Total correlation spectroscopy proteins

H is particularly important in NMR experiments because of its high sensitivity and natural abundance. For macromolecules, 1H NMR spectra can become quite complicated. Even a small protein has hundreds of 1H atoms, typically resulting in a one-dimensional NMR spectrum too complex for analysis. Structural analysis of proteins became possible with the advent of two-dimensional NMR techniques (Fig. 3). These methods allow measurement of distance-dependent coupling of nuclear spins in nearby atoms through space (the nuclear Overhauser effect (NOE), in a method dubbed NOESY) or the coupling of nuclear spins in atoms connected by covalent bonds (total correlation spectroscopy, or TOCSY). 

Using triple resonance and total correlation spectroscopy, Liu et have assigned the H spectra of mutants of horse heart cytochrome c in both the diamagnetic and paramagnetic (S = 1/2) forms of the protein. Chemical shifts of the wild type and mutant proteins were used as a basis for refining the g-tensors. 

In principle, 2D NMR structural determination of proteins in solution involves the linking of information derived from a combination of three complementary experiments - TOCSY (Total Correlated Spectroscopy), DQF-COSY (Double-Quantum Filtered Correlated Spectroscopy) and NOESY (Nuclear Overhauser Effect Spectroscopy). The TOCSY (58) spectra are used to identify spin systems of the amino acids in the protein. COSY (59,60) spectra yield complementary information to the TOCSY, which can be used to obtain the direct scalar connectives. The NOESY (61) spectra yield information on the through space relationship of protons in the protein. It should be noted that because of the vast abundance of hydrogen atoms in a protein molecule proton NMR is the preferred technique to determine the solution structure of a protein. 

A new technique for measuring equilibrium adsorption/desorption kinetics and surface diffusion of fluorescent-labelled solute molecules at surfaces was developed by Thompson et al.74). The technique combines total internal reflection fluorescence with either fluorescence photobleaching recovery or fluorescence correlation spectroscopy with lasers. For example, fluorescent labelled protein was studied in regard to the surface chemistry of blood 75). 

Pero, J. K. Haas, E. M. Thompson, N. L. Size Dependence of Protein Diffusion Very Close to Membrane Surfaces Measurement by Total Internal Reflection with Fluorescence Correlation Spectroscopy. J. Phys. CAew. 5 2006,110, 10910-10918. 

Some of the difficulties experienced when developing spectroscopic techniques to replace traditional wet chemistry have related to the correlation of measurements, despite the intrinsic difference in their origin. Shenk and colleagues [3] discussed this issue in the context of NIR spectroscopy. For example, measurements of protein based on total nitrogen content are not directly equivalent to a spectroscopic approach that only probes the N—H bonds. Similarly, determining individual fatty acids from the fairly broad combination and even broader overtone bands found in the NIR region, is hardly related to the quantification of species isolated on the basis on chain length and level of saturation. 

Purity for a small molecule is a relatively simple concept. Normally, an HPLC method is sufficient to measure the content and impurity levels of a small molecule drug. A macromolecule, such as a protein, has a much more complex behavior. Determining protein concentration by UV absorption spectroscopy can give a measure of the total protein in the product, but it will not necessarily differentiate between active protein and inactive protein (i.e., denatured or otherwise degraded). A validated method or methods to determine the biological activity of the molecule is needed. So, whereas protein concentration is usually tested as part of the specifications, it is also normally accompanied by one or more methods that measure or correlate to biological activity. This is the bioassay. These methods can be animal-based or cell-based, protein interaction assays, binding methods such as surface plas-mon resonance or ELISA (enzyme-linked immunosorbent assay) and immunoblot methods. 

Na NMR spectroscopy permits a direct and non-invasive quantification of sodium ions in cheeses. A linear correlation was found between total sodium concentration as determined by SQ signals and chloride ion concentrations. As expected, relaxation times from SQ experiments were found to correlate with water content. The DQ experiments highli ted the presence of bound sodium ions, or sodium ions with restricted motion, in such food samples. The term bound for sodium ions in these samples could be considered an overstatement, as it is difficult to discriminate between restricted motion induced by the viscosity of the medium, and those induced by sodium binding to proteins. 

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