Inhaled anesthetic binding sites in human serum albumin.

Previous evidence suggests multiple anesthetic binding sites on human serum albumin, but to date, we have only identified Trp-214 in an interdomain cleft as contributing to a binding site. We used a combination of site-directed mutagenesis, photoaffinity labeling, amide hydrogen exchange, and tryptophan fluorescence spectroscopy to evaluate the importance to binding of a large domain III cavity and compare it to binding character of the 214 interdomain cleft. The data show anesthetic binding in this domain III cavity of similar character to the interdomain cleft, but selectivity for different classes of anesthetics exists. Occupancy of these sites stabilizes the native conformation of human serum albumin. The features necessary for binding in the cleft appear to be fairly degenerate, but in addition to hydrophobicity, there is evidence for the importance of polarity. Finally, myristate isosterically competes with anesthetic binding in the domain III cavity and allosterically enhances anesthetic binding in the interdomain cleft.

Investigations of the effects of ethanol on warfarin binding to human serum albumin.

Ethanol effects on warfarin binding to human serum albumin (HSA) have been studied by equilibrium dialysis and fluorescence methods at pH 7.4 in phosphate-buffered saline at 37 degrees C. In the presence of various amounts of ethanol fluorescence intensity of bound warfarin decreased significantly but this intensity reduction was not solely from displacement of bound warfarin from HSA. By comparing fluorescence and equilibrium dialysis data we concluded that fluorescence intensity reduction of warfarin was mainly the result of changes in the surrounding environment of the warfarin binding site by ethanol interaction with HSA and that displacement of bound warfarin was not significant compared to the fluorescence intensity changes. The dissociation constant of warfarin binding to HSA decreased with an increasing amount of ethanol. From the changes in fluorescence intensity upon warfarin binding to HSA with the presence of ethanol ranging from 0 to 5.0% the following dissociation constants (Kd) were determined: 0% ethanol 5.39 +/- 0.2 microM, 0.1% ethanol 5.86 +/- 0.1 microM, 0.3% ethanol 5.83 +/- 0.2 microM, 0.5% ethanol 6.76 +/- 0.1 microM, 1% ethanol 7.01 +/- 0.1 microM, 3% ethanol 9.9 +/- 0.7 microM, 5% ethanol 13.01 +/- 0.1 microM. From the equilibrium dialysis with the same ranges of ethanol presence the following Kd values were obtained: 0% ethanol 6. 62 +/- 1.6 microM, 0.1% ethanol 6.81 +/- 1.1 microM, 0.3% ethanol 8. 26 +/- 2.5 microM, 0.5% ethanol 8.86 +/- 1.9 microM, 1% ethanol 11. 01 +/- 4.2 microM, 3% ethanol 20.75 +/- 2.4 microM, 5% ethanol 21.67 +/- 2.2 microM. The results suggest that warfarin bound to HSA was displaced by ethanol. These data indicate that ethanol influence on warfarin binding to HSA may alter the pharmacokinetics of warfarin.

A dynamic model for bilirubin binding to human serum albumin.

Site-directed mutagenesis of human serum albumin was used to study the role of various amino acid residues in bilirubin binding. A comparison of thermodynamic, proteolytic, and x-ray crystallographic data from previous studies allowed a small number of amino acid residues in subdomain 2A to be selected as targets for substitution. The following recombinant human serum albumin species were synthesized in the yeast species Pichia pastoris: K195M, K199M, F211V, W214L, R218M, R222M, H242V, R257M, and wild type human serum albumin. The affinity of bilirubin was measured by two independent methods and found to be similar for all human serum albumin species. Examination of the absorption and circular dichroism spectra of bilirubin bound to its high affinity site revealed dramatic differences between the conformations of bilirubin bound to the above human serum albumin species. The absorption and circular dichroism spectra of bilirubin bound to the above human serum albumin species in aqueous solutions saturated with chloroform were also examined. The effect of certain amino acid substitutions on the conformation of bound bilirubin was altered by the addition of chloroform. In total, the present study suggests a dynamic, unusually flexible high affinity binding site for bilirubin on human serum albumin.

An algorithm for automated bacterial identification using matrix-assisted laser desorption/ionization mass spectrometry.

An algorithm for bacterial identification using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is being developed. This mass spectral fingerprint comparison algorithm is fully automated and statistically based, providing objective analysis of samples to be identified. Based on extraction of reference fingerprint ions from test spectra, this approach should lend itself well to real-world applications where samples are likely to be impure. This algorithm is illustrated using a blind study. In the study, MALDI-MS fingerprints for Bacillus atrophaeus ATCC 49337, Bacillus cereus ATCC 14579T, Escherichia coli ATCC 33694, Pantoea agglomerans ATCC 33243, and Pseudomonas putida F1 are collected and form a reference library. The identification of test samples containing one or more reference bacteria, potentially mixed with one species not in the library (Shewanella alga BrY), is performed by comparison to the reference library with a calculated degree of association. Out of 60 samples, no false positives are present, and the correct identification rate is 75%. Missed identifications are largely due to a weak B. cereus signal in the bacterial mixtures. Potential modifications to the algorithm are presented and result in a higher than 90% correct identification rate for the blind study data, suggesting that this approach has the potential for reliable and accurate automated data analysis of MALDI-MS.

Familial dysalbuminemic byperthyroxinemia may result in altered warfarin pharmacokinetics.

Two distinct genotypes that result in the amino acid substitutions R218P and R218H in subdomain 2A of human serum albumin (HSA) have been identified as the cause of familial dysalbuminemic hyperthyroxinemia (FDH). These substitutions increase the affinity of subdomain 2A for thyroxine by approximately 10-fold elevating plasma thyroxine levels in affected individuals. While many studies have examined the binding of thyroxine to FDH HSA, the binding of FDH HSA to drugs has not been widely investigated. The widely administered drug warfarin was selected as a model compound to study FDH HSA/drug interactions since it binds to subdomain 2A and its pharmacokinetics are dramatically influenced by HSA binding. Using two independent methods, fluorescence spectroscopy and equilibrium dialysis with radioactive warfarin, the binding of recombinant R218P, R218H, R218M and wild type HSA to warfarin was measured. Both methods showed an approximately 5-fold decrease in the affinity of R218P, R218H and R218M HSA for warfarin relative to wild type HSA. The Kd values determined by fluorescence spectroscopy for wild type, R218H, R218P and R218M HSA binding to warfarin were 1.35, 5.38, 5.61, and 8.34 microM, respectively. The values determined by equilibrium dialysis were 5.36, 29.5, 14.5, and 23.4 microM, respectively. Based on the above findings one would expect the free serum warfarin concentration in homozygous R218P and R218H FDH patients to be elevated about 5-fold, resulting in about a 5-fold reduction in the serum half-life of the drug.

Structural investigations of a new familial dysalbuminemic hyperthyroxinemia genotype.

BACKGROUND: In a previous study, we found that the amino acid substitution R218H in human serum albumin (HSA) was the cause of familial dysalbuminemic hyperthyroxinemia (FDH) in several Caucasian patients. Subsequently the substitution R218P was shown to be the cause of FDH in several members of a Japanese family. This study attempts to resolve discrepancies in the only other study of R218P HSA and identifies two new Japanese R218P FDH patients unrelated to those described previously. METHODS AND RESULTS: Recombinant R218H, R218P, and wild-type HSA were synthesized in yeast, and the affinities of these HSA species for l- and d-thyroxine were determined using fluorescence spectroscopy. The dissociation constants for the binding of wild-type, R218P, and R218H HSA to l-thyroxine were 1.44 x 10(-6), 2.64 x 10(-7), and 2.49 x 10(-7) mol/L, respectively. The circular dichroism spectra of thyroxine bound to R218H and R218P HSA were markedly different, indicating that the structure of the thyroxine/HSA complex is different for either protein. CONCLUSIONS: The K(d) values for l-thyroxine bound to R218P and R218H HSA determined in this study were similar. The extremely high serum total-thyroxine concentrations reported previously for R218P FDH patients (10-fold higher than those reported for R218H FDH patients) are not consistent with the K(d) values determined in this study. Possible explanations for these discrepancies are discussed.

Reproducibility of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for replicate bacterial culture analysis.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to demonstrate the reproducibility of bacterial spectra collected on different days. The reproducibility of analysis by MALDI-MS of intact Escherichia coli and Bacillus atrophaeus is presented as a replicate culture study in which spectra were collected on ten different occasions over a three-month period and by two different operators. The analysis resulted in the detection of specific biomarkers in the m/z 2000-20 000 range. Some of the peaks in the Escherichia coli spectra are identified by comparison with other published work. All of the spectra obtained are reproducible over the course of the experiment, but operator variability does exist. The Escherichia coli spectra show operator variability while the Bacillus atrophaeus spectra do not. This work demonstrates the utility of MALDI in obtaining consistent spectra from bacteria over a period of time.

Extracting and visualizing matrix-assisted laser desorption/ionization time-of-flight mass spectral fingerprints.

We have developed a method for constructing and extracting matrix-assisted laser desorption/ionization (MALDI) fingerprints. This method is fully automated and statistically based, allowing a large number of spectra to be analyzed at a time in an objective manner. This method can be used to extract the fingerprint of a particular analyte from a spectrum containing multiple analytes. Therefore, this method lends itself well to real-world applications where samples to be analyzed are likely to be impure. We illustrate this method on experimental results from a series of studies of E. coli and B. atrophaeus MALDI time-of-flight mass spectrometry (TOFMS) fingerprints.

Expression of a human serum albumin fragment (consisting of subdomains IA, IB, and IIA) and a study of its properties.

Site-directed mutagenesis and a yeast expression system were used to synthesize a human serum albumin (HSA) fragment (amino acids 1-297). The HSA fragment (half HSA) was evaluated with a number of biophysical techniques and found to be similar to the corresponding region in wild-type HSA. Specifically, the circular dichroism spectra of half HSA and wild-type HSA were superimposable, indicating that the highly alpha-helical secondary structure of wild-type HSA is preserved in half HSA. Additionally, half HSA was partially reactive with a polyclonal antibody against authentic HSA. Half HSA, which contains subdomain IIA, had an affinity for thyroxine and several thyroxine analogs, similar to that observed previously for wild-type HSA. This study suggests that the production of recombinant HSA fragments will be useful for the study of HSA ligand interactions.

Mutagenesis studies of thyroxine binding to human serum albumin define an important structural characteristic of subdomain 2A.

The familial dysalbuminemic hyperthyroxinemia (FDH) phenotype results from a natural human serum albumin (HSA) mutant, with histidine instead of arginine at amino acid position 218. This mutation results in an enhanced affinity for thyroxine. In our earlier study, site-directed mutagenesis and a yeast protein expression system were used to synthesize FDH HSA and several other HSA mutants. Measurement of the binding of these HSA mutants to thyroxine and several thyroxine analogs using equilibrium dialysis and quenching of tryptophan 214 fluorescence allowed us to propose a preliminary model of thyroxine binding to the 2A subdomain of wild type and FDH HSA. In this study, we have produced several other HSA mutants. By comparing the binding affinity of these mutants for thyroxine and tetraiodothyroacetic acid to the binding affinity of other mutants, we were able to suggest a new model for thyroxine binding to the 2A subdomain of HSA. We found that the substitution of arginine at position 218 with alanine increased the binding affinity for thyroxine by 2 orders of magnitude relative to the binding affinity of wild type HSA for thyroxine. A more accurate understanding of the mechanism of thyroxine binding to HSA has allowed us to define an important structural characteristic of subdomain 2A, one of the two principal binding sites on HSA for small hydrophobic ligands.

Time-resolved fluorescence studies on site-directed mutants of human serum albumin.

Human serum albumin (HSA) contains a single tryptophan residue at position 214. The emission properties of tryptophan 214 from recombinant albumins, namely, normal HSA, FDH-HSA and a methionine 218 HSA were examined. In all cases, the excited state lifetimes were best described by a two component model consisting mainly of a Lorentzian distribution. The centers of these distributions were 5.60 ns for HSA, 4.23 ns for FDH-HSA, and 6.08 ns for Met-218 HSA. The global rotational correlation times of the three HSAs were near 41 ns while the amplitude and rate of the local motion varied. These changes in the lifetimes and mobilities suggest perturbation in the local protein environment near tryptophan 214 as a consequence of the amino acid substitutions.

Mutations in a specific human serum albumin thyroxine binding site define the structural basis of familial dysalbuminemic hyperthyroxinemia.

The familial dysalbuminemic hyperthyroxinemia (FDH) phenotype results from a natural human serum albumin (HSA) mutant with histidine instead of arginine at amino acid position 218. This mutation results in an enhanced affinity for thyroxine. Site-directed mutagenesis and a yeast protein expression system were used to synthesize wild type HSA and FDH HSA as well as several other HSA mutants. Studies on the binding of thyroxine to these HSA species using equilibrium dialysis and quenching of tryptophan 214 fluorescence suggest that the FDH mutation affects a single thyroxine binding site located in the 2A subdomain of HSA. Site-directed mutagenesis of HSA and thyroxine analogs were used to obtain information about the mechanism of thyroxine binding to both wild type and FDH HSA. These studies suggest that the guanidino group of arginine at amino acid position 218 in wild type HSA is involved in an unfavorable binding interaction with the amino group of thyroxine, whereas histidine at amino acid position 218 in FDH HSA is involved in a favorable binding interaction with thyroxine. Neither arginine at amino acid position 222 nor tryptophan at amino acid position 214 appears to favorably influence the binding of thyroxine to wild type HSA.

Expression of a human serum albumin variant with high affinity for thyroxine.

In this study a protein expression system was used to synthesize recombinant human serum albumin containing a mutation that has been shown to result in familial dysalbuminemic hyperthyroxinemia. Equilibrium dialysis was used to measure the binding of this recombinant human serum albumin with thyroxine. The association constant determined for the binding of this human serum albumin variant with thyroxine was shown to be 65-fold greater than that of recombinant normal human serum albumin.

A point mutation in the human serum albumin gene results in familial dysalbuminaemic hyperthyroxinaemia.

Using DNA samples obtained from two unrelated patients, diagnosed as having familial dysalbuminaemic hyperthyroxinaemia (FDH), exons 1-14 which span the entire coding region of the human serum albumin (HSA) gene were amplified by the polymerase chain reaction. The sequence of each of the 14 DNA fragments was then determined. In each case a point mutation was identified at nucleotide 653 which causes an Arg to His substitution at amino acid position 218. The substitution was confirmed by amino acid sequencing of a mutant peptide resulting from tryptic digestion of the protein. Abnormal affinity of FDH HSA for a thyroxine (T4) analogue was verified by an adaptation of the procedure used in routine free T4 measurement. The location of the mutation is discussed in relation to other studies on the binding properties of HSA.

[Hypothyroidism following intermittent administration of potassium iodide]. / Hypothyreose nach intermittierender Verabfolgung von Kalium Jodatum (DAB).

Iodide-induced hypothyroidism with high TSH and low T4 under long term potassium iodide medication was observed in a toddler suffering from chronic wheezy bronchitis -- in spite of intermittent application. The pathophysiology of iodide-induced hypothyroidism and the apparent immaturity of adaptation to high exogenous iodide in newborn and infants are discussed.

[Isolated abducent nerve paralysis in infectious mononucleosis]. / Isolierte Abduzensparese bei infektiöser Mononukleose.

Infectious mononucleosis in children and adolescents is characterized by a large variability of symptoms and clinical course. Serological tests for Epstein-Barr-Virusspecific antibodies are a valuable aid in differential diagnosis, in particular for pediatricians. - Isolated palsy of the abducens nerve is reported in a 12-year-old girl. The parainfectious etiology of this mononeuritis was determined only by specific serological tests.

Size and shape of the mandibular condyle in primates.

The relationships between the size of the articular surface of the mandibular condyle and masticatory muscle size, tooth size, diet, and biomechanical variables associated with mastication were studied by taking 12 measurements on skulls of 253 adult female anthropoid primates, including three to ten specimens from each of 32 species. In regressions of condylar length, width, or area against body weight, logarithmic transformations substantially improve the fit of the equations compared with untransformed data. There is a strong relationship between condylar measurements and body weight, with all correlations being .94 or higher. The slopes of the allometric regressions of length, width, and area of the condylar head indicate slight positive allometry with body size. Folivorous primates have smaller condyles than frugivorous primates, and colobines have smaller condyles than cebids, cercopithecines, or hominoids. When colobines are eliminated, the differences between frugivores and folivores are not significant. However, the two species with the relatively largest condyles are Pongo pygmaeus and Cercocebus torquatus, suggesting that there may be a relationship between unusually large condylar dimensions and the ability to crack hard nuts between the teeth. Cranial features having strong positive correlations with condylar dimensions include facial prognathism, maxillary incisor size, maxillary postcanine area, mandibular ramus breadth, and temporal fossa area. These data are interpreted as indicating that relatively large condyles are associated with relatively large masticatory muscles, relatively inefficient mandibular biomechanics, and a large dentition. These relationships support the growing evidence that the temporomandibular joint is a stress-bearing joint in normal function.