That was the best, most detailed, extraordinary explanation of this confusing topic. My teachers at the university failed to be as precise as you. Thank you Sir!
Poets and scientists have much in common - we both reach for the stars, but admittedly Michael Rosen's grasp outreaches mine. The very best to you, Gus
It is a nice video. Thank you very much. I kindly ask you how did you calculate epsilon (molarity absorbance) here. you directly put 4600 L per mol per cm. Kindly explain. Thank you
While calculating the umol min-1 why the 4ml volume was considered instead of 1ml? I mean why should we consider the volume of buffer solution used to stop the reaction as the enzyme only acts on the substrate?
Thank you for such a precisely informed video. If I haven't understood it wrong, the change in absorbance here is the difference in absorbance at time = 0 sec and time = 5 mins?
thanks for the very useful video. I have a doubt in calculations part of my experiment. I have got 0.006 OD per minute, after simplifying I have got an answer like 0.017, does that mean I have 0.017 U/mL/minute or I need to convert that value with 0.001 to ge the Units .? I mean I have got 0.017 micro mole/mL/min concentration of the enzyme and the Units are 17Units. is that correct.?
Thank you so much sir.....but if we have volume in ml than is it important to change it. Sir will u please give reference for calculating enzyme activity. By beers lambert law
Does it help you to think of it as adding 2 uL of enzyme and 18 uL of water rather than 20 uL of enzyme? I've a video on dilutions that you might want to watch.
Sir, can u please help me. If I am taking 250 ul of total volume in a microplate reader. should I convert it into? .25ml instead of 250 ul while dividing it by 1000ml (eg. 4ml/1000 ml).
Please i have a question regarding the calculation of relative activity of enzyme over many days, for instance 5, 10 or 20 days. How to do this kind of calculation ???
That's a good question. For biochemists, "purity" is a difficult thing to measure as it implies 100% active protein and while it's relatively easy to determine that no other proteins are present, it's much harder to know if some of "your" protein is denatured, or if there are non-protein molecules present that interfere with the reaction. Normally we purify a protein until we can't get the specific activity any higher and nothing else is present on a SDS-PAGE gel (or Western blot) and leave it at that.
I think not because of dilution factor. since our goal is unit per mL, and the example given is 20uL enzyme, so I think we have to change to 1mL/1000uL
If you don't know the extinction coefficient you can't calculate the concentration of product so you can't calculate the specific activity in enzyme units (umol/min). You could calculate the activity in absorbance change per minute and leave it at that but this is not so useful.
@@guscameron9511 Hello and very nice explanation. To help Veronica, could you not calculate the concentration of the product from your standard curve if you have used for example 4 nitrophenol?
@@stephenaustin2524 Absolutely you can do, but using an extinction coefficient is just as good and less work. The only time an absorbance coefficient won't work is when the product of the reaction gives a non-linear absorbance response (absorbance does not increase linearly with concentration, it fails to obey the Beer-Lambert law), in which case you do need to run a standard curve every time, which is a pain.
Thanks Lisi, unfortunately I don't know if a good book just for these kind of calcs, most enzyme books are desgined for post-grads while books on general biochemical calcuations tend to leave bits out. If anyone knows of a good one I'de be interested to hear too! In the meantime, try searching for "Biochemical calculations" or "Maths for biologists" and see what you can turn up.
There's a few things to consider here. a) there's no such thing as a negative concentration, so are you sure it's not a mistake? On the other hand, b) you could be measuring the disappearance of a substrate rather then the formation of a product, and this would mean the absorbance would reduce over time. If it's the latter, you can calculate the enzyme rate as the disappearance of the substrate, which, and then, assuming you know the reaction stoichiometry, it's simple to convert this to the rate of product formation.
@@gus900111 thank you so much for your reply so my change in absorbance at the optimal wave length is -0.04 per minute how would I go around working out the energy activity here
@@saracirinesa7027 Ask yourself, what is the reaction catalysed by LDH? In which direction are you measuring it, lactate to pyruvate or pyruvate to lactate? Also note the units of your absorbance coefficient are not correct.
how can we convert absorbance into per min per mg protein if we do enzyme assay for polyphenol oxidase in leaves protocol- 1 gm of grounded leaf sample homogenized in 2 ml of 0.1 M sodium phosphate (pH 6.5) incubated in ice for 10 minutes and then centrifuged at 3900 rpm for 20 min at 4 degree C. A reaction mixture of 200 micro L of enzyme extract mixed with 1.5 ml of sodium phosphate buffer. A 200 micro L of 0.01 M catechol was added to start the reaction and the reaction and the enzyme activity was measured at 480 nm. Hope you will help me on this!!
katal/kg? That's an unusual unit to use. A katal is one mole of substrate formed per second. The more-commonly used enzyme unit is one micromole per minute, as above. To convert umol/min into mol/s divide by one million then divide by sixty ((umol into mol and minutes into seconds). Here I’ve given the specific activity as the activity per mg so to get it into kg you’d need to multiply by a million. 0.304 umol/min/mg = 3.04 x 10^-7 mol/min/mg = 5.07 x 10^-9 mol/sec/mg = 5.07 x 10^-3 mol/sec/kg
@@ghazalsafaei5148 Ah! Sorry, yes. That's because we used 20 uL of the enzyme but we want to express the activity per mL of enzyme. We used 20/1000 of a mL so the dilution factor is 1000/20, or 1/50th.
Hi Hymi, the proper answer is to consult the literature! Extinction coefficients are published in scientific journals. But as a student, they are normally provided by your instructor.
If you were correct it would mean you had 250 mL in a cuvette, almost a coke-can's worth, so that's definitely not right - there's only 4 mL, or 4/1000 of a litre
Hi Rohan (and many other viewers), the extinction coefficient varies depending on the reaction being measured. I'm using 4600 as an example. If you are measuring a different reaction you'll need to use a different number. Your teacher will normally supply it. If they don't you should consult the literature. Or Google, but as an academic I'm not supposed to say that ;-) All the best, Gus
![Enzyme Kinetics with Michaelis-Menten Curve | V, [s], Vmax, and Km Relationships](http://i.ytimg.com/vi/kmyR1cYxRL4/mqdefault.jpg)
That was the best, most detailed, extraordinary explanation of this confusing topic. My teachers at the university failed to be as precise as you. Thank you Sir!
For a second I thought that you were the "noice" guy meme.
Poets and scientists have much in common - we both reach for the stars, but admittedly Michael Rosen's grasp outreaches mine.
The very best to you, Gus
HAHHAHAHA I THOUGHT THAT TOO
The best and well explained video. It is the only video through which I can calculate antioxidant enzyme activity of plants. Thank you so much.
Clear, stepwise, and taught by example. Perfect.
Thanks for the feedback,
Gus
Thank you Sir. This has been the most helpful video on RUclips. I was so much stranded on how to go about this.
You're welcome, glad it was helpful. You'll find this kind of stuff it gets much easier with practice.
Thank you so much, I'm currently doing this in my research lab and really needed help.
forget about the enzymes....dude was mirror writing like it was nothing! Respect!
This is the only reasonable explanation I have found so far. Thank you so much for this video.
Nunca lo hago pero debo felicitarlo, excelente explicación. Me salvó de que me retaran en la práctica.....
So glad i found this video...it really helped me with my lab calculation
i am currently failing this man's class lol. He's an excellent lecturer....I just don't belong in biochem 😭
respect for being able to write the wrong directions. purely because of this is subscribed
It is a nice video. Thank you very much. I kindly ask you how did you calculate epsilon (molarity absorbance) here. you directly put 4600 L per mol per cm. Kindly explain. Thank you
Hii, do you get the explanation for that epsilon?
@@arrisyasyafina9586 it's a constant, normally we calculate it with a calibration graph using known concentrations
@@Alireza-fs3ur thank youu
While calculating the umol min-1 why the 4ml volume was considered instead of 1ml? I mean why should we consider the volume of buffer solution used to stop the reaction as the enzyme only acts on the substrate?
Really helped a lot for research calculation! 🙌
Thank you for such a precisely informed video. If I haven't understood it wrong, the change in absorbance here is the difference in absorbance at time = 0 sec and time = 5 mins?
did he... write in reverse for this. props ofc 👏👏
Thank you sir...really help me a lot! May god bless you
This is very helpful. Thank you so much for the video.
Can we calculate the Lipases activity by the use of this formula.
thanks for the very useful video. I have a doubt in calculations part of my experiment. I have got 0.006 OD per minute, after simplifying I have got an answer like 0.017, does that mean I have 0.017 U/mL/minute or I need to convert that value with 0.001 to ge the Units .?
I mean I have got 0.017 micro mole/mL/min concentration of the enzyme and the Units are 17Units. is that correct.?
Thank you so much sir.....but if we have volume in ml than is it important to change it.
Sir will u please give reference for calculating enzyme activity. By beers lambert law
Hi! How to can I calculate the Units of enzyme if I have a dilution of the enzyme (1:10), so I need to compensate for it somewhere?
Does it help you to think of it as adding 2 uL of enzyme and 18 uL of water rather than 20 uL of enzyme? I've a video on dilutions that you might want to watch.
Thank you very much sir. This is what I exactly needed right now.
This video helped a lot, thanks!
Your accent sounds quite different in these older videos, not that you asked.
Thank you so much, it was very helpful, great explanation
You're welcome, glad it helped.
Thank you for your video Sir
Thank you very much, very good video
Very helpful - thank you!
More than welcome, thanks for the feedback
Apakah setiap enzim dapat dihitung dengan cara yang sama?
Hello. How to turn activity 1: 10000 to U / mg? Thank you very much.
Sir, can u please help me. If I am taking 250 ul of total volume in a microplate reader. should I convert it into? .25ml instead of 250 ul while dividing it by 1000ml (eg. 4ml/1000 ml).
how to calculate the e ?
Please i have a question regarding the calculation of relative activity of enzyme over many days, for instance 5, 10 or 20 days. How to do this kind of calculation ???
Thank you!
Thank you for this sir! But how do i know if my enzyme is pure based on the its specific activity?
That's a good question. For biochemists, "purity" is a difficult thing to measure as it implies 100% active protein and while it's relatively easy to determine that no other proteins are present, it's much harder to know if some of "your" protein is denatured, or if there are non-protein molecules present that interfere with the reaction. Normally we purify a protein until we can't get the specific activity any higher and nothing else is present on a SDS-PAGE gel (or Western blot) and leave it at that.
@@guscameron9511 ohh i see. But given your example sir, was your protein/enzyme according to its sepcific activity pure or not?
@@Romil123 We don't have enough information to say one way or the other.
@@guscameron9511 I see. Still, thank you for the information sir! Your video is much appreciated.
the 1000ml/20ul is the concentration of the stock solution, where you multiply the diluted concentration by the dilution factor right?
I think not because of dilution factor. since our goal is unit per mL, and the example given is 20uL enzyme, so I think we have to change to 1mL/1000uL
I am following the protocol of a colorimetric assay to measure ALT activity but I am getting strange values 😭😭😭 I don't know what I'm doing wrong
If the extinction coefficient is unknown how do we go about solving for the specific activity of the enzyme?
If you don't know the extinction coefficient you can't calculate the concentration of product so you can't calculate the specific activity in enzyme units (umol/min). You could calculate the activity in absorbance change per minute and leave it at that but this is not so useful.
@@guscameron9511 Hello and very nice explanation. To help Veronica, could you not calculate the concentration of the product from your standard curve if you have used for example 4 nitrophenol?
@@stephenaustin2524 Absolutely you can do, but using an extinction coefficient is just as good and less work. The only time an absorbance coefficient won't work is when the product of the reaction gives a non-linear absorbance response (absorbance does not increase linearly with concentration, it fails to obey the Beer-Lambert law), in which case you do need to run a standard curve every time, which is a pain.
Thank you so much! Can somebody recommend to me a biochemistry/pharmacology/etc book that contains such calculations?
Thanks Lisi, unfortunately I don't know if a good book just for these kind of calcs, most enzyme books are desgined for post-grads while books on general biochemical calcuations tend to leave bits out. If anyone knows of a good one I'de be interested to hear too! In the meantime, try searching for "Biochemical calculations" or "Maths for biologists" and see what you can turn up.
My favorite is "Biochemical Calculations" by Segel
@@kathleenmcnamara-schroeder7290 thank you very much!
From where I ger exctniction coffient???
hello what if your change absorbance value is a minus
There's a few things to consider here. a) there's no such thing as a negative concentration, so are you sure it's not a mistake? On the other hand, b) you could be measuring the disappearance of a substrate rather then the formation of a product, and this would mean the absorbance would reduce over time. If it's the latter, you can calculate the enzyme rate as the disappearance of the substrate, which, and then, assuming you know the reaction stoichiometry, it's simple to convert this to the rate of product formation.
@@gus900111 thank you so much for your reply so my change in absorbance at the optimal wave length is -0.04 per minute how would I go around working out the energy activity here
@@saracirinesa7027 What's the reaction and the absorbance coefficients?
@@gus900111 the reaction is between LDH and two serum samples the molar coefficient is 6220M-1
@@saracirinesa7027 Ask yourself, what is the reaction catalysed by LDH? In which direction are you measuring it, lactate to pyruvate or pyruvate to lactate? Also note the units of your absorbance coefficient are not correct.
how can we convert absorbance into per min per mg protein if we do enzyme assay for polyphenol oxidase in leaves
protocol- 1 gm of grounded leaf sample homogenized in 2 ml of 0.1 M sodium phosphate (pH 6.5) incubated in ice for 10 minutes and then centrifuged at 3900 rpm for 20 min at 4 degree C. A reaction mixture of 200 micro L of enzyme extract mixed with 1.5 ml of sodium phosphate buffer. A 200 micro L of 0.01 M catechol was added to start the reaction and the reaction and the enzyme activity was measured at 480 nm. Hope you will help me on this!!
Thanks for alot this, though my confusion comes in the part of converting microlitres to ml ( u got 1000/20)ml aint it the other way round (20/1000)ml
its 20/1000 brother. i think you are right
Thankyou os much sir
Thank you so much
Please, how would you express that umol/min/mg into katal/kg ?
katal/kg? That's an unusual unit to use. A katal is one mole of substrate formed per second. The more-commonly used enzyme unit is one micromole per minute, as above. To convert umol/min into mol/s divide by one million then divide by sixty ((umol into mol and minutes into seconds). Here I’ve given the specific activity as the activity per mg so to get it into kg you’d need to multiply by a million.
0.304 umol/min/mg
= 3.04 x 10^-7 mol/min/mg
= 5.07 x 10^-9 mol/sec/mg
= 5.07 x 10^-3 mol/sec/kg
@@guscameron9511 Yeah. It's just that the question requires it to be changed
where does the 1000 ml come from?
1 litre = 1000 mL. Like many biomedical scientists, I tend to jump around between L, mL and uL (and between M, mM and uM). It's a good skill to have.
@@guscameron9511 yes but for the last step would you explain why you divide 1000 to 20 micro liter?
@@ghazalsafaei5148 Ah! Sorry, yes. That's because we used 20 uL of the enzyme but we want to express the activity per mL of enzyme. We used 20/1000 of a mL so the dilution factor is 1000/20, or 1/50th.
from I get extinction coefficient?
Hi Hymi, the proper answer is to consult the literature! Extinction coefficients are published in scientific journals. But as a student, they are normally provided by your instructor.
Well thank you!
You multiply 15.2* (4/1000)..but it is L-1 so i think it is 15.2*(1000/4)
If you were correct it would mean you had 250 mL in a cuvette, almost a coke-can's worth, so that's definitely not right - there's only 4 mL, or 4/1000 of a litre
Molar extinction coefficient of substrate is 4600? i have used a substrate named BANA..how to find molar extinction coeff fr tht?
Hi Rohan (and many other viewers), the extinction coefficient varies depending on the reaction being measured. I'm using 4600 as an example. If you are measuring a different reaction you'll need to use a different number. Your teacher will normally supply it. If they don't you should consult the literature. Or Google, but as an academic I'm not supposed to say that ;-)
All the best,
Gus
bad