Calculation of Specific Activity Recall that Bee law (A ECI gives the relationsh

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Calculation of Specific Activity Recall that Bee law (A ECI gives the relationship between the absorbance of a pound and the concentration. The molar extinction coefficient is a constant and is characteristic of each compound, Thus, if we of solution and the extinction coefficient, we can calculate the know the absorbance a the quantity of product concentration of that substance. Using this information we can determine produced by the enzyme, which is needed for calculation of specific activity. termine the number of moles of product produced by the enzyme. The molar extinction coefficient for pNP 1.88 x 10 L/mole cm. Note the units. 1. Divide Y by 1.88 x 10 L/mole cm moles cm/L 2. The length of the cuvette path is 1 cm, so factor out the cm. amoles/L. 3. Convert to umoles per liter by multiplying by 10 umoles/mole. 4. Calculate how many moles were actually made. You assayed 5 ml. #umoles/L x 0.005 L #umoles 5. This product was made in a quantity of time. Divide by 15 min to reflect this. Rumoles/ 15 min Mumoles/min This quantity is known as the activity of the enzyme: the amount of product formed in a certain amount of time 6. Specific activity is umoles of product/ min mg of protein. So now you must divide by the ma mg of the enzyme present for that particular absorbance, the X of your selected data point. Congratulations! You've determined the specific activity of the enzyme! The Mega-equation for the above calculation is: 000s x 10 umol x 1 x 1 AA10 x mole mg protein 1.88x10 l/mol cm 15 min

Explanation / Answer

Explanation:- Lambert Beer equation is

A = E * c * l ; where

A is the Absorbance at particular wavelength,

E is the molar extinction coefficient or molar absorptivity (It is constant for a particular compound) and

l is the pathlength (if cuvette for absorbance measurement is of 1 cm, then pathlength =1 cm)

Statement 1 – Determine number of moles of product produced by enzyme.

Solution - In the given data, E for PNP is mentioned as 1.88 *104 L/mole cm. The recorded absorbance is mentioned as “Y”.

According to lambert beer equation

Y = (1.88*104) *c *l

Therefore, concentration (mole cm/L) ie:

c = Y/[(1.88*104) * l]                       (Equation 1)

Statement 2 – The length of cuvette pathlength = 1 cm

Now substituting the pathlength 1cm in equation1 we get

c = Y/[(1.88*104)*1]

Therefore, concentration (mole /L) ie:

c = Y/(1.88*104)                                (Equation 2)

Statement 3 –Convert to µmole/L

To convert mole into µmole, we need to multiply c obtained from equation 2 by factor 106

Therefore, Concentration in µmole/L ie;

C =c * 106                                             (Equation 3)

Statement 4 – How many moles were actually made?

In given data, the sample volume assayed is 5 ml ie; 5*10-3 L = 0.005 L

From equation 3, total concentration =C (mole/L)

Therefore, P (concentration is actual sample assayed ie; in 0.005 L)

= C * 0.005

Thus, P (µmole) = C (µmole/L)*0.005(L)                                                (Equation 4)

Statement 5 –The product was made in quantity of time

In given data, time is mentioned as 15 mins.

Therefore, Z (product concentration produced per unit time)

= P/15

Thus, Z (µmole/min) = P (µmole)/ 15 (min)                         (Equation 5)

Statement 6 – Calculate specific activity

Given information- specific activity = product concentration (µmole/min)/mg of protein (enzyme)

In given data, X = mg of protein (enzyme)

Therefore,

Specific Activity (µmole/min mg) = Z (µmole/min) / X (mg)

From Equation2, 3, 4 and 5, combine equation for specific activity is as follows

Specific Activity ={[ (Y/1.88*104)] * 106 * 0.005 * (1/15) *(1/X)}

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