Current Pharmaceutical Biotechnology

ISSN: 1389-2010

Current Pharmaceutical Biotechnology
Volume 6, Number 6, December 2005

Contents


Diversity in the Activity of Individual Enzymes Pp.415-425
A.I. Lee and J.P. Brody
[Abstract]


Colloidal Behavior of Proteins: Effects of the Second Virial Coefficient on Solubility, Crystallization and Aggregation of Proteins in Aqueous Solution Pp.427-436
J.J. Valente, R.W. Payne, M.C. Manning, W.W. Wilson and C.S. Henry
[Abstract]


How the Molecule Number Is Correctly Quantified in Two Color Fluorescence Cross-Correlation Spectroscopy: Corrections for Cross-Talk and Quenching in Experiments Pp.437-444
Z. Földes-Papp
[Abstract]


Monitoring Nucleic Acids Using Molecular Beacons Pp.445-452
C.J. Yang, C.D. Medley and W. Tan
[Abstract]


Single-Molecule Detection and Probe Strategies for Rapid and Ultrasensitive Genomic Detection Pp.453-461
H.-C. Yeh, S.-Y. Chao, Y.-P. Ho and T.-H. Wang
[Abstract]




Abstracts

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Diversity in the Activity of Individual Enzymes
A.I. Lee and J.P. Brody

Although the structure of an enzyme is often depicted as static, it is dynamic. Hence, a population of chemically identical enzymes has not one, but a distribution of structures at any moment in time. Does this have an effect on the activity of the enzyme? This article reviews experiments designed to test the hypothesis that this distribution of structures results in a distribution of enzyme activities. The experiments reviewed here use different enzymes, falvin adenine dinucleotide, β-galactosidase, alkaline phosphatase, exonuclease I, lactate dehydrogenase I, α-chymotrypsin, the 20S proteasome, and horseradish peroxidase. All experiments come to the same conclusion, when measured individually, apparently identical enzymes show a distribution in rates of activity.


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Colloidal Behavior of Proteins: Effects of the Second Virial Coefficient on Solubility, Crystallization and Aggregation of Proteins in Aqueous Solution
J.J. Valente, R.W. Payne, M.C. Manning, W.W. Wilson and C.S. Henry

There has been an increasing awareness that proteins, like other biopolymers, are large enough to exhibit colloidal behavior in aqueous solution. Net attractive or repulsive forces have been found to govern important physical properties, such as solubility and aggregation. The extent of intermolecular interactions, usually expressed in terms of the osmotic second virial coefficient, B, is most often measured using static light scattering. More recently, self-interaction chromatography (SIC) has emerged as a method for rapid determination of B in actual formulations, as it uses much less protein and has higher throughput. This review will summarize the relationship of B to crystallization, solubility, and aggregation of proteins in aqueous solution. Moreover, the capability of SIC to obtain B values in a rapid and reproducible fashion will be described in detail. Finally, the use of miniaturized devices to measure B is presented.


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How the Molecule Number Is Correctly Quantified in Two Color Fluorescence Cross-Correlation Spectroscopy: Corrections for Cross-Talk and Quenching in Experiments
Z. Földes-Papp

Fluorescence correlation spectroscopy (FCS) and two-color fluorescence cross-correlation spectroscopy (FCCS) are among the cutting-edge technologies for measuring molecule numbers at the single-molecule level in liquid phases. Yet, even after single molecule technologies caught up with theory, the techniques remained tools only for specialists able to navigate the formulas that give meaning to their observations. This original article aims at the derivations of relevant and useful quantification of molecule numbers for researchers with more diverse backgrounds who have begun probing questions previously unanswerable, except on the level of the molecule.

The quantitation depends on the exact conditions of measurement. To some extent these are arbitrary, so that standard procedures are necessary in for valid comparisons of measurements among different data sets. To agree on and specify such procedures is one of the further aims here.

No matter what fluorophores, which have, of course, to meet photophysical and photochemical requirements for FCS/FCCS, and optical setups/devices are used, the primary measurement signal arises from fluctuations of the mean molecule number in a confocal femtoliter or smaller probe region. Since FCS/FCCS relies on fluorescence emission measurements of rare events, one is looking for small signals on essentially zero background. Optical separation by FCCS setups is usually defined in terms of cross-talk and cross-excitation/cross-emission, respectively, which can be calculated and minimized by the experimenter from readily measurable quantities of the absorption/emission scenario for single labels and multiple labels n and m bound to or incorporated into the two-color molecules. Furthermore, this article derives relevant formulas for the quantification of molecule numbers under different experimental conditions with substantial quenching of the two-color molecules such as single labels and multiple labels n and m bound to or in-corporated into the two-color molecules, high-density labeling of two-color molecules with multiple n green labels and one red label. Here, we summarize and extend the formulas to make them more generally applicable


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Monitoring Nucleic Acids Using Molecular Beacons
C.J. Yang, C.D. Medley and W. Tan

The ability to observe the dynamic of RNA in single living cells offers many exciting opportunities in biology and medicine. In the last few years, molecular beacons (MBs) have shown great potential in monitoring RNA synthesis, transportation, and localization with good sensitivity and selectivity. A hairpin structure probe, MB is a dual-labeled single stranded oligonucleotide that only fluoresces in the presence of target sequences. In this paper, the basic principle and design of MB will be described. The application of MB for RNA imaging in living cells will be reviewed. The limi-tations of MB for in vivo application will be identified. In the last section of the article, the efforts on designing better MBs for highly sensitive and selective RNA imaging will be discussed.


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Single-Molecule Detection and Probe Strategies for Rapid and Ultrasensitive Genomic Detection
H.-C. Yeh, S.-Y. Chao, Y.-P. Ho and T.-H. Wang

This paper reviews the current state-of-the-art development of single-molecule detection (SMD)-based methods for ultrasensitive and specific analysis of genomic sequences. We first discuss several newly devised single fluorescent probe strategies that allow separation-free detection of low-abundance DNA sequences, such as quantum dot (QD)-mediated fluorescence resonance energy transfer (FRET) technology and dual-color fluorescence coincidence and colocalization analysis. Various schemes toward single DNA sizing and sequencing in solutions or on surfaces are also reviewed. In the end, we summarize the different microfluidic approaches developed for use with SMD to facilitate rapid, low-volume and quantitative analysis, such as electrokinetic and hydrodynamic single-molecule manipulation techniques.