BIOCHEMISTRY AND HUMAN NUTRITION

Module 2. Enzymes

Lesson 15
DETERMINATION OF ENZYME ACTIVITY

15.1 Introduction

• Enzyme activity may be expressed as μmol of substrate transformed per minute (μmol min^-1).
• The standard unit of enzyme activity is enzyme unit and katal (kat).
• An enzyme unit is that amount of enzyme which catalyzes the transformation of 1 μmol of substrate per minute at 25⁰C under optimal conditions for that enzyme.
• The Katal is the SI unit of enzyme activity and is defined as that catalytic activity which will raise the rate of reaction by one mole per second in a specified system. 1U=16.67 nanokatal.
• The term activity refers to total units of enzyme in the sample, whereas the specific activity is the number of enzyme units per milligram of protein (Units/mg).

15.2 Effect of Enzyme Concentration on Enzyme Activity

15.2.1 Alkaline phosphatase : E.C3.1.3.1 (Orthophosphoric-monoester phosphohydrolase)

15.2.1.1 Principle

Alkaline phosphates hydrolyzes p-nitrophenyl phosphate, which is colourless, to p-nitrophenol which is yellow in alkaline medium. Measurement of O.D, at 400-420 nm helps to determine the concentration of p-nitrophenol, which in turn is directly proportional to p-nitrophenyl phosphate. In alkaline solution, p-nitrophenol absorbs at 405 nm. The substrate p-nitrophenyl phosphate does not absorb at this wavelength, so that the progress of the enzyme catalyzed reaction can be readily followed by measuring the change in extinction at 405 nm. The optimum pHs for acid and alkaline phosphatases are 5.3 and 10.5 respectively.

15.2.1.2 Reagents

• 50 mM Carbonate-bicarbonate buffer (pH 10.5)
• Standard Solution: p-nitrophenol solution 0.5 mM, prepared in above buffer.
• Calculation: 1mM = 139 mg/l or 0.5mM = 69.5mg/l or 34.75 mg/500ml buffer.
• Substrate Solution : p-nitrophenyl phosphate (PNP)

10 mM, dissolved in above buffer (M.W. of PNP disodium salt = 371.16).

15.2.1.3 Calculation

1mM = 371.16 mg/l
10mM = 3711.6 mg/l or 371.16 mg/100 ml buffer

• 2% Na2CO3 solution – Dissolve 2g sodium carbonate in 100ml distilled water.
• Seminal Plasma: Buffalo or cattle semen is kept at room temperature for 10-15 minutes and then centrifuged at 1000 g* for 15 minutes, at room temperature. The sperms are pelleted at the bottom, while the clear supernatant obtained is the seminal plasma. This is diluted 1:30 with carbonate-bicarbonate buffer, 0.05 M, pH = 10.5.

• Method

• Preparation of Standard curve of p- Nitrophenol

Concentration of Std. PNP = 0.5 mM

Add p- Nitrophenol and distilled water to all the tubes as shown below.

Table 15.1 Preparation of standard curve of p- Nitrophenol

• Plot Graph of absorption against PNP concentration

Fig. 15.1 Plot Graph of absorption against PNP concentration

• Enzyme Assay

Table 15.2 Effect of enzyme concentration on enzyme activity

• Absorbance (405 nm)

From the standard curve for p- nitrophenol, convert the absorbance readings into μ moles of substrate, converted by the enzyme and calculate the enzyme activity.

Plot a graph of absorbance against enzyme concentration

Enzyme activity may be calculated as “Number of micromoles of the substrate converted into product under defined conditions”.

Specific activity is defined as “micromoles of p-nitrophenol released per min per mg protein.”