Covalent labeling

Tosyl-L-phenylalanine chloromethyl ketone (TPCK) specifically inhibits chymotrypsin by covalently labeling His ". 

Definitive identification of lysine as the modified active-site residue has come from radioisotope-labeling studies. NaBH4 reduction of the aldolase Schiff base intermediate formed from C-labeled dihydroxyacetone-P yields an enzyme covalently labeled with C. Acid hydrolysis of the inactivated enzyme liberates a novel C-labeled amino acid, N -dihydroxypropyl-L-lysine. This is the product anticipated from reduction of the Schiff base formed between a lysine residue and the C-labeled dihydroxy-acetone-P. (The phosphate group is lost during acid hydrolysis of the inactivated enzyme.) The use of C labeling in a case such as this facilitates the separation and identification of the telltale amino acid. 

Candidates for the renal brush border Na /H exchanger transport protein identified by covalent labeling, affinity chromatography, or other methods... 

Covalent labelling of these two polypeptides was also specifically blocked by other PCP analogues such as TCP, by some K channel blockers (4 - A P and tetrabutylammoni urn ions), and stereoselectively by certain PCP-like "sigma opiates" (dexoxadrol levoxadrol)... 

Griffin BA, Adams SR, Tsien RY (1998) Specific covalent labeling of recombinant protein molecules inside live cells. Science 281 269-272... 

Chattopadhaya S, Srinivasan R, Yeo DSY et al (2009) Site-specific covalent labeling of proteins inside live cells using small molecule probes. Bioorg Med Chem 17 981-989... 

The commercially available dicyanomethylene squaraine dye Seta-670-mono-NHS showed extremely low blinking effects and good photostability when used in single-molecule studies of multiple-fluorophore labeled antibodies [113]. Seta-670-mono-NHS and Seta-635-NH-mono-NHS were covalently labeled to antibodies and used in a surface-enhanced immunoassay [114]. From the fluorescence intensity and lifetime changes determined for a surface that had been coated with silver nanoparticles, both labeled compounds exhibited a 15- to 20-fold... 

ABPP is only applicable to targets that possess a nucleophilic active-site residue (Ser, Cys, Lys) susceptible to covalent labeling by an electrophile. When this is lacking, an alternative is to add a photoaffinity group to an inhibitor scaffold so that a covalent adduct with the target can be created by exposure to UV light. 

Fig. 6.14. Quenched activity based probe for the imaging of cathepsins. Upon covalent binding of the sensor to histidine and serine residues in the active site of the enzyme, the quencher is released and increased fluorescence indicates the now covalently labeled enzyme of interest. 

Boyer, T.D. (1986) Covalent labeling of the nonsubstrate ligand-binding site of glutathione S-transferase with bilirubin-Woodward s reagent K./. Biol. Chem. 261, 5363. 

Howard, A., de La Baume, S., Gioannini, T.L., Hiller, J.M., and Simon, E.J. (1985) Covalent labeling of opioid receptors with human b-endorphin./. Biol. Chem. 260,10833-10839. 

The excited state or radical should react preferably with unreactive C-H groups yielding a uniform and stable covalent label rather than interact with nucleophiles including solvents of that type. 

Pankey et al.21 described a rapid, reliable, and specific enzyme multiplied immunoassay technique (EMIT ) for amitriptyline, nortriptyline, imipramine, and desipramine in sera. To overcome crossreactivity, solid phase extraction was included in sample pretreatment. Disposable 1 mL columns packed with covalently labeled silica gel were conditioned with HPLC-grade methanol (1 mL) and then with de-ionized or distilled water (1 mL). Serum (calibrator, control, or patient sample, 500 L) was applied onto the column, eluted to waste, washed with 900 /uL of wash solution containing acetonitrile (236.1 g/L) and ion-pairing reagent in acetate buffer, pH 4.2, washed with 500 fiL of mobile phase solution containing acetonitrile (393.5 g/L) in methanolic phosphate buffer, pH 7.0,... 

Silverman has pointed out that several criteria must be met to demonstrate that a compound is a true suicide substrate 1101 (1) Loss of enzyme activity must be time-dependent, and it must be first-order in [inactivator] at low concentrations and zero-order at higher concentrations (saturation kinetics), (2) substrate must protect the enzyme from inactivation (by blocking the active site), (3) the enzyme must be irreversibly inactivated and be shown to have a 11 stoichiometry of suicide substrate active site (dialysis of enzyme previously treated with radiolabeled suicide substrate must not release radiolabel into the buffer), (4) the enzyme must unmask the suicide substrate s potent electrophile via a catalytic step,1121 and (5) the enzyme must not be covalently labeled with the activated form of the suicide substrate following its escape from the active site (the presence of bulky scavenging thiol nucleophiles in the buffer must not decrease the observed rate of inactivation). 

These inactivators typically have negligible reactivity toward cellular nucleophiles, in contrast to the classic affinity labels and the activated (escaped) form of suicide substrates (I ). However, all classes of irreversible inactivators - even in the ideal case of covalently labeling only their target enzymes - suffer from the possibility of eliciting an undesired immune response against the inactivator-derivatized protein following protein denaturation and degradation.1171... 

Figure 5.17 Stability of optical encoding towards oligonucleotide reagents. Organosilica microspheres covalently labelled with ATTO 550 dye are stable towards each of the reagents used in phosphoramidite oligonucleotide synthesis. In contrast, optically encoded polystyrene-divinylbenzene (DVB) beads are unstable in most steps, in particular those involving dichloromethane and tetrahydrofuran. (Reproduced from ref. 28, with permission.)...

Non-Covalent Labeling of Primary Antibodies with Labeled Fab Fragments... 

The cross-reaction of secondary species-specific antibodies with primary antibodies from the same species is obviously avoided by direct (one antibody layer) methods. The direct method offers an easy way for simultaneous labeling of a pair or more antigens, even when using primary antibodies from the same species. Recently, a direct technique with primary antibodies that are covalently labeled by different fluorophores was described for a simultaneous detection of up to seven... 

There are two basic considerations when attempting SDM. One is to determine the amino acids that should be mutated and the other is to decide what to replace them with. The first question is, of course, dependant upon information gathered from previous experimentation in order to target residues that are appropriate. Such information may be derived from biochemical techniques. For instance, in our binding site studies, we have specifically mutated amino acids that had previously shown to be covalently labeled by photoactive ligands. Additionally, we have used comparisons between the sequences of different receptor subunits that correlate with receptor function to identify domains of interest. Chimeragenesis, the technique described in the first half of this chapter, can provide important information in this regard. Obviously, those proteins for which a detailed structural model is available will lend themselves to more rational substitutions. 

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