Dear Subash,

İ have already used spectrophotometer, but i want to know that how using absorbans value for enzym activity.

is there formula for enzym activity?

thanks alot

Muzaffer, I can provide you help with that, I've worked with cell wall enzymes and done the respective calculus, feel free to contact me for further information at [email protected]

Cheers

You can calculate enzyme activity of enzyme by using this

enzyme activity= change in OD/time taken (min) x 1/extinction cofficient of enzyme x total reaction volume/ volume of enzyme extrct taken x total volume of enzyme extract/ Fresh wt of tissue (g) x total protein x 1000 = nano moles of enzyme present per g of sample tissue. for catalase ext coff is 39.4 mM-1cm-1and for peroxidases 26.6 mM-1cm-1. In case of SOD % inhibition = control OD- treatment OD/ control x 100 =X% inhibition. 50% inhibition is equal to 1 unit of enzyme. then X% is equal to 1/50 x X= Y unit. Calculate activity by inserting value of Y in above formula of activity in place of change in OD w.r.t. time. Rest of formula will be same.

Dear Shena,

I was glad to see your message but i didnt undersatant "total protein" in formula. what value do you use instead of total protein? Also you gave activity result in nano moles but generally results were given micro moles per gram fresh leaf tissue.

Dear Muzaffer Ipek,

You need to know the "total protein" content in your substrate, this is a first step to calculate enzyme activity because only a part of this total protein is enzyme and the enzyme activity obteined refers to this part.

Kind regards

Rosa

You can use the Bradford method to determine "total protein"

I agree with Rosa. First you have to find out the total protein content then only you can proceed for enzyme activity. Protein content can be calculated either by lowry's or bradford method.

Dear Ipek, you can use any of the standard units for enzyme activity calculation. I have used nano moles for the same.

Can anyone give me example activity measurement?

For example CAT activity;

Reading ABS at 240nm along 3 minute

CAT EC: 246,000 cm-1 M-1

Extraction total volume: 2 ml

sample :100 microliter

buffer :1.9 ml

dilition factor :1/10

first readinh (0. minute) 0,250 (ABS-1)

last reading (3.minute) 0,200 (ABS-2)

Expressing enzyme activity on a total protein basis is ONE way of expressing enzyme activity; better yet if you purify your enzyme and express on a per gram of enzyme protein basis; I have more simply expressed enzyme activity on a per gram of fresh weight basis; if you work with desiccation stress, expressing activity on a gram of DRY weight basis would be more meaningful and still simple.

P.S.: 1 micromol= 1000 nanomoles; just depends on how active is youe enzyme with respect to its concentration.

Hope it was helpful

Dear Bianco,

i use fresh tissue, and only i want to know how calculate total enzyme in per gram fresh tissue.

would you calculate example for me please?

For example CAT activity;

Reading ABS at 240nm along 3 minute

CAT EC: 246,000 cm-1 M-1

Extraction total volume: 2 ml

sample :100 microliter

buffer :1.9 ml

dilition factor :1/10

first readinh (0. minute) 0,250 (ABS-1)

last reading (3.minute) 0,200 (ABS-2)

It is hard to answer because I am not sure what you mean by Extraction total volume, but I assume the calculation should be:

(|ABSchange|*TRVol)/(SampleVol*EC*min*gFW)=(0.05*2)/(0.1*EC*3*gFW)

Of course, you need to know the fresh weight (FW) you extracted and the extinction coefficient for CAT in the right units. For Sorbitol dehydrogenase at 340 nm I was using EC=0.00622. Volumes are all expressed in the same unit (ml).

I hope this helps

Dear Bianco,

if you are not tired, would you give me example of calculate of Catalase in your study?

firstly you can give formula and then give companents of your formula.

it is important for me this calculating.

thanks your helps.

I do not work with catalase, but the principle is the same. In your example above, provide grams of fresh tissue extracted, extinction coefficient in the right units, and total reaction volume in the cuvette you load on the spectrophotometer. With those values we can simulate a calculation for your example.

Dear Bianco,

i think so i understood that what you can say.

Your formula is

(|ABSchange|*Total Reac. Vol(ml))/(SampleVol(ml)*EC(?)*min*gFW)= enzym activity

is it ok?

But i have a last question. what is result value? Forexample (UI), (micromoles mg-1), (nanomoles mg-1) etc...

which is your formula result value? or yours?

that depends on the EC you use. The one I was using for SDH was 0.00622 to get nanomoles/min*gFW or 6.22 to get micromoles/min*gFW.

However, be careful when you express on a per gram of fresh weight and work with drought vs well watered. People may argue that your plant material was different in terms of hydration and FW is not appropriate.

Activity = (ΔE X Vf) / (Δt x € x Vs x d)

Where: ΔE is change in absorbance,

Vf final reaction volume

Vs volume of enzyme extract used

Δt is time of hydrolysis

€ is extinction coefficient

d is diameter of cuvette used.

The final answer is divided by mg or g protein of the tissue.

Activity = (ΔE X Vf) / (Δt x € x Vs x d)

Where: ΔE is change in absorbance,

Vf final reaction volume

Vs volume of enzyme extract used

Δt is time of hydrolysis

€ is extinction coefficient

d is diameter of cuvette used.

The final answer is divided by mg or g protein of the tissue.

the enzymatic activity should be reported on the amount of proteins present in the extract

You need to know the the extinction coefficient  (epsilon: e) of your product  then you apply the Beer Lambert Abs= e c l (l is the pathlength if you use cuvette of 1 cm then you can calculate  c (concentration of product that appeared or substrate that disappeared) by Abs/el .  Be careful with the units of e, to determine the C (usually in mM). If you have c in mM for instance and you are working in 1 mL you will know that you have let say if c = 0.2 mM 0.2 µMol in 1 mL . If now you know that you have a delta Abs in 1 min then means you have 0.2 µmol (200 nmol) per 1 min and you have to know how much enzyme you put in your cuvette (let say 2 nM) then your kcat  (catalytic constant) will be 100 min-1. You can also work out activity as nmol/min/mg (then you need to know how much you put in the cuvette let say 1 µg in the 1 mL then meaning that you got 200 nmol/min for 100 µg so you mutliply by 10 to get  2000 nmol/min/mg or 2 µmol/min/mg that is also the enzyme activity.

Sonam Sneha

Hi!

for the extinction coefficient of for catalase ext coff is 39.4 mM-1cm-1

and for peroxidases 26.6 mM-1cm-1 you've anwsered. is it given?

and if not how to compute the extinction coefficient of for catalase and ascorbate peroxidase

thank you

Activity (u/l) = Change Abs x V x 1000 divided by d x t x E x v

Bamidele Ajilore

Could you provide some reference where I can find this equation?

Dear Sonam Sneha , Do you have any excel sheet containing formulas for the calculation of enzyme activity? If yes, could you please share it with us?