of direct and indirect structural and dynamic effects.

Probing protein structure using biochemical and biophysical methods. Proteolysis, matrix-assisted laser desorption/ionization mass spectrometry, high-performance liquid chromatography and size-exclusion chromatography of p21Waf1/Cip1/Sdi1.

The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53 tumor suppressor-dependent cell growth control in humans and other organisms, mediates G1/S-phase arrest through inhibition of cyclin-dependent kinases (Cdks). The enzymatic activity of these kinases is essential for progress through the cell division cycle and one level of cell cycle regulation is exerted through inhibition of Cdks by a family of small proteins, including p21. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2-terminus. Using proteolytic mapping, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, HPLC and size-exclusion chromatography, we show that p21, active as a Cdk inhibitor, exists in an extended, non-globular conformation in the absence of its biological target and that p21 lacks the hallmarks of stable secondary and tertiary structure. We have developed an efficient approach to obtain detailed proteolytic maps that takes advantage of the high accuracy and sensitivity of MALDI mass spectrometry. Our method allows a proteolytic map to be obtained from a single mass spectrum for fragments produced from a single proteolytic reaction.

Paralysis of the preterm rabbit fetus inhibits the pulmonary uptake of intraamniotic iron dextran.

OBJECTIVE: Whether fetal breathing movements or gasping result in the movement of amniotic fluid substances into the distal airways remains controversial. We evaluated the effect of paralysis of the preterm rabbit fetus on the pulmonary distribution of iron dextran. STUDY DESIGN: Laparotomy was performed on 10 New Zealand White rabbits of 25 days' gestation (term 31 days) under general anesthesia. Fetuses in one uterine horn were given an intramuscular injection of pancuronium (1.5 mg/kg) and fetuses in the other horn were given an equal volume of normal saline solution as controls. A 1 ml volume of iron dextran (100 mg/ml) was injected into the amniotic sac of all fetuses. The laparotomy was closed, and 20 to 24 hours later the fetuses were removed by hysterotomy and assessed for paralysis. Necropsy was performed. Lungs were stained with prussian blue and evaluated histologically for the presence of iron. RESULTS: A total of 92 pups were delivered (49 given pancuronium, 43 given normal saline solution), of which 64 were born alive. There were no differences between groups for live births (31 pancuronium, 33 normal saline solution), pup body weight, or lung weight. Pups given normal saline solution demonstrated more breathing motions, spontaneous movement, and brown (color of iron dextran) stomach contents than did the pups given pancuronium (p < 0.001). At necropsy a greater number of control pups (31/33) had brown lungs grossly compared with pups given pancuronium (2/31, p < 0.001). Lung histologic examination showed that more control pups (29/29) had iron in the trachea and main bronchi compared with pancuronium pups (0/27, p < 0.001), and more control pups (29/29) had iron in the distal lung airways compared with pancuronium pups (0/27, p < 0.001). With use of the Optimas Image Analysis System, iron in the lungs of control pups was found to be equally distributed between right versus left lungs, upper half versus lower half lungs, and anterior versus posterior lung sections. More iron was identified in the central airways than in the periphery (p < 0.001). CONCLUSION: We conclude that paralysis prevents the uptake of iron dextran into the main and distal airways of the rabbit fetus. Although lung fluid production results in a net efflux of fluid, we speculate that fetal breathing movements can result in the movement of fluid into distal airways and potentially provide fetal therapy.

Effects of buried charged groups on cysteine thiol ionization and reactivity in Escherichia coli thioredoxin: structural and functional characterization of mutants of Asp 26 and Lys 57.

To investigate the role of Asp 26 and Lys 57, two conserved, buried residues, in the redox mechanism of Escherichia coli thioredoxin (Trx), three mutant proteins, Asp 26 --> Ala (D26A), Lys 57 --> Met (K57M), and the double mutant D26A/K57M, were prepared, replacing the charged amino acids with hydrophobic residues with similar sizes. Both the oxidized (Trx-S2) and reduced [Trx-(SH)2] forms of the mutant thioredoxins are fully folded and similar in overall structure to the wild-type protein (wt). The structure of the active site hydrophobic surface is unchanged by the mutation of Asp 26 and Lys 57, since DNA polymerase activity in the 1:1 complex of the T7 gene 5 protein and mutant Trx-(SH)2 shows similar Kd values (approximately 5 nM) for both mutants and wt. In contrast, redox reactions involving thioredoxin as a catalyst of the reduction of disulfides or oxidation of dithiols are strongly affected by the mutations. In the reaction of Trx-S2 with thioredoxin reductase at pH 8.0, the kcat/Km value for the D26A mutant is decreased by a factor of 10 from that of wt, while the value for the D26A/K57M mutant is reduced 40-fold. The activity of Trx-(SH)2 as a protein disulfide reductase was measured with insulin, using fluorescence to detect oxidation of thioredoxin. At 15 degrees C and pH 8.0, both the D26A and K57M mutants showed 5--10-fold decreases in rates of reaction compared to those of the wild type, and the pH-rate profiles for the mutants were shifted 1 (K57M) and 2 (D26A) units to higher pH compared with the wt curve. NMR measurements for the three mutant proteins indicate that the proteins have the same global fold as that of the wild type, although changes in the chemical shifts of a number of resonances indicate local structural changes in the active site region. The resonances of oxidized D26A and D26A/K57M are pH-independent between pH 6.0 and 10.0, confirming the identification of the active site group titrating with a pKa of 7.5 in wt Trx-S2 as Asp 26. A profound change in the pKa of Asp 26, from 7.5 in the wild type to 9.4 in the mutant, is observed for K57M Trx-S2. The pH-dependent behavior of the resonances is affected in all mutant Trx-(SH)2 proteins. A single pKa shifted to higher values is observed on both the Cys 32 and Cys 35 Cbeta resonances. Ultraviolet absorbance measurements (A240) as a function of pH for wt Trx-(SH)2 demonstrate that the cysteine thiols titrate with apparent pK(a)s of about 7.1 and 9.9. The mutant proteins each show a single transition in the A240 measurements, with a midpoint at pH 7.8-8.0, consistent with the NMR results. The change in absorbance at 240 nm with increasing pH indicates that the number of thiols titrating in each mutant is greater than one but less than two. It is clear that both thiol pK(a)s have been significantly shifted by the mutations. The Cys 32 pKa is moved from 7.1 in wt to 7.8-8.0 in the mutants. The value of the Cys 35 pKa either is indistinguishable from that of Cys 32, thus accounting for more than one thiol titrating in the UV absorbance measurements or else is shifted to much higher pHs (> 10) where its transition is masked in both UV and NMR measurements by the effects of ionization of the tyrosine residues and unfolding of the protein. Our results strongly suggest that the buried Asp 26 carboxyl and Lys 57 epsilon-amino groups significantly affect the pK(a)s of the active site thiols, particularly that of the exposed low-pKa thiol Cys 32, thereby enhancing the rates of thiol-disulfide reactions at physiological pH.

Maximizing performance feedback effectiveness through videotape replay and a self-controlled learning environment.

This study was designed to examine whether participants who could control the schedule of performance feedback (KP) would learn differentially from those who received a rigid feedback schedule while learning a complex task. Participants (N = 48) were randomly assigned to self-controlled KP (SELF), summary KP (SUMMARY), yoked control (YOKE), or knowledge of results only (KR) conditions. Data collection consisted of an acquisition phase and a 4-day retention phase during which right-handed participants performed a left-handed ball throw. Overall, throwing form improved across trial blocks during acquisition, with the SUMMARY, SELF, and YOKE groups showing more improvement than the KR group. During retention, the SELF group retained a higher level of throwing form and accuracy in comparison to the other groups. Results suggest that when given the opportunity to control the feedback environment, learners require relatively less feedback to acquire skills and retain those skills at a level equivalent to or surpassing those who are given more feedback but receive it passively.

Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity.

The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53-dependent cell cycle control, mediates G1/S arrest through inhibition of Cdks and possibly through inhibition of DNA replication. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2 terminus. We show, using proteolytic mapping, circular dichroism spectropolarimetry, and nuclear magnetic resonance spectroscopy, that p21 and NH2-terminal fragments that are active as Cdk inhibitors lack stable secondary or tertiary structure in the free solution state. In sharp contrast to the disordered free state, however, the p21 NH2 terminus adopts an ordered stable conformation when bound to Cdk2, as shown directly by NMR spectroscopy. We have, thus, identified a striking disorder-order transition for p21 upon binding to one of its biological targets, Cdk2. This structural transition has profound implications in light of the ability of p21 to bind and inhibit a diverse family of cyclin-Cdk complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4. Our findings suggest that the flexibility, or disorder, of free p21 is associated with binding diversity and offer insights into the role for structural disorder in mediating binding specificity in biological systems. Further, these observations challenge the generally accepted view of proteins that stable secondary and tertiary structure are prerequisites for biological activity and suggest that a broader view of protein structure should be considered in the context of structure-activity relationships.

Dextrality and eye position in putting performance.

The relationship between eye and hand dominance and the relative positioning of the ball with respect to the subjects' eyes on putting performance was investigated. Twenty-four pure dextral (right-eyed and right-handed) and 24 cross-dextral (left-eyed and right-handed) novice golfers were randomly assigned to putt at a target 3.66 m away in two conditions, eyes focused directly over the ball and eyes positioned midway between their feet and the ball, i.e., eyes positioned 5 cm closer to their feet. The analysis indicated a significant interaction for dextrality and the relative position of the eyes during putting. Pure dextral golfers demonstrated less absolute error and less variable error in their putting performance when they focused their eyes midway between the ball and their feet than when they positioned their eyes directly over the ball. No differences in error scores were found for cross-dextrals across the two putting conditions.

1H, 15N and 13C resonance assignments for the first three zinc fingers of transcription factor IIIA.

The first three zinc fingers (ZF1-3) of transcription factor IIIA (TFIIIA) from Xenopus have been shown to contribute the majority of the binding energy to the intact TFIIIA-DNA interaction [Liao et al. (1992) J. Mol. Biol., 223, 857-871]. We have expressed a 92-amino acid polypeptide containing the three N-terminal zinc fingers of TFIIIA. This three-fingered polypeptide has been isotopically labeled with 15N and 13C in E. coli and purified to homogeneity. Assignment of backbone 1H, 15N, aliphatic 1H and 13C and aromatic 1H and 13C resonances of delta NZF1-3 has been obtained using a combination of single-, double- and triple-resonance multidimensional NMR experiments. The secondary structures for each finger have been determined from NOE connectivities, 3JNH alpha values and chemical shifts. The results show that each finger folds into a canonical beta-sheet-helix zinc finger structural motif, while the linkers adopt an extended structure. The helix between the two histidine ligands in ZF3 is distorted by zinc coordination, to accommodate the presence of four intervening amino acids instead of three as in ZF1 and ZF2.

Effects of visualization and Danjeon breathing on target shooting with an air pistol.

This investigation was conducted to assess which technique, visualization, Danjeon breathing, or a combination of these, would be most effective in enhancing target shooting with an air pistol. 48 college students (28 men, 20 women) were selected and randomly assigned to one of four groups (ns = 12): control, visualization, Danjeon breathing, and visualization plus Danjeon breathing. Data were analyzed by a 4 x 5 (groups x days) analysis of variance with repeated measures on days. Both the combination of visualization plus Danjeon breathing and the Danjeon breathing alone were significantly better than the control on most of the measures of shooting performance.

Electrostatic calculations of side-chain pK(a) values in myoglobin and comparison with NMR data for histidines.

Site-specific titration curves for 12 histidine residues in carbon monoxy sperm whale myoglobin (MbCO) have been determined from two-dimensional (2D) double quantum NMR experiments. Eight of these histidine residues are observed to titrate over the accessible pH range, and pK(a) values have been determined; bounds on the titration midpoints of the remaining four histidines are also reported. Results for residues 48, 81, and 119 differ significantly from those estimated from earlier, one-dimensional studies, but they are in good agreement with values recently determined for metaquomyoglobin. These experimental values (plus those determined earlier for tyrosine titrations) are compared to predictions from crystal structures of myoglobin using a numerical Poisson-Boltzmann model and a Monte Carlo treatment of the multiple-site titration. An extension of existing models is described that accounts for alternate tautomers for histidines. Calculations are reported using several choices for radii and charges, and for five crystal structures, in order to assess the sensitivity of the results to details of the calculations. In general, the agreement between calculated and observed titration behavior suggests that this theoretical model captures much of the electrostatic behavior in this system, even though it ignores conformational fluctuations and the differences in mean structures that may exist between crystal and solution. Interactions among titrating groups are often important; in general, these interactions lead to more gradual individual site titrations (the mean Hill coefficient is about 0.8), and in several cases the interactions are so strong that two side chains need to be considered as a unit and single residues may participate in two-step titrations. It is suggested that histidines involved in such two-step titrations and carboxylic acid residues with abnormally low pK(a) values in the native conformation may be involved in the acid-induced partial unfolding of MbCO.