FUNDAMENTALS OF DIAGNOSTIC ENZYMOLOGY

Dr. Gangadhar ChatterjeeMBBS;MD

Assistant ProfessorRCSM Govt. Medical college, Kolhapur, MH, India

FUNDAMENTALS OF ENZYME ESTIMATION

You are NOT measuring the enzyme molecule or its concentration like sugar or urea……….

Because- Enzymes are biocatalysts……and catalysts in reaction remains in VERY MINUTE quantity……….even less than picogram per liter………making it almost impossible to measure directly.

FUNDAMENTALS OF ENZYME ESTIMATION

- enzymes, being very delicate molecule and extreme minute presence in body fluids, it is very difficult to extract it in pure form to prepare STANDARDS. ( without which it’s NOT possible to estimate)

………………………..Hence ESTIMATION OF ENZYME is a WRONG term…..as we are not doing that.

then …………what's the possible way out???????

We can measure the SUBSTRATE or PRODUCT of an enzyme appreciably.

So what actually we can do………..

• We can look for the enzyme ACTIVITY in terms of decreasing SUBSTRATE or an increasing PRODUCT of certain enzyme mediated reaction.

…………….So actually we are estimating the ENZYMATIC ACTIVITY.

*******right term is Estimation of ABC enzyme ACTIVITY in XYZ sample*******

Control tube

Used to nullify effect of the INDIGENOUS (S/P) molecule in the final colour in the TEST tube………………………………………………………………………………………………………………………………………………………………………………….as the colour produced by the indigenously present molecule is NOT evolved due to enzyme action……………………………………………………………………………………………………………………….NOTE some modification in the equation for this CONTROL TUBE.

Some facts about ALP- Alkaline phosphatase ( EC 3.1.3.1; orthophosphoric

monoester phosphohydrolase) catalyzes the alkaline hydrolysis of organic phosphates.

- Zn2+ is a constituent metal ion of this enzyme…………………Metalozyme

- Mn2+ , Co2+ , Mg2+ are activator of this enzyme.- Phosphate, Borate, Oxalate and Cyanide are inhibitor

of this enzyme.

PRINCIPLE OF THE METHOD

Alkaline phosphatase (ALP) catalyzes the hydrolysis of p-nitrophenyl phosphate at pH 10.4, liberating p-nitrophenol and phosphate, according to the following reaction:

ALPp-Nitrophenylphosphate + H20 p-Nitrophenol + Phosphate

The rate of p-Nitrophenol formation, measured photometrically, is proportional to the catalytic concentration of alkaline phosphatase present in the sample

Isoenzymes of ALP

Hepatic, bone, intestinal and placental are major subtypes of ALP isoenzymes.

On electrophoresis, Liver form is fastest moving. Bone and intestinal forms give a diffuse large band, little slow moving than liver form.