Reagent affinity labeling

FOURIER TRANSFORM IR/PHOTOACOUS-TIC SPECTROSCOPY TO ASSESS SECONDARY STRUCTURE PHOTOAFFINITY LABELING AZIDO PHOTOAFFINITY REAGENTS AFFINITY LABELING Photobleaching,... 

Herbicidal Inhibition of Enzymes. The Hst of known en2yme inhibitors contains five principal categories group-specific reagents substrate or ground-state analogues, ie, rapidly reversible inhibitors affinity and photo-affinity labels suicide substrate, or inhibitors and transition-state, or reaction-intermediate, analogues, ie, slowly reversible inhibitors (106). 

An affinity label is a molecule that contains a functionality that is chemically reactive and will therefore form a covalent bond with other molecules containing a complementary functionality. Generally, affinity labels contain electrophilic functionalities that form covalent bonds with protein nucleophiles, leading to protein alkylation or protein acylation. In some cases affinity labels interact selectively with specific amino acid side chains, and this feature of the molecule can make them useful reagents for defining the importance of certain amino acid types in enzyme function. For example, iodoacetate and A-ethyl maleimide are two compounds that selectively modify the sulfur atom of cysteine side chains. These compounds can therefore be used to test the functional importance of cysteine residues for an enzyme s activity. This topic is covered in more detail below in Section 8.4. 

Bergmann, K.E., Carlson, K.E., and Katzenellenbogen, J.A. (1994) Hexestrol diazirine photo-affinity labeling reagent for the estrogen receptor. Bioconjugate Chem. 5, 141-150. 

Having an increased or elevated reactivity. This term has been used in reference to the relative activity of amino acyl residues at the active sites of enzyme. The immediate environment (Le., the microenvironment) may allow simple reagents to react faster with the amino acid than would normally be expected. Thus, in labeling of proteins with active site-directed reagents, an investigator should always consider the basis of increased reactivity Is it due to facilitation of the reaction by increased affinity (Le., affinity labeling), or is it due to increased activity of the amino acyl side chain (e.g., perhaps increased nucleophilicity due to the microenvironment). 

Procedures " for distinguishing between the two means of facilitation include (1) Use of a reagent that does not bind as an affinity label. If facilitation is due to hyperreactivity, this reagent should also be more reactive. (2) Use of transition-state analysis. A favorable change in the entropy of activation (A ) would imply facilitation via affinity labeling whereas a more favorable change in the enthalpy of activation (AH ) implies hyperreactivity. However, a certain caution should always be exercised since other factors, e.g. differential solvation effects, can result in a certain degree of compensation between AH and AS. ... 

Affinity Labeling of Catalytic ATP Sites. Residues involved in ATP binding are potentially revealed by the use of affinity labels that are based on ATP s structure. Perhaps the most systematically studied of these compounds is 5 -fluorosulfonylbenzoyladenosine (5 -FSBA) (Figure 3a), which has been reported to label at least six kinases (32-A1). In the case of rabbit muscle pyruvate kinase such work has Indicated the presence of a tyrosine residue within the metal nucleotide binding site and an essential cysteine residue located at or near the free metal binding site (32). A similar reagent, 5 -FSBGuanosine, revealed the presence of two cysteine residues at the catalytic site of this same enzyme, both distinct residues from those modified by 5 -FSBA (33,34). With yeast pyruvate kinase both tyrosine and cysteine residues were modified by 5 -FSBA at the catalytic site ( ), and with porcine cAMP-dependent protein kinase a lysine residue was labeled at the active site (36). 

The principles of the method are very nicely illustrated by one of the first affinity labeling experiments, the reaction of /exs-i.-phenylalanine chloro-methyl ketone (TPCK) with chymotrypsin.1 TPCK resembles substrates like fexs-L-pheny 1 alanine methyl ester, but the chloromethyl ketone group of TPCK is an alkylating reagent. 

In the preceding section, four diagnostic tests of affinity labeling were listed (inactivation inhibited by substrates, pH dependence of inactivation similar to that of catalysis, labeled inhibitor covalently bound in 1 1 stoichiometry, and saturation kinetics obeyed). The same criteria may be used to diagnose suicide inhibition. In addition, tests must be made to detect any diffusion of the activated intermediate I into solution. For example, the addition of —SH reagents that rapidly react with electrophiles and hence scavenge them should not slow down the rate of reaction. The suicide inhibitor should not, in any case, react with the thiol at an appreciable rate in the absence of enzyme. 

An important consequence of the chemical reaction taking place in the confines of an enzyme- substrate complex is that not only is the binding specific, but the rate of the chemical step may be unusually rapid because it is favored en-tropically over a simple bimolecular reaction in solution, in the same way as is a normal enzymatic reaction. Thus, reagents that are normally only weakly reactive may become very reactive affinity labels. 

Creatine kinase - [MEDICALDIAGNOSTIC REAGENTS] (Vol 16) -affinity label for [ENZYME INHIBITORS] (Vol 9)... 

The orientation of the j8-polypeptide has been explored by the use of lipophilic affinity labelling reagents generated photochemically inside the membrane. This shows the region of the polypeptide embedded in the lipid bilayer. The probe 0-hexanoyl-3,5-diiodo-JV- 4-azido-2-nitro-phenyl)tyramine undergoes photochemical conversion into the reactive nitrene, and has been used to label the (Na+, K+)-ATPase from Bufo marinus toad kidney. This shows that the j8-polypeptide is also a transmembranous polypeptide,49 a view that is in accord with immunochemical evidence.50... 

The low-molecular-weight vitamin biotin is easily conjugated to antibodies and enzyme markers. Up to 150 biotin molecules can be attached to one antibody molecule, and the strong affinity of the biotin for the glycoprotein avidin allows its use as complex-ing secondary reagents. Biotin labeling of the primary (direct) or secondary (indirect) antibody can be used in the avidin-biotin methods. In the labeled avidin method the tracer is attached directly to the avidin molecule. In the avidin-biotin bridge method a biotinylated enzyme such as peroxidase is allowed to bind after attachment of avidin to the biotin-labeled antibody. 

Publications on the synthesis of aryl azides as photoactivatable reagents selected for their experimental procedures are Fleet et al. (1972) Galardy et al. (1974) Katzenellenbogen et al. (1973) Schwyzer and Calviezel (1971) Bridges and Knowles (1974) Huang and Richards (1977). The reader should also consult the detailed description of Smith and Boyer (1963) and the Methods in Enzymology volume on Affinity Labeling... 

---