Question

Steps: ( HRP )

1. put the sample in 15ml in to the tube.

2. took 200.0 microliter of the sample and added to 500 microliter of 100mM phosphate buffer at pH 7 and mix it.

3. after mixing it, tool 100.0 microliter of first dilution and added to 500microliter buffer.

4. pipette 300.0 microliter of the second dilution into standard 1cm cuvette( contained 800 microliter of buffer and 1.00mL stock h2o2, 1.00mL stock guaiacol)

( coefficient of guaiacol=26600M^-1cm^-1 @ 470nm)

5. got absorbance at 470nm.

6. I.U= 0.752

- find the number of units of HRP activity in cuvette

- find the total number of unites of HRP sample ( total volume-11ml)

Answer 1

			vol taken	mixed in	sum	dilution factor	fraction factor
step 1	volume of sample	11ml					11/0.2	55
step 2	dilution 1		200 or 0.2 ml	500	700	3.5
step 3	dilution 2		100	500	600	6
step 4	dilution 3		300	2800	3100	10.33333
	molar extinction coef	26600
step 5	absorbance	0.752	conc	absorbance/molar extinction coeff	2.82707E-05	unit is M	convert it into µ mole	MxV/ 1000 contt.. to below table

V is volume of solution in cuvette

3100 µL

3.1 mL

8.76391E-08

covert it into no of micromoles*

0.00876

considering time of enzymatic reaction

10 mins

0.000876

µ mol /min

activity in cuvette

total number of units

total dilutions x fraction factor * activity in cuvette

3.5*6*10.33*55*0.000876

10.45169