Radioenzymatic [Transferase] Assays

RADIOENZYMATIC [TRANSFERASE] ASSAYS

The ‘radioenzymatic assays’ have gained their abundant acceptance and recognition for the assay of aminoglycoside antibiotics e.g., amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, doxorubicin, cephalosporins, cephamycins, thienamycin, lincomycin, clindamycin, eryth-romycin, clarithromycin, azithromycin, oleandomycin, spramycins etc ; and chloramphenicol (or Chloromycetine). Importantly, the radioenzymatic assays are exclusively based upon the fact that the prevailing inherent microbial resistance to the said aminoglycoside antibiotics and chloramphenicol is predominantly associated with the specific as well as the critical presence of certain highly specialized enzymes* that particularly render the ‘antibiotics’ absolutely inactive via such biochemical means as : acetylation, adenylation, and phosphorylation.

It has been duly proved and established that :

·        aminoglycoside antibiotics—are susceptible to prominent attack by these critical and specific enzymes as :

Aminoglycoside acetyltransferases (AAC) ;

Aminoglycoside adenylyltransferases (AAD) ;

Aminoglycoside phosphotransferases (APH).

·        Chloramphenicol—is prone to predominant attack by the enzyme :

Chloramphenicol acetyl transferases (CAT).

Mechanism of Action : The mechanism of action of these enzymes viz, AAC, AAD, and APH are not the same :

Acetyltransferases [i.e., AAC]—invariably attack the most susceptible amino moieties (–NH₂), and to accomplish this critical function may require acetyl coenzyme A (AcCoA).

Adenylyltransferases [i.e., AAD] and Phosphotransferases [i.e., APH]—these enzymes usu-ally attack the most susceptible hydroxyl moieties (–OH), and specifically requires adenosine triphosphate [ATP] i.e., another nucleotide triphosphate.

Applications : As to date quite a few AAC and AAD enzymes have been judiciously employed for the radioenzymatic assays.

Example : Both the enzyme and the suitable radiolabelled cofactor [1 – ¹⁴C]** acetyl coenzyme A, or [2 – ³H]*** ATP are used frequently in order to specifically radiolabel the ‘drug substance’ under investigation.

Method— The various steps involved in the assay are as follows :

(1) Enzymes are normally prepared by anyone of the following two techniques,

(a) Osmotic Shock i.e., by breaking the cells of an appropriate microbial culture by exposing than to a change of strength of solution therby affording a definite perceptible alteration in the ‘osmotic pressure’, and

(b) Ultrasonic Sound-waves i.e., by breaking the cells of a suitable bacterial culture by means of the high-frequency ultrasonic sound waves.

Thus, the said two methods do break open the cells to a considerable extent, and no purification is required at all.

(2) Radiolabelled drug substance is subsequently separated from the ensuing reaction mixture soonafter the said reaction has attained completion duly. Thus, the exact quantum of the extracted radioactivity is observed to be directly proportional to the exact quantum of the drug substance present in the given sample.

Note : Separation of two types of antibiotics are accomplished duly as stated under :

(a) Aminoglycoside Antibiotics—by binding them suitably to phophocellulose paper, and

(b) Chloramphenicol—by making use of an organic solvent.

1. Calibration

In a particular situation when the reactants are adequately present in enough quantum, and the prevailing reaction attains completion in due course, one may conveniently plot a graph of the counts per minute (min^–1) Vs concentration of calibrator, which is found to be linear, as illustrated in Fig. 10.4.

2. Non-Isotopic Modification

The calibration accomplished by using the radiolabelled drug essentially needs either a Gei ger Müller Counter or a Scintillation Counter, for measuring the ensuing radio activity (in mC) of the ra dioactive chemicals, which being an enormously ex pensive equipment, and a skilled technician. There- fore, in order to circumvent these glaring untoward se- rious problems one may adopt a photometric varia tion of the aminoglycoside acetyltransferases [AAC] assay meticulously. For this the sulphydry rea-gent viz., 5, 5′-dithiobis (2-nitrobenzoic acid) is incorporated carefully into the on-going assay-sys-tem. Thus, the said reagent specifically interacts with the corresponding coenzyme A (reduced form) duly generated thereby producing a distinct yellow-coloured product that may be quantitatively as-sayed by using a previously standardized UV-Visible Spectrophotometer.

(a) Reactions : The two reactions are as follows :

(b) Aminoglycoside + Acetyl CoA —→ Acetyl – Aminoglycoside + CoASH CoASM + DTNB —→ Yellow Product