Enzyme Purification, Activity Assay and Enzymes Kinetics.

 Enzyme Purification, Activity Assay and Enzymes Kinetics.

                                                        i.            Purification of α-amylase from saliva.

                                                      ii.            Quantification of proteins by Bradford method.

                                                    iii.            Assay for α-amylase activity in saliva.

Objective:

o   To get familiar with the protein purification protocols.

o   To study about quantifying methods of enzymes (proteins).

o   To Study about enzyme kinetics.

 

Introduction:

            Enzymes are proteins which are catalyzing (or regulate) most of biological reactions. All the enzymes are proteins. Those enzymes (proteins) are made with amino acids. All those natural enzymes are produced by according to DNA sequence, and primarily proteins has liner amino acids chain which can be called as polypeptide chain and the working enzyme or protein is build up with the 3D structure of the polypeptide.

In the practical session enzyme was purified and this purification methods are important to get the pure form of enzyme or protein to detect any disease conditions, mutations of genes industrial productions etc.  

           

i.                    Purification of α-amylase from saliva.

Alpha-Amylase (1.4-α-D-glucan glucanohydrolase ) is calcium-binding protein present In both salivary and pancreatic secretions. This cans clave polysaccharides. It catalyzes the hydrolysis of alpha-1.4-glycosidic linkages of polysaccharides.

This method is based on the highly specific binding of the enzyme with the glycogen. Once bound to this, the resulting complex is precipitated by the addition of ethanol, Because of the ethanol cause for change of the dielectric constant of the solutions. This difference is cause for precipitation of the complex.

	Dielectric constant (25 ºC)
Water	80.1
Ethanol	24.55

Then the separated complex allows to hydrolysis and subsequently the pure form of enzyme was precipitated likewise changing the polarity of the solution using ethanol.

            Purification is not enough for denoting an enzyme, quantification steps should have be done also. There are certain method for quantification like spectroscopic method, Chemiluminescence etc. In this lab session spectroscopic method was used.

ii.                  Quantification of proteins by Bradford method.

In this experiment Bradford method was used to quantify enzyme as a spectroscopic method. ‘Coomassie Brilliant Blue G-250’ (Figure 2.1) is the dye that was used in this experiment this form colored solutions with proteins. This dye makes strong non covalent complex with proteins by electrostatic interactions with amino and carboxyl groups via Van der Waals forces.

 So using this attribute the concentrations were measured using the spectroscopy and the highest absorbance can be seen in 465 nm to 595nm.

According to Beer–Lambert law,

A = ε.l × c

y =   m × x 

A – Absorbance

ε – Molar coefficient

l – Path length

c – concentration 


This liner relationship exists only in 0.2 to 0.7 absorbance values.

i.                    Assay for α-amylase activity in saliva. To justifying the quantified enzyme is as amylase the activity was assessed.              There are several ways to assay the enzyme activity of α-amylase. Those are enzymatic assay, turbidometric assay and assay using chromogenic substrates. Enzymatic assays are two type saccharogenic which measures the rate of appearance of reducing sugars and amyloclastic which measures the hydrolysis of starch and the rate of its disappearance. The amyloclastic assay type was used in this experiment. The rate of disappearance of starch was determined by using an iodine solution which is form a blue color complex with starch and appears in brown without starch. The reaction is considered to have reached its endpoint when samples produce reddish brown color with iodine. Activity of an enzyme can be expressed in Somogyi units. Somogyi Unit The α-amylase activity can be expressed in Somogyi Units. [Salivary amylase] (somogyi units/dL)  = (temprature factor)/(Endpoint time(min)) 1 Somogyi Unit           =  The amount of amylase required to produce the equivalent of 1mg of glucose in free aldehyde groups in 30 minutes at 40°C Somogyi Units/dL may be converted to International Units (µmol minute-1 L-1) by multiplying by 1.85. Enzyme kinetics Michaelis – Menten equation Plotting a graph of  1/Vo   vs  1/[s] Michaelis – Menten constant and theoretical maximum velocity can be found.

Calibration plot should be prepared and the concentrations are obtained from the plot.