Labeling biomolecules

This section covers early indirect fiber optic chemical sensors (FOCS) for species that cannot be sensed directly but require the use of indicators, probes, labeled biomolecules, or color-forming reactions. 

The following sections describe the most important fluorescein derivatives having reactive groups commonly used to label biomolecules. 

The iodoacetamido derivatives of fluorescein possess a sulfhydryl-reactive iodoacetyl group (Chapter 1, Section 4.2 and Chapter 2, Section 2.1) at either the 5- or 6-carbon position on their lower ring. The isomers are commercially available in purified form, since some reactivity and specificity differences between the 5- and 6-derivatives toward various sulfhydryl sites in proteins may be observed. Both iodoacetamido derivatives are among the most intense fluoro-phores available for labeling biomolecules due to high QY. 

BODIPY fluorophores are a class of probes based on the fused, multi-ring structure, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (Figure 9.27) (Invitrogen) (U.S. patent 4,774,339). This fundamental molecule can be modified, particularly at its 1, 3, 5, 7, and 8 carbon positions, to produce new fluorophores with different characteristics. The modifications cause spectral shifts in its excitation and emission wavelengths, and can provide sites for chemical coupling to label biomolecules. 

These factors make 125I the iodine label of choice for radiolabeling biological molecules. Its commercial availability from a number of suppliers at relatively low cost further adds to its popularity. Even though it has lower specific activity than 131I, iodine-125 still provides much greater sensitivity than 14C, 32P, 35S, or 3H in labeling biomolecules. In fact, the use of a radioactive iodine label can create probes that have 150-fold more sensitivity than tritiated molecules and as much as 35,000 times the detectability of 14C-labeled molecules (Bolton and Hunter, 1986). 

Figure 18.15 NHS-chromogenic-PEG3-biotin contains an amine-reactive NHS ester that can be used to label biomolecules through an amide linkage. The chromogenic bis-aryl hydrazone group within the spacer arm of the reagent allows the degree of biotinylation to be quantified by measuring its absorbance at 354 nm. The compound also contains a hydrophilic PEG spacer, which provides greater water solubility.

Gee, K.R., Archer, E.A., and Kang, H.C. (1999) 4-Sulfotetrafluorophenyl (STP) esters New water-soluble amine-reactive reagents for labeling biomolecules. Tetrahedron Lett. 40, 1471. 

Measurement of cross-correlated relaxation has been described for homo-nuclear cases [10,11], and is widely used in soUd-state NMR [12-14]. It is the availability of isotopically labelled biomolecules and its appHcation to solution-state NMR that makes the method so interesting. The first application of CCR in solution-state NMR with a N, C labelled protein, was the determination of the torsion angle in the small protein rhodniin [7]. This torsion angle is difficult to obtain by traditional methods. 

The distance measured by EPR is the point dipole distance between the two paramagnetic centres. When applied to structures of spin-labelled biomolecules the desired distance is between sites on the biomolecules. A key question is the conformation and conformational flexibility of the spin label. X-ray crystal structures of four spin-labelled derivatives of T4 lysozyme have been reported.54 Preferred rotational conformations of the linkage between the cysteine introduced by site-directed mutagenesis and the spin label were observed. The electron density associated with the nitroxide ring in different mutants was inversely correlated with the mobility of the label observed in fluid solution EPR spectra. 

Because of concerns about the safety of radioisotope use, researchers are developing fluorescent and chemiluminescent methods for detection of small amounts of biomolecules on gels. One attractive approach is to label biomolecules before analysis with the coenzyme biotin. Biotin forms a strong complex with enzyme-linked streptavidin. Some dynamic property of the enzyme is then measured to locate the biotin-labeled biomolecule on the gel. These new methods approach the sensitivity of methods involving radiolabeled molecules, and rapid advances are being made. 

The following sections describe some of the most popular fluorescent probes for use in labeling biomolecules. These fluorophores are available from a number of manufac-... 

Semiconductor nanoparticles are powerful fluorescent probes, which can be used for labelling biomolecules. Quantum dots have several advantages over... 

Scheme 18 Metal-mediated cleavage of "mTc-labelled biomolecules from a solid-phase support (grey spheres). Unlabelled biomolecules remain bound, whereas labelled ones only are partially cleaved...

Scheme 19 First preparation of a [Cp99mTc(CO)3] labelled biomolecule by the so-called double ligand transfer approach...

Scheme 20 Fully aqueous preparation of a [Cp99mTc(CO)3] labelled biomolecule, enabled by the carbonyl group directly attached to the cyclopentadienyl-ring a two-step approach, b direct one-step procedure and c observed by-product from carbonyl reduction by bora-nocarbonate...

The Tc-6-hydrazinonicotinamide (HYNIC) core for labeling biomolecules, in particular peptides, was introduced by Schwartz et al. [41]. However, since... 

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