William E. Blumberg

Behavioral indicators of lead neurotoxicity: Results of a clinical field survey

SummaryCentral nervous system dysfunction in workers occupationally exposed to lead was investigated by means of performance tests. The test scores of lead-exposed workers were compared with those of control groups (steel workers, papermill workers and farmers). It was found that secondary lead smelter workers showed significantly poorer performance scores than the nonexposed, control groups. The group differences between steel workers and lead workers in test scores were not attributable to differences in age or education. In the lead-exposed workers correlations between test scores and indicators of lead absorption (particularly blood lead and zinc protophyrin levels) were analyzed. Increases in zinc protoporphyrin levels were found to be highly correlated with decreases in test scores. Lower performance test scores were consistent with a sizeable prevalence of central nervous system symptoms among secondary lead smelter workers. Moreover, lead workers without central nervous system symptoms also showed decrements in performance test scores which were also correlated with elevated zinc protoporphyrin levels. The data indicate that certain behavioral tests might be important tools for studying subclinical central nervous system dysfunction due to lead toxicity; significant correlations between zinc protoporphyrin levels and behavioral test scores are considered to be consistent with an etiologic relationship between decrement in performance scores and lead effects on the central nervous system.

Nuclear magnetic resonance determination of ligand-induced conformational changes in myoglobin.

Abstract We report the results of a high-resolution proton nuclear magnetic resonance at 220 MHz of deoxymyoglobin and three diamagnetic complexes of myoglobin with oxygen, carbon monoxide and ethylisocyanide. We emphasize the resonances which are shifted from their normal positions by ring currents from aromatic rings, particularly the porphyrin ring, because these ring current shifts are very sensitive to changes of the relative co-ordinates of the observed protons and the ring. A specific resonance at −6.1 p.p.m., with intensity of about three protons, has been attributed to the protons of an aromatic residue, shifted upfield by the ring currents of the heme group. A possible origin of this resonance is Phe CD1. The observed change of this resonance of 0.2 p.p.m. upon oxygenation corresponds to a movement of a few tenths of an angstrom unit. Slightly larger shifts upon oxygenation of resonances attributed to methyl groups of aliphatic residues are interpreted in terms of movements of about 0.5 A. A comparison of the complete spectra of Mb ‡ and MbO2 shows that there are many small differences of 0.1 to 0.2 p.p.m. which indicate widespread small structural changes upon oxygenation. Differences in the ring-current-shifted resonances amongst various ligated forms including cyanoferrimyoglobin, indicate the existence of structural differences in the protein when the ligand is changed.

The demonstration of ferrihemochrome intermediates in heinz body formation following the reduction of oxyhemoglobin A by acetylphenylhydrazine

Interaction of acetylphenylhydrazine with oxyhemoglobin A in a hemolysate or in intact red cells resulted in the formation of ferrihemochromes as shown by a characteristic optical spectrum. The same optical spectrum was observed in a suspension of red cell ghosts containing numerous Heinz bodies. Electron paramagnetic resonance of actylphenylhydrazine-incubated red cells disclosed the presence of previously identified reversible ferrihemochromes, which can be reduced to functional hemoglobin, and irreversible ferrihemochromes, which cannot be reduced to functional hemoglobin. (Ferrihemochromes are defined as low spin forms of ferric hemoglobin having heme ligands endogenous to the protein structure). In contrast, only irreversible ferrihemochromes could be observed in ghosts containing Heinz bodies. In addition both optical and magnetic features of sulfhemoglobin were observed in an acetylphenylhydrazine-treated red cell hemolysate. Similar optical features are produced by the interaction of aromatic nitrogen-containg reductants with purified oxyhemoglobin in the presence of (NH4)2S. This reaction is not effected by the presence of catalase, suggesting that H2O2 is not an intermediate of the reaction. It is concluded that the mechanism of action of acetylphenylhydrazine with oxyhemoglobin is two-fold, ultimate reduction to high spin ferric hemoglobin followed by ferrihemochrome formation. Thus it appears that the pathway of denaturation of hemolytic anemias and thalassemia or induced by chemical reagents, entails a common route involving the formation of ferric hemoglobin by a reductive mechanism, followed by reversible ferrihemochromes, irreversible ferrihemochromes, and ultimately, precipitation.

Fluorometric study of the partition of bilirubin among blood components: Basis for rapid microassays of bilirubin and bilirubin binding capacity in whole blood

Abstract Bilirubin binds to many sites in blood, the strongest binding being to a single site on albumin. Secondary sites on albumin, most sites on other plasma proteins, and sites on erythrocyte membranes have affinities for bilirubin that are at most one-hundredth as great. Bilirubin binds to hemoglobin in red cells with an effective affinity that is less than one-thousandth that of the primary albumin site. Essentially the only bilirubin present in blood which fluoresces is that bound to the primary albumin site. Almost all the other bilirubin in blood fluoresces with a yield no more than one-fiftieth as large. Quantitative fluorometry of whole blood is possible using the “front-face” technique. The concentration of bilirubin bound to the primary albumin site can be determined in this way. The albumin binding capacity of a blood specimen can be similarly assayed upon titration of the specimen with bilirubin. The nonionic detergent dodecyldimethylamine oxide (DDAO) scavenges bilirubin from all sites in blood, and, since bilirubin is fluorescent in DDAO micelles, the total blood bilirubin can be assayed fluorometrically after addition of DDAO to the specimen. This detergent method also allows facile assay of red-cell-bound bilirubin. These fluorometric assays for total blood bilirubin, albumin-bound bilirubin, and albumin binding capacity are simple and rapid and use very small volumes of blood. They should be of great value in the research on neonatal jaundice and in its clinical management.

High resolution EPR studies of the fine structure of heme proteins. Third harmonic detection approach

When third harmonic detection is applied in EPR studies of the superhyperfine structure of nitrosyl derivatives of a number of human hemoglobin variants, significant resoltuion enhancement is obtained. This has allowed a detailed analysis of the number of superhyperfine lines, their g-values and their splittings, and has led to a more complete understanding of the interaction between the axial ligands of the heme iron. Sudies of the effect of the modulation amplitude on EPR line-shapes revealed that the amplitude required to resolve fine structure in the third harmonic mode is 3-10 times larger than that used to record an undistorted first derivative spectrum. The application of this approach for other systems is discussed, and practical guidelines for its use are given.

Isotope effect of nuclear magnetic resonances in metallic mercury

Abstract Accurate measurements of the nuclear magnetic resonances of Hg 199 and Hg 201 in metallic mercury are reported. The Knight shifts were (2.724 ± 0.005) per cent and (2.722 ± 0.005) per cent respectively. From these measurements the hyperfine structure anomaly of these nuclei was shown to be very small i.e. (0.07 ± 0.11) per cent. The experimental techniques required to measure the resonance positions accurately are discussed. Corrections for the effects of mode mixing and integrating time constant are developed in the appendix. An analysis is made of the influence of hyperfine, quadrupole and exchange effects on the resonance line.

The electron paramagnetic resonance of molybdenum in rat liver and in rat liver mitochondria

Abstract 1. 1. A spectral feature of the EPR spectrum of rat liver and rat liver mitochondria observed near g = 1.96 arises from a paramagnetic form of molybdenum. Liver mitochondria prepared from untreated control animals, from animals treated with 92 Mo ( I = o) or from animals treated with the naturally occurring mixture of molybdenum isotopes show a doublet in the g = 1.96 region while liver mitochondria from rats treated with 95 Mo ( I = 5 2 ) show no such EPR absorptions. 2. 2. Resuspension of mitochondria from control animals in sucrose- 2 H 2 O solution converts the EPR doublet to a singlet suggesting interaction of paramagnetic molybdenum with protons. 3. 3. The addition of NADH to rat liver mitochondria which had been frozen diminishes the resonance ascribable to molybdenum. 4. 4. It is believed that the EPR of molybdenum in rat liver mitochondria, which cannot be observed under the same conditions in beef heart mitochondria, arises from molybdoproteins previously isolated from liver and not from proteins involved in mitochondrial electron transport.

X-Ray Absorption Spectroscopy: Probing the Chemical and Electronic Structure of Metalloproteins

Recently high fluxes of X-rays have become available in the form of synchrotron radiation from electron storage rings such as at the Stanford Synchrotron Radiation Project (SSRP). At SSRP one has 104 to 105 times more intensity of X-rays with a continuous spectrum than that available from standard X-ray tubes. This increase in intensity has allowed X-ray absorption to be observed successfully in dilute materials such as metalloproteins and metal complexes in solution. While X-ray absorption measurements have been made sporadically since 1931 (1), the measurements, their interpretation and their applications to dilute systems have only recently been extensively developed due to the work of Sayers, Stern, and Lytle (2) and the use of synchrotron sources (3).

A statistical analysis of the publications and collaborations of Alessandro Rossi Fanelli.

In the three and a half decades since World War II, the scientific career of Alessandro Rossi Fanelli has included study of a wide variety of subject matter and has involved a great number of other scientists. The scale of the effort has progressed from very modest beginnings, with minimal personnel, funding and equipment, to spacious, well-equipped laboratories in Rome and a world-wide network of former students and collaborators. It is the purpose of this article to examine the nature of this evolving structure through statistical and graphical analysis of Professor Rossi Fanelli’s publications.

Electron paramagnetic resonance study of nitrosyl haemoglobin M Iwate

Abstract The nitrosyl derivative of haemoglobin M Iwate, a haemoglobin variant which has functional beta chains and non-functional alpha chains, is known to exist only in a low affinity form. It shows a nine-line superhyperfine (SHF) pattern in the low temperature electron paramegnetic resonance (e.p.r.) spectrum, even in the presence of inositol hexaphosphate. This provides further support for the suggestion (Chevion, M., Stern, A., Peisach, J., Blumberg, W. E. and Simon, S. Biochemistry 1978 bd17 , 1745) that the beta chains of tetrameric, fully ligated, nitrosyl derivatives of all haemoglobins invariably exhibit an e.p.r. spectrum with a nine-line SHF pattern regardless of the affinity state of the molecule. On the other hand, nitrosyl ferrous alpha chains within the haemoglobin tetramer can exhibit an e.p.r. spectrum with either a nine-line or a three-line SHF pattern which is dependent upon the affinity state of the molecule.