Irvin Isenberg

Studies on the Analysis of Fluorescence Decay Data by the Method of Moments

The method of moments, as presented by Isenberg and Dyson (1969; Biophys. J. 9:1337) has been shown to be a reliable way of obtaining the amplitudes and time constants of several simultaneously emitting species, even in the presence of an overlapping excitation. Recent improvements in the method include (a) a component incrementation test for determining the number of relaxations, (b) a procedure, which we call exponential depression, for dramatically improving convergence, and (c) a new algorithm for implementing the method of moments on a digital computer with a high degree of flexibility and efficiency. These improvements, as well as new general theory, are described and tested using both synthetic and real experimental data. Component incrementation consists of examining models with increasing numbers of exponential terms. Given adequate precision, we find that an analysis for N + 1 components, of data that are actually represented by N components, provides the correct amplitudes and time constants plus an N + 1 term with an insignificant amplitude. Exponential depression is a transformation in which the original excitation and fluorescence, E(t) and F(t), are multiplied by exp (-lambdat), where lambda is an arbitrary parameter. While the convolution is invariant to this transformation, the proper choice of lambda greatly reduces the number of iterations necessary to obtain the amplitudes and time constants and may even improve their accuracy. In addition, an appendix by John P. Mullooly presents a statistical analysis of the effect of counting error on the method of moments estimates of fluorescence decay parameters, applicable when data are obtained by the monophoton technique. Formulas are derived that give the approximate precision of the decay parameters for the general case of N exponential components, with calculational details for one and two component systems.

A Monophoton Fluorometer with Energy Discrimination

A recently constructed monophoton time decay fluorometer having several novel features is described. A number of tests of the instruments performance are presented. A method of moments is used to analyze the data. Single photon energy discrimination permits high collection rates and with coincidence circuitry improves the signal‐to‐noise ratio. Subnanosecond resolution has been achieved and double exponential decays resolved.

Chromosomal HMG proteins occur in three eukaryotic kingdoms

Abstract We report that yeast and wheat contain chromosomal proteins belonging to the high mobility group (HMG). This establishes that the HMGs are widely distributed throughout eukaryotes. We further suggest that prokaryotes may also contain HMGs.

Interaction of renatured histones f3 and f2a1

Abstract Ultracentrifuge measurements show that the individual histones f2a1 and f3 interact to form tetramers in solution. The conditions which facilitate renaturation are discussed.

Preparative gel electrophoresis: Detection, excision, and elution of protein bands from unstained gels

Methods are described for localizing proteins in unstained gels and accurately excising the regions containing them. A gel elutor, which is all glass with Lucite fittings, is also described. The elutor removes proteins from gels with a high yield and concentrates the protein in a small volume. The elutor is simple and very easy to use. A way is presented for avoiding the oxidation of methionine and cysteine during preparative electrophoresis.

On moment index displacement

Moment index displacement can determine the amount of light scatter and zero point displacement in fluorescence time decay data. Two different types of light scatter are considered and shown to have significantly different effects on the analysis of the data. The amplitude correction factor determined by Eisenfeld and Mishelevich is experimentally verified.

DNA‐POLYLYSINE INTERACTION AS STUDIED BY POLARIZATION OF FLUORESCENCE*

In multicellular organisms, DNA exists in the form of a nucleoprotein complex. Most of these proteins are histones, which are basic and carry a large positive charge. The electrostatic interaction between negatively charged DNA and positively charged histone undoubtedly accounts for a considerable part of the binding. Other interactions may exist, however, and probably do. In recent years, much attention has been given to the study of histoneDNA and basic polyamino acid-DNA complexes (Ohlenbusch et al., 1967; Ohba, 1966 a, b; Olins et al., 1967; Leng & Felsenfeld, 1966; Tsuboi et al., 1966; Huang & Bonner, 1964; Huang et al., 1964; Johns & Butler, 1964). In particular, the polylysine-DNA complex has been examined by a variety of techniques, and the following properties are known: a) At low salt concentrations the complex is irreversible in the sense that dynamic equilibrium does not exist (Tsuboi et al., 1966). Alteration of the site of binding of the polylysine does not occur over relatively long periods of time. b) The complex has a definite stoichiometry of one lysine per one DNA phosphate (Tsuboi et al., 1966; Olins et al., 1967). This implies that a definite structural basis for the complex exists. c ) The polylysine stabilizes the DNA. The complexed DNA melts at a considerably higher temperature than uncomplexed DNA (Ohba, 1966 b; Olins et al., 1967; Tsuboi et al., 1966). d ) The binding shows a degree of cooperativity e) In the presence of NaCI, polylysine binds preferentially to A-T rich DNA, but the specificity is reversed in tetramethylammonium chloride solutions. (Leng & Felsenfeld, 1966; Olins et al:, 1967). In this paper, we shall describe studies on the salt dissociation of polylysine-DNA by measurements of the intensity and polarization of fluorescence of dye-labeled polylysine and also report the results of turbidity measurements on such samples. There has been surprisingly little use of polarization of fluorescence for quantitative investigations of DNA complexes. To our knowledge, the only prior quantitative study is an investigation of lysozyme-DNA and bovine serum albumin-DNA interactions (Steiner, 1953; Steiner & Edelhoch, 1962).

Exponential depression as a test of estimated decay parameters

A new test for judging the goodness of estimated decay parameters is presented. The test is based on the fact that a convolution is invariant under exponential depression. In the absence of significant error the estimated parameters will then remain constant as the degree of depression is varied over a finite range. In the presence of error, the parameters will vary. Up to now, no test has existed to see if moment index displacement corrects errors to a satisfactory extent in any given analysis. It has always been necessary to have some a priori knowledge of the type of error that limited the analysis. The test presented here removes that requirement. In addition, it is shown that the test performs better than a visual inspection of residual and autocorrelation plots in judging analyses when decays are closely spaced, even in the absence of nonrandom errors. The test is useful in accepting or rejecting analyses, with or without automatic error correction, in helping to discriminate between different model...

Construction and tuning of a monophoton decay fluorometer with high‐ resolution capabilities

A fluorescence decay instrument, assembled largely from commercially available components, is described. This system incorporates a tunable picosecond laser system as an excitation source, energy windowing to minimize artifacts due to pulse pile up and sample alternation to compensate for long‐term instrumental drift. The instrument was tested for its ability to resolve multiple exponential decays using methods of moments analysis. By examining the flatness of λ‐invariance plots and the accuracy of recovered decay parameters, proper tuning of electronic components and definition of a range of reasonable counting rates was possible. Errors of significance to the resolution of closely spaced decays are discussed. A number of examples are shown for resolutions that are generally regarded as difficult, including two, three, and four components.

The effect of temperature on histone GRK aggregation

Abstract The aggregation of histone GRK, induced by the addition of salt, is found to be highly dependent on temperature. The rate of this aggregation decreases with decreasing temperature and can be essentially stopped at low temperatures.