RESUMEN
This article describes an interesting case of a patient presenting with congestive heart failure found to have restrictive cardiomyopathy with initial laboratory evaluation showing hypogammaglobuminemia without a monoclonal band on serum and urine electrophoresis. This case highlights the clinically significant cardiac manifestation caused by protein misfolding, a defect in protein homeostasis. In addition, the utility of a relatively newer laboratory test, serum free light chains as well as the importance of clinical and pathophysiologic correlation is also discussed. We present a relatively uncommon cause of heart disease, cardiac amyloidosis in a patient with a systemic plasma cell dyscrasia, and multiple myeloma.
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Amiloidosis/etiología , Insuficiencia Cardíaca/etiología , Mieloma Múltiple , Deficiencias en la Proteostasis/complicaciones , Anciano , Amiloidosis/diagnóstico , Insuficiencia Cardíaca/diagnóstico , Humanos , Masculino , Deficiencias en la Proteostasis/diagnósticoRESUMEN
BACKGROUND: Human serum albumin is the principal protein in human serum. It participates in regulation of plasma oncotic pressure and transports endogenous and exogenous ligands such as thyroxine, free fatty acids, bilirubin, and various drugs. Therefore, studying its ligand binding mechanism is important in understanding many functions of the protein. SCOPE OF REVIEW: This review discusses the pleiotropic biochemical effects and their relevance to physiologic functions of albumin. MAJOR CONCLUSIONS: Although HSA is traditionally recognized for its ligand transport and oncotic effects in human circulation, our studies have revealed its participation in several other important physiological functions. In some instances, it may function as a catalyst. Pleiotropic properties of HSA have been exploited by development of recombinant HSA and its mutants, and the use of these recombinant proteins in studies with various biochemical and biophysical techniques. These studies allowed us to obtain new insights on the diverse roles of HSA in human physiology. The following aspects of HSA were discussed in this review: 1) HSA and its mutants' role in thyroxine transport, 2) structural details of the ligand binding functions of HSA to ligands such as warfarin, digoxin, halothane anesthetics, nitric oxide, bilirubin, free fatty acids, etc, and 3) the formation of modified albumin during myocardial ischemia, its diagnostic significance, and HSA's role in cardiovascular disease. GENERAL SIGNIFICANCE: The appreciation and understanding of structural details and new physiological roles has provided a renewed interest in HSA research. Specific structural information gained on various mechanisms of HSA-ligand interaction can be used to develop a model to better understand protein-drug interactions, aid in the development of new drugs with improved pharmacokinetic effects, and ultimately be used to improve the quality of healthcare. This article is part of a Special Issue entitled Serum Albumin.
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Albúmina Sérica/fisiología , Transporte Biológico , Enfermedad de la Arteria Coronaria/metabolismo , Cristalografía por Rayos X , Ácidos Grasos/metabolismo , Humanos , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/fisiología , Ligandos , Modelos Moleculares , Infarto del Miocardio/fisiopatología , Unión Proteica , Albúmina Sérica/química , Albúmina Sérica/metabolismo , Tiroxina/metabolismoRESUMEN
Fatty acids are endogenous ligands of human serum albumin (HSA) that induce conformational changes and participate in allosteric ligand binding to HSA. In a previous study, we showed that, when myristate (MYR) is present, the binding of [(14) C]ketoprofen (KP) to subdomain IA of HSA was increased, indicating that, when MYR binds to HSA, a new binding site in formed in that region. Meanwhile, an N-B transition has been reported to increase the binding of ligands at alkaline pH when the status of albumin is the B-conformer. Six histidine single mutants of HSA, H9A, H39A, H67A, H105A, H128A and H146A were produced and photolabeled with [(14) C]KP at pH 6.5, 7.4 and 8.2 and the role of each histidine in causing the N-B transition induced allosteric ligand binding was examined. Cyanogen bromide cleavage of the photolabeled native HSA showed that subdomain IA was the site of the allosteric binding of KP at pH 8.2. From the photolabeling results, H146 was found to play a prominent role whilst H128 played little or no role in the allosteric binding. However, the remaining 4 mutants did not show a clear photolabeling pattern that was similar to either native HSA or H146A and, as a result, no firm conclusions can be made. An additional histidine mutant, H146I, was produced to confirm the results for H146A. A similar experiment using H146I showed that a benzene ring-like structure at position 146 is required for the allosteric ligand binding to occur.
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Sitio Alostérico , Histidina/química , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Albúmina Sérica/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Dicroismo Circular , Humanos , Concentración de Iones de Hidrógeno , Cetoprofeno/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/genética , Albúmina Sérica/genéticaRESUMEN
BACKGROUND: S-nitrosation--the formation of S-nitrosothiols (RSNOs) at cysteine residues in proteins--is a posttranslational modification involved in signal transduction and nitric oxide (NO) transport. Recent studies would also suggest the formation of N-nitrosamines (RNNOs) in proteins in vivo, although their biological significance remains obscure. In this study, we characterized a redox-based mechanism by which N-nitroso-tryptophan residues in proteins may be denitrosated. METHODOLOGY/PRINCIPAL FINDINGS: The denitrosation of N-acetyl-nitroso Trp (NANT) by glutathione (GSH) required molecular oxygen and was inhibited by superoxide dismutase (SOD). Transnitrosation to form S-nitrosoglutathione (GSNO) was observed only in the absence of oxygen or presence of SOD. Protein denitrosation by GSH was studied using a set of mutant recombinant human serum albumin (HSA). Trp-214 and Cys-37 were the only two residues nitrosated by NO under aerobic conditions. Nitroso-Trp-214 in HSA was insensitive to denitrosation by GSH or ascorbate while denitrosation at Cys-37 was evident in the presence of GSH but not ascorbate. GSH-dependent denitrosation of Trp-214 was restored in a peptide fragment of helix II containing Trp-214. Finally, incubation of cell lysates with NANT revealed a pattern of protein nitrosation distinct from that observed with GSNO. CONCLUSIONS: We propose that the denitrosation of nitrosated Trp by GSH occurs through homolytic cleavage of nitroso Trp to NO and a Trp aminyl radical, driven by the formation of superoxide derived from the oxidation of GSH to GSSG. Overall, the accessibility of Trp residues to redox-active biomolecules determines the stability of protein-associated nitroso species such that in the case of HSA, N-nitroso-Trp-214 is insensitive to denitrosation by low-molecular-weight antioxidants. Moreover, RNNOs can generate free NO and transfer their NO moiety in an oxygen-dependent fashion, albeit site-specificities appear to differ markedly from that of RSNOs.
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Nitrógeno/química , Oxidación-Reducción , Proteínas/química , Antioxidantes/química , Cisteína/química , Glutatión/metabolismo , Humanos , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Nitrosaminas/química , Oxígeno/química , Procesamiento Proteico-Postraduccional , S-Nitrosotioles/química , Albúmina Sérica/química , Transducción de Señal , Superóxido Dismutasa/metabolismoRESUMEN
We report a perinatal death due to medium-chain acyl-CoA dehydrogenase deficiency, which was referred to the Coroner's Physician as sudden unexplained infant death. Detailed death investigation including the autopsy findings, and newborn biochemical and molecular studies revealed the cause and natural manner of death. This disorder affects fatty acid oxidation and results in decreased tolerance for fasting, which can be life threatening. This case illustrates the critical role of newborn screening in the investigation of perinatal death. A brief historical perspective of the origins of newborn biochemical screening is also presented.
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Acil-CoA Deshidrogenasa/deficiencia , Errores Innatos del Metabolismo Lipídico/diagnóstico , Tamizaje Neonatal , Muerte Súbita del Lactante/etiología , Carnitina/análogos & derivados , Carnitina/sangre , Eliminación de Gen , Humanos , Recién Nacido , Errores Innatos del Metabolismo Lipídico/genética , Masculino , Mutación PuntualAsunto(s)
Síndrome Coronario Agudo/diagnóstico , Biomarcadores/sangre , Ácidos Grasos/sangre , Infarto del Miocardio/diagnóstico , Albúmina Sérica/análisis , Albúmina Sérica/metabolismo , Biomarcadores/metabolismo , Ácidos Grasos/metabolismo , Humanos , Isquemia Miocárdica/diagnóstico , Proyectos Piloto , Unión ProteicaRESUMEN
Statins reduce cholesterol biosynthesis by inhibiting HMG-CoA reductase and thereby lower total cholesterol and LDL cholesterol levels in serum, which in turn lower the incidence of cardiovascular disease (CVD). Statins are also known to modulate various cellular functions such as gene expression, cell proliferation, and programmed cell death through inhibition of downstream intermediates in cholesterol synthesis. In this study, we have investigated the possible effects of statins on the secretion of serum albumin from cultured HepG2 cells since high levels of serum albumin are associated with reduced risks for CVD and statins are effective in lowering the risk of CVD through other effects in addition to their effects on serum total cholesterol and LDL cholesterol levels, known as pleiotropic effects. Our results showed that simvastatin increased HSA secretion up to 32.3% compared to the control group. Among 3 statin analogs we tested, simvastatin exhibited the highest stimulatory effects on HSA secretion compared to the control group. Our study also showed that the increased HSA secretions from HepG2 cells by simvastatin treatments were due to the increased rate of HSA synthesis, not due to the reduced posttranslational degradation rate of HSA. Our finding suggests another added benefit of statins' treatments in preventing CVD through stimulation of HSA biosynthesis.
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Línea Celular/efectos de los fármacos , Línea Celular/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Albúmina Sérica/metabolismo , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/prevención & control , Línea Celular/citología , Relación Dosis-Respuesta a Droga , Humanos , Lovastatina/farmacología , Pravastatina/farmacología , Albúmina Sérica/genética , Simvastatina/farmacologíaRESUMEN
The objective of this study is to investigate the relationship between lowlevels of human serum albumin (HSA) and the incidence of coronary heart disease (CHD) in a cohort of elderly Japanese-American men. Using data from the Honolulu Heart Program's fourth examination (1991-1993), HSA levels of 998 Japanese American men aged 71-93 years was compared with plasma levels of fibrinogen, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, diastolic BP, BMI, and fasting blood glucose. HSA was significantly negatively associated with age and fibrinogen, and significantly positively associated with total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, diastolic BP, BMI and fasting blood glucose. After adjusting for age, tertiles of HSA were significantly positively associated with total cholesterol, HDL cholesterol and triglycerides, and significantly negatively associated with fibrinogen. Using multivariate stepwise regression, significant correlations were seen between HSA and fibrinogen, cholesterol, age, HDL cholesterol and triglycerides, and a borderline correlation was seen with systolic blood pressure. However, the model R-square for all variables was only 0.10. In conclusion, HSA levels are significantly associated with several traditional cardiovascular risk factors, particularly serum lipid levels.
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Colesterol/sangre , Enfermedad Coronaria/sangre , Enfermedad Coronaria/epidemiología , Albúmina Sérica/análisis , Anciano , HDL-Colesterol/sangre , LDL-Colesterol/análisis , Fibrinógeno/análisis , Hawaii/epidemiología , Humanos , Masculino , Factores de RiesgoRESUMEN
Epidemiologic studies have shown an inverse relationship between human serum albumin (HSA) levels and coronary heart disease (CHD). However, no mechanisms have been identified to explain this relationship. We hypothesized that this relationship is due to differences in binding affinity of fatty acids to HSA and subsequent atherogenic lipoprotein synthesis and secretion from hepatocytes. To test our hypothesis we undertook the current study. Using HepG2 cells, we demonstrated that oleic acid (OA) bound to HSA in a molar ratio of 4:1 and after incubation for 24 h stimulated apolipoprotein B (apoB) secretion. We also tested whether mutant forms of HSA could alter the binding affinity for fatty acids and change the availability of substrate for lipoprotein secretion. Based on the results obtained in this study using 11 HSA mutant proteins complexed with OA, we were able to classify into three major mutant groups based on their effects on apoB secretion. One group in particular (R410Q/Y411W, R410A/Y411A, and W214L/Y411W) showed a significantly diminished effect on apoB secretion when compared to the wild type HSA/OA complex. Furthermore, the amount of free OA internalized in HepG2 cells in the presence of HSA mutant proteins was in good agreement with the effects seen on apoB secretion by the various HSA mutants. This suggests that some mutant forms of HSA might potentially bind fatty acids with a much higher binding affinity and thus deprive fatty acids available for lipoprotein assembly in hepatocytes. In conclusion, our data illustrate that certain HSA polymorphic forms may be protective against the development of CHD and warrants further investigation.
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Apolipoproteínas B/metabolismo , Ácido Oléico/metabolismo , Albúmina Sérica/metabolismo , Línea Celular Tumoral , Enfermedad Coronaria/etiología , Enfermedad Coronaria/metabolismo , Hepatocitos/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Albúmina Sérica/química , Albúmina Sérica/genética , Relación Estructura-ActividadRESUMEN
Site-directed mutagenesis was used to study the role of histidine residues located in domain I in the neutral-to-basic (N-B) transition of human serum albumin (HSA). Based on a previous study of the N-B transition by means of proton NMR, the following recombinant HSA species were synthesized in the yeast species, Pichia pastoris: H9F, H9S, H39F, H39S, H67F, H67S, H105F, H105S, H128F, H128S, H146F, H146S, and wild type HSA. By monitoring the fluorescent intensity of warfarin bound to the above recombinant human serum albumin species as a function of pH, the mutational effect of individual histidine residues on the N-B transition was examined. While H9, H67, H105, H128 and H146 contribute to the transition significantly, H39 appears to have virtually no contribution to the transition. Based on the X-ray crystallographic structure, it is suggested that electrostatic interactions are the principal factor in determining the histidine pK shifts.
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Histidina/química , Albúmina Sérica/química , Histidina/genética , Humanos , Concentración de Iones de Hidrógeno , Mutagénesis Sitio-Dirigida , Mutación , Pichia/química , Pichia/genética , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Albúmina Sérica/análisis , Albúmina Sérica/genéticaRESUMEN
We report a case of Insulinoma, a rare neuroendocrine tumor with an incidence of approximately four per 5 million. This case demonstrates the characteristic clinical, biochemical and histological features of an insulinoma, a rare benign neuroendocrine tumor where early recognition is important to ensure proper surgical treatment and prevent serious adverse consequences.
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Insulinoma , Neoplasias Pancreáticas , Femenino , Humanos , Técnicas para Inmunoenzimas , Insulinoma/diagnóstico , Insulinoma/patología , Insulinoma/cirugía , Persona de Mediana Edad , Páncreas/patología , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/cirugía , Resultado del TratamientoRESUMEN
Multiple binding sites for anaesthetics in HSA (human serum albumin) make solution studies difficult to interpret. In the present study, we expressed the wild-type HSA domain 3 (wtHSAd3), a peptide with two known anaesthetic binding sites in a yeast expression system. We also expressed a site-directed mutant of domain 3 (Y411Wd3). The stability and secondary structure of the constructed fragments were determined by HX (hydrogen-tritium exchange) and CD spectroscopy. The binding of two general anaesthetics, 2-bromo-2-chloro-1,1,1-trifluoroethane and propofol, to wtHSAd3 and Y411Wd3 was determined using isothermal titration calorimetry, HX and intrinsic tryptophan fluorescence quenching. Although the expressed fragments are less stable than intact wtHSA as indicated by both CD and HX, they retain the secondary structure and anaesthetic-binding characteristics of an intact HSA molecule, but with fewer binding sites. Y411Wd3 had decreased affinity for propofol but not for 2-bromo-2-chloro-1,1,1-trifluoroethane, consistent with steric hindrance. Retention of structural features and anaesthetic binding properties with fewer binding sites in this truncated protein provide feasibility for using scaled-down models of otherwise intractable systems to gain an understanding of anaesthetic binding requirements and binding-stability relationships.
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Anestésicos Generales/química , Halotano/química , Propofol/química , Albúmina Sérica/química , Sitios de Unión , Humanos , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , TermodinámicaRESUMEN
Tryptophan 214, the only tryptophan residue in human serum albumin, is located in the physiologically important subdomain 2A ligand binding site. In the present study the fluorescence lifetime of tryptophan 214 in the following human serum albumin (HSA) mutants with substitutions in subdomain 2A were determined: K195M, K199M, F211V, R218M, R218H, R218A, R222M, H242V, and R257M. An HSA mutant in which tryptophan was moved from subdomain 2A to subdomain 3A (W214L/Y411W) was also examined. Additionally, the fluorescence lifetime of tryptophan 214 in an HSA fragment consisting of subdomains 1A, 1B, and 2A (1A-1B-2A HSA) was determined. For those species expected to have the most dramatic changes in tryptophan microenvironment, W214L/Y411W and 1A-1B-2A HSA, clear changes in tryptophan lifetimes were observed. Significant changes were also seen for those species with mutations at position 218, which is next to tryptophan in the X-ray structure of HSA. However, significant changes were also observed for H242V and R257M, which contain substitutions at positions not immediately adjacent to tryptophan 214, highlighting the conformational flexibility of subdomain 2A.
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Modelos Químicos , Modelos Moleculares , Albúmina Sérica/análisis , Albúmina Sérica/química , Espectrometría de Fluorescencia/métodos , Triptófano/análisis , Triptófano/química , Sustitución de Aminoácidos , Sitios de Unión , Simulación por Computador , Humanos , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Relación Estructura-ActividadRESUMEN
Propofol and halothane are clinically used general anaesthetics, which are transported primarily by HSA (human serum albumin) in the blood. Binding characteristics are therefore of interest for both the pharmacokinetics and pharmacodynamics of these drugs. We characterized anaesthetic-HSA interactions in solution using elution chromatography, ITC (isothermal titration calorimetry), hydrogen-exchange experiments and geometric analyses of high-resolution structures. Binding affinity of propofol to HSA was determined to have a K(d) of 65 microM and a stoichiometry of approx. 2, whereas the binding of halothane to HSA showed a K(d) of 1.6 mM and a stoichiometry of approx. 7. Anaesthetic-HSA interactions are exothermic, with propofol having a larger negative enthalpy change relative to halothane. Hydrogen-exchange studies in isolated recombinant domains of HSA showed that propofol-binding sites are primarily found in domain III, whereas halothane sites are more widely distributed. Both location and stoichiometry from these solution studies agree with data derived from X-ray crystal-structure studies, and further analyses of the architecture of sites from these structures suggested that greater hydrophobic contacts, van der Waals interactions and hydrogen-bond formation account for the stronger binding of propofol as compared with the less potent anaesthetic, halothane.
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Anestésicos Generales/metabolismo , Halotano/metabolismo , Propofol/metabolismo , Albúmina Sérica/metabolismo , Anestésicos Generales/farmacocinética , Anestésicos Generales/farmacología , Sitios de Unión , Calorimetría , Cromatografía Liquida , Halotano/farmacocinética , Halotano/farmacología , Humanos , Hidrógeno/metabolismo , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Propofol/farmacocinética , Propofol/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Albúmina Sérica/química , Electricidad Estática , Tritio/metabolismoRESUMEN
Human serum albumin (HSA) is the major protein component of blood plasma and serves as a transporter for thyroxine and other hydrophobic compounds such as fatty acids and bilirubin. We report here a structural characterization of HSA-thyroxine interactions. Using crystallographic analyses we have identified four binding sites for thyroxine on HSA distributed in subdomains IIA, IIIA, and IIIB. Mutation of residue R218 within subdomain IIA greatly enhances the affinity for thyroxine and causes the elevated serum thyroxine levels associated with familial dysalbuminemic hyperthyroxinemia (FDH). Structural analysis of two FDH mutants of HSA (R218H and R218P) shows that this effect arises because substitution of R218, which contacts the hormone bound in subdomain IIA, produces localized conformational changes to relax steric restrictions on thyroxine binding at this site. We have also found that, although fatty acid binding competes with thyroxine at all four sites, it induces conformational changes that create a fifth hormone-binding site in the cleft between domains I and III, at least 9 A from R218. These structural observations are consistent with binding data showing that HSA retains a high-affinity site for thyroxine in the presence of excess fatty acid that is insensitive to FDH mutations.
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Albúminas/metabolismo , Hipertiroxinemia/metabolismo , Tiroxina/metabolismo , Sitios de Unión , Humanos , Hipertiroxinemia/genética , Modelos Moleculares , Estructura Molecular , Unión Proteica , Difracción de Rayos XRESUMEN
In the present study, we used the human EA.hy926 endothelial cell line as the model system to investigate the effect of human serum albumin (HSA) and its structural variants on cholesterol efflux. Initial studies showed that HSA promoted cholesterol efflux in a dose- and time-dependent manner, reaching a plateau at 10 mg/ml at 90 min. As a control, gelatin displayed no significant effect on efflux, while HSA was significantly more efficient than ovalbumin and bovine serum albumin (BSA) in promoting cholesterol efflux. Equal molar concentrations of HSA and apolipoprotein A-I (apoA-I) showed that apoA-I had considerably higher efficiency in efflux. However, the prevailing high plasma concentrations of HSA may compensate for its lower efflux rate compared to apoA-I. To characterize the mechanism of HSA-mediated cholesterol efflux, we studied the effects of cAMP and temperature on efflux using both EA.hy926 endothelial cells and murine RAW 264.7 macrophages. We found that HSA-mediated efflux occurred via a cAMP-independent and relatively temperature-insensitive pathway. We next examined the nature of HSA-cholesterol interaction by comparing the effects of various HSA mutants to wild-type HSA on cholesterol efflux. We found specific interactions between subdomains 2A and 3A and cholesterol, as indicated by the changes in the efflux rate of various HSA mutants. In conclusion, our study provides evidence for the role of HSA in cholesterol efflux, and shows that the substitution of specific amino acid residues in subdomains of 2A and 3A may be important structural determinants in its ability to bind to cholesterol and participate in cholesterol efflux.
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Colesterol/metabolismo , Endotelio Vascular/efectos de los fármacos , Albúmina Sérica/farmacología , Apolipoproteína A-I/fisiología , Sitios de Unión , Células Cultivadas , AMP Cíclico/fisiología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/metabolismo , Humanos , Albúmina Sérica/química , Albúmina Sérica/metabolismo , Relación Estructura-Actividad , Temperatura , Factores de TiempoRESUMEN
BACKGROUND: Clinical diagnoses were correlated with results of a Co(II)-albumin binding assay in 167 patients treated at an emergency department of a health maintenance organization. METHODS: Patients were evaluated as being nonischemic or potentially ischemic through standard coronary disease indicators [creatine kinase (CK), CK-MB, cardiac troponin I, and electrocardiographic findings] and were tested by a Co(II)-albumin binding assay. Samples were tested anonymously, and the study was double-blinded. The sensitivity and specificity of this assay for the detection of ischemia were evaluated by ROC curve analysis. Known Co(II) binding sites on albumin were analyzed by N-terminal amino acid sequencing. RESULTS: The mean absorbance units (ABSU) +/- 2 SD for non-myocardial ischemic and myocardial ischemic individuals measured at 470 nm were 0.43 +/- 0.10 and 0.63 +/- 0.25, respectively (P <0.0001). The area under the ROC curve was 0.95 [95% confidence interval (CI), 0.92-0.99], and at a cutoff value of 0.50 ABSU, sensitivity and specificity were 88% (78-94%) and 94% (86-98%), respectively, suggesting a high distinction between the two groups. When we compared non-acute myocardial infarction (AMI) and AMI ischemic individuals, the area under the ROC curve was 0.66 (95% CI, 0.53-0.79) and was considered a poor discriminator between these two groups. N-Terminal amino acid sequencing data for purified albumin showed normal amino acid residues for six of seven high-ABSU (> or =0.70) individuals and one nonischemic individual tested. However, only one individual with a high ABSU (0.80) had two missing amino acid residues (DA) from the N-terminal region. Clinical diagnosis for this patient did not reveal an ischemic event. CONCLUSIONS: The Co(II)-albumin binding test may serve as a useful diagnostic tool in emergency facilities for the assessment of myocardial ischemia. High and low ABSU were associated with myocardial ischemic individuals and non-myocardial ischemic individuals, respectively. However, the Co(II)-albumin binding was a poor discriminator between ischemic individuals with and without MI.
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Cobalto/química , Isquemia Miocárdica/diagnóstico , Albúmina Sérica/química , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Sitios de Unión , Biomarcadores/sangre , Femenino , Humanos , Indicadores y Reactivos , Masculino , Persona de Mediana Edad , Infarto del Miocardio/diagnóstico , Unión Proteica , Curva ROCRESUMEN
Electrostatic interactions have been proposed as a potentially important force for anesthetics and protein binding but have not yet been tested directly. In the present study, we used wild-type human serum albumin (HSA) and specific site-directed mutants as a native protein model to investigate the role of electrostatic interactions in halothane binding. Structural geometry analysis of the HSA-halothane complex predicted an absence of significant electrostatic interactions, and direct binding (tryptophan fluorescence and zonal elution chromatography) and stability experiments (hydrogen exchange) confirmed that loss of charge in the binding sites, by charged to uncharged mutations and by changing ionic strength of the buffer, generally increased both regional (tryptophan region) and global halothane/HSA affinity. The results indicate that electrostatic interactions (full charges) either do not contribute or diminish halothane binding to HSA, leaving only the more general hydrophobic and van der Waals forces as the major contributors to the binding interaction.
Asunto(s)
Anestésicos por Inhalación/farmacología , Halotano/farmacología , Albúmina Sérica/química , Albúmina Sérica/metabolismo , Amidas/química , Sitios de Unión , Clonación Molecular , ADN Complementario/metabolismo , Humanos , Hidrógeno/metabolismo , Concentración 50 Inhibidora , Iones , Hígado/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Electricidad Estática , Factores de Tiempo , Tritio/metabolismo , Triptófano/metabolismoRESUMEN
The binding of warfarin to the following human serum albumin (HSA) mutants was examined: K195M, K199M, F211V, W214L, R218M, R222M, H242V, and R257M. Warfarin bound to human serum albumin (HSA) exhibits an intrinsic fluorescence that is approximately 10-fold greater than the corresponding signal for warfarin in aqueous solution. This property of the warfarin/HSA complex has been widely used to determine the dissociation constant for the interaction. In the present study, such a technique was used to show that specific substitutions in subdomain 2A altered the affinity of HSA for warfarin. The fluorescence of warfarin/mutant HSA complexes varied widely from the fluorescence of the warfarin/wild-type HSA complex at pH = 7.4, suggesting changes in the structure of the complex resulting from specific substitutions. The fluorescence of the warfarin/wild-type HSA complex increases about twofold as the pH is increased from 6.0 to 9.0 due to the neutral-to-base (N-B) transition, a conformational change that occurs in HSA as a function of pH. Changes in the fluorescence of warfarin/mutant HSA complexes as a function of pH suggests novel behavior for most HSA species examined. For the HSA mutants F211V and H242V, the midpoint of the N-B transition shifts from a wild-type pH of 7.8 to a pH value of 7.1-7.2.