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how to calculate the activity of this enzyme ...????????

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Topic Started by fatma_elzahraa
on 10/15/2009 14:47 PM   
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Dear all,

I have a real problem in calculating the activity of tartrate resistant acid phosphatase by a kinetic assay illustrated in a paper in the following link:

www.clinchem.org/cgi/content/full/46/4/469

and this is a part of the paper:

chemicals
2,6-Dichloro-4-acetylphenyl phosphate (DCAPP) was obtained from Nitto Boseki Co. Ltd. Sodium L-(+)-tartrate and heparin were obtained from Sigma, and polybrene was obtained from Aldrich. Carboxymethyl-agarose (CM-Sepharose) was obtained from Pharmacia Fine Chemicals. All other analytical grade chemicals were purchased from Wako Pure Chemical Co.

reaction solution
The reaction solutions we reported previously (12) were used with some modification. Briefly, buffer solution I consisted of 150 mmol/L 2-(-hydroxy-3-morpholino) propanesulfonic acid, 60 mmol/L sodium L-(+)-tartrate, 23 kIU/L heparin, and 5 g/L bovine albumin (pH 6.6). Buffer solution II consisted of buffer solution I plus sodium fluoride (45 mmol/L). The substrate solution consisted of 45 mmol/L DCAPP and 50 mmol/L Tris (pH 4.0).

serum samples
Blood samples collected from 516 apparently healthy Japanese subjects, ages 5–79 years (210 males and 306 females), by clean venipuncture were allowed to clot at room temperature for 2–4 h and centrifuged at 1000g for 10 min at room temperature. Serum thus separated was transferred into 1.5-mL tubes and stored at -80 °C until use (within 6 months).

bone extract
Bone extract samples were prepared as described previously (12). In brief, bovine tibia after the removal of soft tissue was cut into small cubes. The marrow and blood were removed, and the bone was ground to powder and homogenized in a solution containing Triton X-100, potassium chloride, phenylmethylsulfonyl fluoride, benzamidine, and aminocaproic acid. The extract was collected by centrifugation at 10 000g for 20 min at 4 °C and stored at -80 °C until use.

assay procedure
TrACP activity was measured using a centrifugal automatic analyzer (Cobas Fara; Hoffmann-La Roche). Briefly, 150 µL of buffer solution I was added to 15 µL of sample, and the mixture was incubated at 37 °C for 5 min. The enzyme-substrate reaction was initiated by the addition of 60 µL of substrate solution. The change in absorbance at 340 nm was monitored at 20-s intervals for 5 min. The millimolar absorptivity of the hydrolysis product (2,6-dichloro-4-acetylphenol) is 21.49 L · mmol-1 · cm-1 at 340 nm. One IUB unit (1 U) of TrACP activity is defined as 1 µmol of DCAPP hydrolyzed per minute at 37 °C in the presence of 40 mmol/L sodium L-(+)-tartrate and 23 kIU/L heparin at pH 6.6. Tartrate-resistant fluoride-resistant acid phosphatase activity was assayed using buffer solution II instead of buffer solution I. Band 5b TrACP activity was estimated by subtracting tartrate-resistant fluoride-resistant acid phosphatase activity from TrACP activity.

The italic part is the procedure of the assay... I need to know how to calculate the activity using many absorbances at 20 second intervals till 5 min... I couldn't get it simply...

I hope if there is anyone can help or direct me, please tell me.....


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Pippuri
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Posted By Pippuri
on 10/15/2009 16:06 PM   
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Hi Fatma_elzahraa,

First, you have to do your enzymatic assay by using such concentration of your enzyme that when you measure absorbance during let's say for 5 minutes (every 20 seconds), you can draw a straight line through all the absorbance points. If you have too much enzyme, you won't get a linear relationship but a curved one. If this occurs, you have to use less enzyme in your assay. On the other hand, you need to use enough to have the absorbance at 5 minutes around 0.5 - 1.5. In this case, you have high enough signal (good signal to noise ratio) and still in the linear region of the spectrophotometer detector.

At this point you have a graph with absorbance as a function of time. The slope of this line (absorbance per time) is really an enzymatic activity value in disguise.

The only problem here seems to be to convert your absorbance measurement to product concentration. You can do it by using the Beer-Lambert law:

Absorbance per time = Absorptivity * Concentration per time * Path length.

Solve for Concentration:

Concentration per time = Absorbance per time / (Absorptivity * Path length)

Concentration per time is the velocity of the enzymatic reaction. If its value is 1 micromolar per minute, the activity of the enzyme in the reaction cuvette is 1 U.

You can then calculate the specific activity by dividing the total activity by the amount of enzyme. For example, if you use 10 micrograms of enzyme you divide the velocity by 10 micrograms to get the specific activity.

Best of luck,


Last edited Oct 15, 2009, 18:16 PM by Pippuri


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Posted By fatma_elzahraa
on 10/16/2009 8:09 AM   
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Thanx alot for your reply... but iam still had some unclear points... i returned to my results and drew a curve time vs absorbance and get the slope... but as you said, the curve is not straight but curved... so as you told, i should dilute my samples... but what is the dilution factor best for my results... and can't i take the absorbances of the first 3 min only as they gave a straight line.... and there are other enzymes i will measure kinetically too, can i treat the results in the same way... and dilute the samples if there is a small change in absorbance... also, i calculated the slope but there is an intercept, how can i deal with it in my equation.... do you think that this kinetic procedure cannot be converted into colorimetric one by adding a chemical to stop the enzyme activity at certain time.... i hope i can get answers for my questions.... Best regards



Pippuri
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Posted By Pippuri
on 10/16/2009 8:58 AM   
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fatma_elzahraa,

First of all, you don't have to use the whole 5 minute data if the last data points are not on the straight line. You can use the early part of the data. For example, you can use only the first 3 minutes, if they fall on a straight line. If you want to repeat the experiment and use whole 5 minutes, you have to reduce the amount of the enzyme so that the reaction graph does not curve. You can estimate the dilution factor by looking at how long the linear part of the graph is. If the linear part of your current graph is 2.5 minutes, you can double the linear part to 5 minutes by halfing the amount of enzyme.

The intercept of your line doesn't matter because even if you subtract some value from each data point, the slope is still the same. And it is the slope that matters. You should think about this issue, understanding the meaning of your data is very important so you can design and interpret your own experiments in the future.

And, yes, same principles of experiment design and data analysis apply for other enzyme reactions as well.

Your experiment is already colorimetric, if you are measuring absorbance with a spectrophotometer.  Maybe you mean to convert your experiment from time-point experiment to end-point experiment. You can do that, but you get more reliable and accurate results by doing rigorous time-point analysis.

Good luck to you.





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Posted By fatma_elzahraa
on 10/16/2009 12:43 PM   
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dear Pippuri,

Really, tooooo many thanx for ur great help...

I appreciate your cooperation...

Best regards



boyscientist
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Posted By boyscientist
on 4/24/2010 7:15 AM   
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Hiii...Could u tell me the easiest and the best way to calculate the activity of a protein...also plz tell me if there is any assay that can be developed easily.. thanks heaps ..   :)




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Posted By fatma_elzahraa
on 4/24/2010 15:12 PM   
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hi boyscientist what do u mean by activity of protein?.... did u mean an enzyme? or protein concentration... if it is an enzyme... what is it? as each enzyme has its own assay.... if u want to measure protein conc. u can use buiret or bradford assay if u wanna more info. about anything... just clarify what u want to search about..... best regards



boyscientist
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Posted By boyscientist
on 4/25/2010 9:01 AM   
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Hii..thanks a lot for ur reply.well, its an enzyme. my enzyme is thermostable phytase. I would like to know, how to calculate the acitivity of that perticular enzyme.If there is an assay for it, could u plz tell me the easiest way to do it. or, if there is a method to develop my own assay for it, how to do it?

thanks heaps



boyscientist
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Posted By boyscientist
on 4/25/2010 9:16 AM   
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Hii fatma,..is there is a place where is can upload my resume..
thanks heaps




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Posted By fatma_elzahraa
on 4/25/2010 14:44 PM   
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hi i didn't deal with such an enzyme before... u can upload ur resume using www.rapidshare.com



sadia khan
India

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Posted By sadia khan
on 4/27/2010 2:22 AM   
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could u plz tell me how to calculate the specific enzyme activity of superoxide dismutase by marklund method  and its reference too.
how do i make 0.2 mM PYROGALLOL solution for 3 ml assay and i have to add only 0.1 ml pyrogallol.if i have to make a stock of 5 ml




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Posted By zareenk19@gmail.com
on 3/31/2013 2:32 AM   
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 Hi,

I read your reply to fatma_elzahraa for calculation enzyme activity. I am facing a similar kind of problem. I will be very grateful if you can help me solve that how I should calculate enzyme activity in my case.

Kindly help me proceed to analyse Polyphenol Oxiadse(PPO) enzyme activity in grapes.I have extracted PPO enzyme using Triton-X detergent. (Reference: Kinetic characterisation and thermal inactivation study of polyphenol oxidase

and peroxidase from table grape, the reference is attached). 

Polyphenol oxidase (PPO; EC 1.14.18.1) is a copper-containing enzyme, which, in the presence of oxygen, catalyses the hydroxylation of monophenols to o-diphenols (cresolase activity) and the oxidation of o-diphenols to their corresponding o-quinones (catecholase activity)


 For analysis of enzyme activity, The protocol for enzyme analysis is as follows:
 
The reaction mixture consisted of 1.25 ml of catechol (20mM) in 0.2 M acetate buffer (pH=3.4) and 0.25 ml of freshly prepared enzyme solution.
The increase in absorbance was measured at 410nm at 30ºC for duration of 5 minutes at every 30 seconds interval.
The increase in absorbance per time will be used to calculate the enzyme activity.

The enzyme reaction was followed by absorbance measurement of the quinone or derivative, formed as a result of substrate oxidation, at 475 nm for 10–40 min in a Spectrophotometer.
 
Observed values: 
Time (sec) Abs at 410nm ΔA/min
0 0.557  
30 0.557  
60 0.584 0.027
90 0.608 0.051
120 0.628 0.044
150 0.647 0.039
180 0.664 0.036
210 0.681 0.034
240 0.714 0.05
270 0.728 0.047
300 0.745 0.031

How should I calculate Molar absorptivity/ molar extinction co-efficient? 
A = Molar absorptivity *concentrationof substrate * pathlength

Please help....

Thanking you in anticipation,
Zareen


















































 




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Posted By zareenk19@gmail.com
on 4/1/2013 4:11 AM   
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 Hi, ther were some mistakes in the question that i had asked yesterday. Hence I have reframed my query today. Sorry for the inconvenience


I need help to proceed with analysis of Polyphenol Oxiadse(PPO) enzyme activity in grapes. I have extracted PPO enzyme using Triton-X detergent. (Reference: Kinetic characterisation and thermal inactivation study of polyphenol oxidase
and peroxidase from table grape, the reference is attached). 

Polyphenol oxidase (PPO; EC 1.14.18.1) is a copper-containing enzyme, which, in the presence of oxygen, catalyses the hydroxylation of monophenols to o-diphenols (cresolase activity) and the oxidation of o-diphenols to their corresponding o-quinones (catecholase activity)


 For analysis of enzyme activity, The protocol for enzyme analysis is as follows:
 
The reaction mixture consisted of 1.25 ml of catechol (20mM) in 0.2 M acetate buffer (pH=3.4) and 0.25 ml of freshly prepared enzyme solution.
The increase in absorbance was measured at 410nm at 30ºC for duration of 5 minutes at every 30 seconds interval.
The increase in absorbance per time will be used to calculate the enzyme activity.

The enzyme reaction was followed by absorbance measurement of the quinone or derivative, formed as a result of substrate oxidation, at 410nm for 5 min in a Spectrophotometer.
 
Observed values: 
Time (sec) Abs at 410nm ΔA/min
0 0.557  
30 0.557  
60 0.584 0.027
90 0.608 0.051
120 0.628 0.044
150 0.647 0.039
180 0.664 0.036
210 0.681 0.034
240 0.714 0.05
270 0.728 0.047
300 0.745 0.031



ΔA/min values were calculated by subtracting the absorbance every 60 secs for 1 minute. I think the average of these values should give the rate of change of absorbance per minute for the complete reaction.

This value obtained by calculation(0.040) is not matching with the slope (0.0007)value obtained in Excel by plotting a curve of linear regression. According to the literature, the values of the slope for a graph of Absorbance plotted against time should be ΔA/min.
Inline image 1
This value obtained would be velocity or rate of reaction. 
 
This can be converted into enzyme activity by using Beer Lambert's law. 
Absorbance = Molar absorptivity (Epselon) * Path length of cuvette * Concentration.
 
So, Epselon = (Absorbance) / (Path length of cuvette * Concentration).
 
To find out the constant Epselon, here I want to confirm whether I can take concentration of the substrate (20Millimolar)? Which absorbance should I use for calculating the Epselon?
 
Further, I have to use this value to calculate concentration of the product formed per time. Hence, by rearranging the equation for Beer Lambert's law,
 
Concentration =  (change in absorbance / min) / (Path length of cuvette *Epselon)
 
Hence this can be written as,
Concentration =  (slope) / (Path length of cuvette *Epselon).
 
Suppose, this concentration value is obatined as 5 micro mol/min,
the corresponding units for enzyme activity will be 5 units.
 
I need help in solving all these obstacles in reaching the final result.
 
In search of an answer,
Zareen
 
 



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