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2.
J Biol Chem ; 264(11): 6029-32, 1989 Apr 15.
Article in English | MEDLINE | ID: mdl-2703478

ABSTRACT

X-ray absorption spectroscopy has been used to investigate the local environment of the copper sites in bovine dopamine beta-hydroylase, the enzyme that catalyzes the conversion of dopamine to norepinephrine in the adrenal medulla and noradrenergic nerve cells. The marked similarity of the x-ray absorption edge features of the oxidized and ascorbate-reduced forms of the enzyme with those of the corresponding Cu(imidazole)4 complexes suggests that the ligation in both cases is very similar. Furthermore, this similarity is found for the extended x-ray absorption fine structure data, and analysis shows only nitrogen (or oxygen) ligation for both enzyme forms. Thus, four nitrogen atoms provide the best fit to the data at an average distance of 1.97 +/- 0.02 A for the oxidized enzyme and four nitrogen atoms at 2.05 +/- 0.02 A for the ascorbate-reduced form. The present data analysis also indicates that there is little change in the average copper ligand environment upon reduction of the enzyme-bound copper from Cu(II) to the Cu(I). The data for the oxidized form of the enzyme are in agreement with previous spin-echo EPR experiments that show three to four imidazole nitrogen ligands for each copper (McCracken, J., Desai, P. R., Papadopoulos, N. J., Villafranca, J. J., and Peisach, J. (1988) Biochemistry 27, 4133-4137). In addition, the data do not indicate the presence of any heavy atom (sulfur or chlorine) ligation to the ascorbate-reduced form of the enzyme as reported by Scott et al. (Scott, R. A., Sullivan, R. J., DeWolf, W. E., Jr., Dolle, R. E., and Kruse, L. I. (1988) Biochemistry 27, 5411-5417).


Subject(s)
Dopamine beta-Hydroxylase , Adrenal Medulla/enzymology , Animals , Cattle , Copper , Imidazoles , Oxidation-Reduction , Spectrum Analysis , X-Rays
4.
J Biol Chem ; 261(19): 8805-10, 1986 Jul 05.
Article in English | MEDLINE | ID: mdl-3722175

ABSTRACT

The properties of sulfhemoglobin (sulfHb) were investigated using disc gel isoelectric focusing and optical spectrophotometry. Laboratory-prepared samples, which contained a high yield of sulfHb (70-85%), and a patient-derived sample, which contained a low yield (12%), contain a tetrameric population that reflects a random distribution of modified (sulfurated) subunits. Hybrid tetramers, i.e. those containing sulfurated and unmodified subunits, were resolved from fully sulfurated and unmodified hemoglobin upon electrofocusing of ferric or ferrous sulHb samples. The electrophoretic differences between ferric sulfHb and metHb arise from a difference in the pK for the met to hydroxymet conversion of sulfurated subunits (Carrico, R., Peisach, J., and Alben, J. O. (1978) J. Biol. Chem. 253, 2386-2391). Both partially and fully sulfurated tetramers, when in the deoxy form and when in the fully ligated CO form, co-focus with their unmodified deoxy-Hb and Hb CO counterparts. Thus, these sulfurated tetramers exhibit normal ligand-dependent ionization of Bohr protons. In air-equilibrated samples, hybrid tetramers are partially ligated as a result of the reduced O2 affinity of the sulfurated subunits. These partially ligated tetramers exhibit a pI intermediate between oxy-Hb and deoxy-Hb due to the fractional ionization of Bohr protons. The partially sulfurated, partially ligated tetramers as well as deoxy-sulfHb tetramers were found to bind 2,3-diphosphoglycerate in disc gels. Despite the preservation of these heterotropic effects, which are confirmed in CO binding studies in solution, fully sulfurated tetramers recovered using preparative isoelectric focusing exhibit little or no cooperativity.


Subject(s)
Sulfhemoglobin/metabolism , Carbon Monoxide/blood , Humans , Isoelectric Focusing , Kinetics , Macromolecular Substances , Molecular Weight , Protein Binding , Spectrophotometry , Sulfhemoglobin/isolation & purification
5.
Eur J Biochem ; 145(3): 549-54, 1984 Dec 17.
Article in English | MEDLINE | ID: mdl-6096143

ABSTRACT

Autoxidation and chemically-induced oxidation of hemoglobin Zurich (beta 63 E7 Arg) have been investigated by electron paramagnetic resonance and optical absorption spectroscopy. The results show that the replacement of the distal histidine of the hemoglobin beta chains by an arginine greatly enhances the susceptibility of the heme-iron to oxidative challenge. Both the kinetics and the products of the oxidation are pH dependent. Thus, at acidic and neutral pH, treatment of the protein with ferricyanide leads to a fast conversion of the oxy-protein to aquo-methemoglobin, which, eventually, is slowly converted to hemichromes. In contrast, the hydroxy-met derivative, formed upon chemical oxidation at high pH, is rapidly converted to hemichromes. The electron paramagnetic resonance features of the ferric derivatives of hemoglobin Zurich are somewhat singular, reflecting the modifications of the heme environment in the distal region of the abnormal chains. However, they can be related to heme complexes having their structural counterparts in oxidation products of hemoglobin A.


Subject(s)
Hemoglobins, Abnormal , Chemical Phenomena , Chemistry , Electron Spin Resonance Spectroscopy , Erythrocytes/analysis , Humans , Hydrogen-Ion Concentration , Kinetics , Magnetic Resonance Spectroscopy , Oxidation-Reduction , Spectrophotometry
6.
Eur J Biochem ; 132(1): 165-9, 1983 Apr 15.
Article in English | MEDLINE | ID: mdl-6840080

ABSTRACT

Results of experiments based upon circular dichroic spectra suggest that configurationally (Z leads to E) isomerized bilirubin (photobilirubin) binds to human serum albumin at the primary bilirubin binding site with an affinity only 2-3 times lower than that of bilirubin. The high affinity of photobilirubin for albumin, comparable to that of bilirubin, supports the roles of albumin in the stabilization and transport of the isomerized pigment in vivo and strongly suggests that albumin also functions to sequester photobilirubin effectively, reducing its toxic potential. The high affinity of photobilirubin for albumin predicts that the isomerized pigment, formed in large amounts during phototherapy for neonatal hyperbilirubinemia, should not appear in the fast-diazo-reacting ('direct') bilirubin pool nor should it interfere with nonspectroscopic bilirubin binding tests. These predictions were confirmed for the Evelyn and Malloy diazo assay for 'direct' bilirubin and a Sephadex chromatography method for assessing 'loosely bound' plasma bilirubin.


Subject(s)
Bilirubin/blood , Serum Albumin/metabolism , Binding Sites , Circular Dichroism , Humans , Protein Binding , Stereoisomerism
7.
Biophys J ; 38(3): 277-85, 1982 Jun.
Article in English | MEDLINE | ID: mdl-7104439

ABSTRACT

Stellacyanin is a mucoprotein of molecular weight approximately 20,000 containing one copper atom in a blue or type I site. The metal ion can exist in both the Cu(II) and Cu(I) redox states. The metal binding site in plastocyanin, another blue copper protein, contains one cysteinyl, one methionyl, and two imidazoyl residues (Colman et al. 1978. Nature [Lond.]. 272:319-324.), but an exactly analogous site cannot exist in stellacyanin as it lacks methionine. The copper coordination in stellacyanin has been studied by x-ray edge absorption and extended x-ray absorption fine structure (EXAFS) analysis. A new, very conservative data analysis procedure has been introduced, which suggests that the there are two nitrogen atoms in the first coordination shell of the oxidized [Cu(II)] protein and one in the reduced [Cu(I)] protein; these N atoms have normal Cu--N distances: 1.95-2.05 A. In both redox states there are either one or two sulfur atoms coordinating the copper, the exact number being indeterminable from the present data. In the oxidized state the Cu--S distance is intermediate between the short bond found in plastocyanin and those found in near tetragonal copper model compounds. Above -140 degree C, radiation damage of the protein occurs. At room temperature the oxidized proteins is modified in the x-ray beam at a rate of 0.25%/s.


Subject(s)
Metalloproteins , Plant Proteins , Binding Sites , Spectrum Analysis , X-Rays
8.
Clin Chem ; 28(3): 432-9, 1982 Mar.
Article in English | MEDLINE | ID: mdl-7067082

ABSTRACT

The apparent unbound bilirubin concentration by the "peroxidase" method (U) and the total unconjugated bilirubin in blood (T), albumin-bound bilirubin (B), and reserve bilirubin binding capacity (R) by the bilirubin hematofluorometer were measured in 164 specimens from 98 neonates and in a series of artifactual specimens, made by adding bilirubin to the blood of a single adult donor. Linear correlations between U and (B/R) were found for both the prepared specimens (r = 0.99) and the clinical specimens (r = 0.87), with the slopes of the regression lines being close to the reciprocal of the albumin-bilirubin binding constant, a prediction of the mass action law. An excellent linear correlation was observed for the prepared specimens (r = 0.96) between U and (T--B), the concentration of bilirubin bound to low-affinity secondary sites ("loosely bound bilirubin"). A simple model for low-affinity binding of bilirubin in blood predicts this simple relation. A significant linear correlation between U and (T--B) was found for the clinical specimens, although the correlation was less good (r = 0.72), as one would expect. The demonstrated simple linear relationships between U, (B/R), and (T--B) support the hypothesis that both the hematofluorometer and peroxidase methods provide valid measurements of bilirubin binding status.


Subject(s)
Bilirubin/blood , Blood Proteins/metabolism , Adult , Binding Sites , Chromatography, Gel , Fluorometry , Humans , Infant , Protein Binding , Regression Analysis , Serum Albumin/metabolism
10.
Proc Natl Acad Sci U S A ; 78(3): 1882-6, 1981 Mar.
Article in English | MEDLINE | ID: mdl-6940196

ABSTRACT

A pigment different from (Z,Z)bilirubin-IX alpha was detected by fluorometric methods in blood specimens from newborn infants undergoing blue-light therapy for unconjugated hyperbilirubinemia; it was not detected in specimens from infants not under therapy. The phototherapy-associated pigment has fluorescence, solubility, and photochemical properties that are identical to those exhibited by what are thought to be configurational (Z leads to E) isomers of bilirubin. It is concluded that isomerized bilirubin in the blood of neonates under phototherapy can reach as high as 15% of the total.


Subject(s)
Bilirubin/blood , Hyperbilirubinemia/therapy , Infant, Newborn, Diseases/therapy , Phototherapy , Humans , Infant, Newborn , Isomerism , Spectrometry, Fluorescence , Spectrophotometry
12.
Pediatrics ; 66(3): 411-6, 1980 Sep.
Article in English | MEDLINE | ID: mdl-7422430

ABSTRACT

The concentrations of total blood bilirubin, albumin-bound bilirubin, and the reserve and total bilirubin binding capacities of 35 neonatal blood samples (28 patients) were determined by automated front-face fluorometry ((hematofluorometer). These values were compared to results of diazo determinations, Sephadex gel filtration, and peroxidase-oxidation methods. Total blood bilirubin level by fluorometry agreed well with the total plasma bilirubin level by diazotization (r = .96, sigma = 1.7 mg/100 ml). Albumin-bound bilirubin concentrations by fluorometry also correlated well with diazo values (r = .95, sigma = 1.9 mg/100 ml) and were slightly lower than the total blood bilirubin concentrations. Values for total bilirubin binding capacity determined by fluorometry agreed well with results obtained for the same specimens by Sephadex gel filtration (n = 28, r = .97, sigma = 1.8 mg/100 ml) and by peroxidase-catalyzed oxidation (n = 25, r = .97, sigma = 1.7 mg/100 ml). The agreement among the results obtained by the three methods indicates a well-defined in vitro end point at which available primary or "tight" binding sites on albumin are saturated with bilirubin. In this clinical experience the coefficient of variation of results with the hematofluorometer was 8.4% for total blood bilirubin and 6.5% for total binding capacity. A comparison of "sick" with "well" infants revealed that the fraction of bilirubin not bound to albumin was significantly different for these two groups. The assays made with the hematofluorometer are quick (10 to 15 minutes) and require only a small quantity (approximately 150 microliters) of blood.


Subject(s)
Bilirubin/blood , Fluorometry/methods , Jaundice, Neonatal/blood , Serum Albumin/metabolism , Binding Sites , Chromatography, Gel , Horseradish Peroxidase , Humans , Infant, Newborn , Protein Binding
13.
Am J Public Health ; 70(7): 697-700, 1980 Jul.
Article in English | MEDLINE | ID: mdl-6966895

ABSTRACT

The results of a cross-sectional clinical field survey of 90 telephone cable splicers are presented. Despite the rare occurrence of clinically overt lead poisoning among cable splicers, the observed prevalence of symptoms was 29% for lead-associated central nervous system symptoms and 21% for gastrointestinal symptoms. These two groups of symptoms were directly related to zinc protoporphyrin (ZPP) levels but no relationship was found between them and blood lead concentrations. Only 5% of the workers had significantly elevated blood lead levels (greater than 40 microgram/100ml). Because of the intermittent lead exposure encountered in this trade, individuals were identified with "normal" blood lead levels associated with "elevated" zinc protoporphyrin concentrations, indicating the difference in biological significance between exposure-(blood lead) and biological-response tests (ZPP). Suggestion is made that both types of diagnostic tests be utilized in the medical surveillance of lead-exposured workers.


Subject(s)
Lead Poisoning/epidemiology , Occupational Diseases/epidemiology , Adult , Aged , Central Nervous System Diseases/diagnosis , Cross-Sectional Studies , Gastrointestinal Diseases/diagnosis , Health Status , Humans , Lead/blood , Lead Poisoning/diagnosis , Middle Aged , Occupational Diseases/chemically induced , Protoporphyrins/blood , United States , Zinc/blood
16.
Pediatrics ; 65(4): 767-76, 1980 Apr.
Article in English | MEDLINE | ID: mdl-7367084

ABSTRACT

A simple, rapid fluorometric method for determining the albumin-bound bilirubin concentration, total blood bilirubin concentration, and the bilirubin reserve-binding capacity of albumin was clinically evaluated using blood specimens from 79 neonates. This study showed that these bilirubin determinations, made by means of the Bell Laboratories hematofluorometer, correlated well with plasma bilirubin levels obtained by a diazotization (Jendrassik-Grof) method. Hematofluorometer reserve-binding capacities correlated very well with 2-(4'-hydroxybenzene)azobenzoic acid (HABA) dye reserve-binding capacities for specimens of artificially jaundiced adult blood. For specimens of neonatal blood the HABA dye reserve capacity was, on the average, higher than the hematofluorometer reserve-binding capacity, particularly for specimens from low-birth-weight babies (less than 2,000 gm). Comparison of HABA reserve capacity and hematofluorometer reserve capacity for high-birth-weight babies (greater than 2,000 gm) gave data very similar to those for adult blood specimens. The specific bilirubin-binding capacity of albumin was found to be greater for infants whose birth weight exceeded 2,000 gm than for the lower birth weight group. The total blood bilirubin concentration obtained by the hematofluorometer is shown to be significantly higher than the concentration of bilirubin bound to albumin, an indication of other important compartments of bilirubin in blood.


Subject(s)
Bilirubin/blood , Infant, Newborn , Spectrometry, Fluorescence/methods , Adult , Azo Compounds , Binding, Competitive , Birth Weight , Fluorescent Dyes , Humans , Kernicterus/blood , Male , Serum Albumin/metabolism
17.
Clin Chem ; 26(3): 409-13, 1980 Mar.
Article in English | MEDLINE | ID: mdl-7363461

ABSTRACT

We describe the rapid, inexpensive fluorometry of hemoglobin in undiluted whole blood. The procedure consists only of adding a standard quantity of a fluorescent dye to a measured volume (approximately 15 microL) of blood, together with some solubilizing detergent. The assay is based on the attenuation of the dye's fluorescence (excited within the region 400--440 nm) that results from the competitive absorption of exciting light by the hemoglobin present--the "inner filter effect." The wavelength that one can use is optional and will determine which dyes can be used. Measurements are made with the hematofluorometer, a "front-face" filter fluorometer (Blumberg et al., J. Lab. Clin. Med. 89: 712--723, 1977). We demonstrate the validity of the method for two dyes, rhodamine B and fluorescein dibutyrate, which we used with hematofluorometers that were designed to determine blood zinc protoporphyrin and bilirubin, respectively. Our method exhibited a standard error of about 4 g of hemoglobin per liter vs the comparison method (Coulter Counter method, for which the CV is 1.2%). The CV is about 3%. The method seems appropriate for "field" use (i.e., use outside the laboratory) in anemia-screening programs.


Subject(s)
Hemoglobins/analysis , Colorimetry/methods , Humans , Indicators and Reagents , Microchemistry , Spectrometry, Fluorescence/methods
18.
20.
J Biol Chem ; 254(21): 10906-12, 1979 Nov 10.
Article in English | MEDLINE | ID: mdl-91607

ABSTRACT

Despite extensive structural dissimilarities, iron . bleomycin complexes and heme-containing oxygenases display remarkable similarities in binding oxygen antagonists and in spectral properties deriving from bound iron. Fe(II)-bleomycin reversibly forms a complex with either CO or isocyanide (lambda max = 384 and 497 nm, respectively), either of which interfere with its oxygen-dependent cleavage of DNA. A similar but paramagnetic complex forms with NO (lambda max = 470 nm; AN = 24 G). In contrast, cyanide enhances bleomycin activity against DNA. Complexes of bleomycin and FE(III), formed either by direct association or by autoxidation of the Fe(II) . bleomycin complex, exhibit indistinguishable EPR and visible spectra, which change characteristically with pH. At neutral pH, Fe(III) . bleomycin is a low spin complex (g = 2.45, 2.18, 1.89; lambda max = 365, 384 nm) and, at low pH, it is a high spin rhombic complex (geff = 9.4, 4.3; lambda max = 430 nm). These complexes are interconvertible (pK 4.3). Fe(II) . bleomycin oxidation, although reversible by spectral criteria, is accompanied by drug inactivation unless DNA is present.


Subject(s)
Bleomycin , Iron , Oxygen , Animals , Azides , Carbon Monoxide , Cattle , Chemical Phenomena , Chemistry , Cyanides , DNA , Electron Spin Resonance Spectroscopy , Malondialdehyde , Nitric Oxide , Oxidation-Reduction , Spectrophotometry
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