Diisopropylfluorophosphate , labeling

The absolute number of essential residues, either of the same or different types of amino acids, can frequently be determined. One of the simplest and best methods is the use of one of the several types of active-site labeling reagents which have an easily monitored group attached. Such a group could be labeled with a radioactive element or with a distinct optical absorption band. One that has frequently been used with the serine -type enzymes is diisopropylfluorophosphate labeled with 32P. 

CMV protease is a serine protease, and labeling with diisopropylfluorophosphate (DFP) has identified Serl32 as the active site serine (4,5). The structure of the CMV protease containing the diisopropylphosphorylserine at residue 132... 

Amino acid residues of the A.c. are identified by specific labeling with coenzyme, or by reaction with inhibitors or reagents specific for particular side chains. Some widely used irreversible inhibitors of the catalytic center of serine proteases are tosyllysine chloromethyl ketone (TLCK), which reacts with histidine, and diisopropylfluorophosphate (DFF) and phenylmethane sulfonyl fluoride (PMSF), which forms esters with serine residues. 

It has been shown by means of labelled diisopropylfluorophosphate that one end-plate contains 1-2 x 10 active centres of AChE, which means that one active centre of AChE corresponds to an area of about 1 - 3 x 10 A. Thus the number of active centres of AChE on the subsynaptic membrane is several times greater than that of ChR. 

Hjort, P.F., Paputchis, C.B., 1960. Labeling of red blood cells with radioactive diisopropylfluorophosphate PPjDFP evidence for an initial release of label. J. Lab. Clin. Med. 55, 416-424. 

Vorob eva, E.N., 1976. Study of the kinetics of blood granulocytes labelled with radioactive diisopropylfluorophosphate (DFP- P). Med. Radiol. (Mosk) 21 (8), 62-73. (In Russian). 

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