Azide enzyme inhibitor

Phosphate buffer, azide free, 0.1-0.5 M, is suitable for the iodination reaction, but the enzymes and hormone should be diluted in 10—50 mM buffer, as the higher molarity may cause aggregation. There must be no azide or other enzyme inhibitors in the buffers used for the iodination reagents. 

All of the azides investigated were time-dependent inhibitors at millimolar concentrations and the inhibition was reversible in each case, with hepatic glutathione 5-transferase proving the most sensitive enzyme. Inhibitor potency appears to depend upon the substrate employed, -heptyl and allyl azides (60) and (62) being the most potent with NBC, and -butyl and -hexyl azide (57) and (59) when DNCB was included in the assay. Kinetic studies, where the GSH and DNCB concentrations were independently varied, indicated that compounds (61),(63) and (64) were noncompetitive inhibitors, while allyl azide (62) and the n-alkyl azides (56)-(60) inhibited the enzyme in a competitive manner. From these observations, the authors speculate that, in a process reminiscent of that known to occur with alkyl and aryl halides, glutathione 5-transferase may catalyse the conjugation of azides with GSH in vivo. 

An unprecedented example of the application of an organic azide as an enzyme inhibitor derives from the elegant studies of Stubbe and coworkers at MIT, who have investigated the mechanism of action of ribonucleotide reductase (RNR) using several mechanism-based inhibitors including 2 -azido-2 -deoxyuridine-5 -diphosphate (NjUDP) (71) [82]. RNR plays a... 

Organic azides are used for photoaffinity labelling (see Special Topic 6.16) because the azide group is small and, when attached to enzyme inhibitors, they often retain their activity as inhibitors. Azides are readily synthesized and the light-induced denitrogenation... 

Neutrophil-mediated HA degradation was shown to be increased in the presence of the MPO inhibitor azide [104] and is inhibited by dimethylsulf-oxide [105] but not by metal chelators such as deferoxamine (DFO) or diethy-lenetriamine pentaacetic acid (DTPA). Selected specific enzymes are able to prevent the degradation of HA by stimulated neutrophils [106]. Interestingly, the ability to degrade HA seems to be specific to PMA-stimulated PMNs and is not observed with neutrophils stimulated by other compounds such as formyl-methionyl-leucyl-phenylalanine, concanavalin-A or digitonin. This may be related to the increased concentrations of H O generated in response to these stimuli, or a greater release of specific enzymes that consume the relevant reactive species [107]. 

Click chemistry, as introduced by Kolb and Sharpless in 2001 relates mainly to the Cu(i) eatalyzed [3+2] cycloaddition reaction of azides with alkynes. This copper catalyzed cycloaddition reaction is highly useful for attaching fluorescent or other markers to a wide variety of biomolecules. Although azides are often unstable at elevated temperatures, they are stable at physiological conditions, have no intrinsic toxicity and have extraordinary chemical selectivity. Proteins and glycans have already been labeled with azides in laboratory mice using enzyme inhibitors and sugar azides. 

Azide, thiocyanate, and cyanate also have been shown to bind to Cluster C (152). Although they are weak inhibitors of the enzyme, they cause marked changes in the EPR spectrum of Cluster C (153), converting the Credi state (with all g-values below 2) to a state in which all g-values are above 2. 

Notes. When using biotin-labeled secondary antibodies instead of enzyme-labeled antibodies, you have first to detect biotin with enzyme-labeled (strept) avidin and proceed further with the Substrate Step (9). Do not add normal serum, non-fat dried milk, culture media or other potential sources of biotin to (strept)avidin-containing reagents. This may result in reduced sensitivity. Solutions containing sodium azide or other inhibitors of peroxidase activity should not be used in diluting the peroxidase substrate. 

Sodium azide is a powerful inhibitor of HRP, but sodium azide can be used with alkaline phosphatase conjugated antibodies without harmful effects. In addition, tap water or water deionized with polystyrene resins may inactivate the enzyme conjugate. Only use distilled, deionized water Tween-20 may interfere with some antibody-antibody reactions or may wash the protein of interest off the membrane. Tween-20 may be left out of the washes, but this may result in increased background... 

Dissolve 4.25 g Tris (35 mMoles), 7.6 gNaCl (130 mMoles), 0.47 g KCl (2.5 mMoles), and 0.2 g NaNs (should be substituted by other biocides, because azide is an inhibitor for iron-containing enzymes) in 800 ml ddH20. Adjust the wanted pH between 7.6 and 8.0 with 1N hydrochloric acid, then fill up to 1000 ml. 

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