Josef Eisinger

The orientational freedom of molecular probes. The orientation factor in intramolecular energy transfer

The measurement of the efficiency of Förster long-range resonance energy transfer between donor (D) and acceptor (A) luminophores attached to the same macromolecular substrate can be used to estimate the D-A separation, R. If the D and A transition dipoles sample all orientations with respect to the substrate (the isotropic condition) in a time short compared with the transfer time (the dynamic averaging condition), the average orientation factor less than K2 greater than is 2/3. If the isotropic condition is not satisfied but the dynamic averaging condition is, upper and lower bounds for less than K2 greater than, and thus R, may be obtained from observed D and A depolarizations, and these limits may be further narrowed if the transfer depolarization is also known. This paper offers experimental protocols for obtaining this reorientational information and presents contour plots of less than K2 greater than min and less than K2 greater than max as functions of generally observable depolarizations. This permits an uncertainty to be assigned to the determined value of R. The details of the D and A reoreintational process need not be known, but the orientational distributions are assumed to have at least approximate axial symmetry with respect to a stationary substrate. Average depolarization factors are derived for various orientational distribution functions that demonstrate the effects of various mechanisms for reorientation of the luminophores. It is shown that in general the static averaging regime does not lend itself to determinations of R.

Front-face fluorometry of liquid samples.

Abstract Quantitative analytical spectrofluorometry is usually performed with solutions which are optically dilute at the excitation wavelength and employs square cuvettes for observation at right angles to the excitation beam. In many biochemical applications, particularly when studying fluorophores in blood, front-face fluorometry of optically dense samples offers certain advantages which include fluorescence intensities which are an order of magnitude larger. The features which characterize quantitative fluorometry employing these two geometries are compared and a design for a front-face cell and cell holder, suitable for use in any standard spectrofluorometer, is presented.

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.

Complex formation between transfer RNA's with complementary anticodons

Abstract tRNA phe (anticodon GAA) and tRNA glu (anticodon presumably UUC) have been found to form a complex with an association constant of about 5×10 5 M −1 at 0°C. This binding is much stronger than the binding of trinucleotide UUC to tRNA phe but has a weaker temperature dependence. This suggests that the anticodon regions of tRNA have similar and complementary structures, such as Watson and Crick helices.

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.

Interpretation of intramolecular energy transfer experiments

The measurement of the efficiency of resonance energy transfer between two luminophores attached to the same macromolecular substrate is a promising tool for determining intramolecular distances if reasonable bounds can be placed upon the orientation factor k2, To date, the analysis of such experiments has been based upon assumed average values of k2. A critical review of this procedure is offered along with a method for estimating upper and lower limits for k2 which derive from the freedom of motion of the luminophores as determined by polarized emission spectroscopy.

The anticodon-anticodon complex

Abstract Gel electrophoresis has been used to measure the binding between two tRNAs with complementary anticodons, tRNA Val ( Escherichia coli ) (anticodon X,A,C) and tRNA Tyr ( E. coli ) (anticodon Q,U,A). The association constant K at 0 °C was found to be 4 × 10 5 m −1 which is about three orders of magnitude greater than the association constant for tRNA Tyr ( E. coli ) binding its trinucleotide codon UAC. The temperature dependence of K suggests that this results from the rigidity of the anticodon loop. tRNA Tyr ( E. coli ) binds an order of magnitude more weakly to tRNA Val (yeast) than to tRNA Val ( E. coli ), presumably because it contains the wobble base pair A · I. The relationship between the anticodon-anticodon complex and codon recognition is discussed.

Binding of complementary pentanucleotides to the anticodon loop of transfer RNA.

Abstract The conformation of the anticodon loop of tRNA (yeast) was studied by detecting the most strongly binding pentanucleotide among the pentamers obtained by digestion of ribosomal RNA with T1 RNase. This pentamer was identified as UUCAG which is complementary to the anticodon and the two pyrimidines on the 5′ side of the anticodon loop. Gel electrophoresis was used to detect binding. Control experiments employing other tRNAs showed that UUCAG formed a five base-pair complex with the tRNA. This indicates that the pentamer binds to the anticodon and the two pyrimidines to the 5′ side of it and lends support to a model for the tRNA loop which was recently proposed by Woese (1970).

Energy transfer in poly dAT.

The difference in the absorption spectra of adenine and thymine is used to show that at 80°K light absorbed by either of these bases in poly dAT leads to an excimer singlet state and to the triplet state of thymine.

Visible gel electrophoresis and the determination of association constants

Summary When a polyacrylamide gel is formed between a quartz plate and a fluorescent glass plate and is illuminated by an ultraviolet lamp, zones of molecules in the gel appear as dark bands on the fluorescent plate. This permits the visual observation and photography of gels during electrophoresis and obviates the need for staining or the making of a fresh gel for each run. The use of gel electrophoresis for the determination of binding constants between interacting molecules is described and illustrated.