The data are somewhat noisy, but there is a clearly a dependency of the reaction rate on the substrate concentration, and there is some evidence of saturation. With some improvement in reproducibility, you should be able to use these data for M-M kinetics. Try doing every measurement in triplicate and taking the averages of the triplicates. You may have to go to some higher substrate concentrations to see the saturation.

Dear Adam

Thanks for your valuable information. You said "Try doing every measurement in triplicate and taking the averages of the triplicates." I have done three trials for each concentration. Do you mean that?

Yes. Did you try averaging the results of the three trials?

yes I did Adam. I have included that in the document. It is called average of three trials. It is in the last. Thanks.

Dear Rony,

there is to small amount of points at hieger substrate concentration. And they are very very noisy. Can you not done much more experiments?

This last curve must be fitted to Hill rate equation, but not Michaelsi one. It doesnt look to hiperbolic curve but fore sigmoidal one. You obtain K0.5, kcat=Vmax/[E] and Hill copperaytivity coefficient h. If h=1, it means thta it is michaelsian kinetics. If h.1 the positive coopeartivity is observed and homotropic activation by substrate itself.

"In order to determine the kinetic constants (K0.5, kcat and h) the Hill equation (1) was fitted to the experimental data with EZ-Fit (Perrella Scientific, Inc.; http://www.JLC.net/~fperrell [35] or Sigma Plot (Jandel Scientific) software:

                                        vo = kcat [E] [So] h  / K0.5 h  + [So] h                         (1)

where: vo is the initial reaction rate, [E]o and [S]o – the initial concentration of enzyme and of substrate, h is the Hill cooperativity coefficient, kcat is the turnover number and K0.5 is the half saturation constant."

I attach my papers.

ewa luchter-wasylewska

I have done another kinetics. Here are the results. It looks like michaelis Menten curve now but suddenly at higher concentration the absorbance goes down.I am attaching the file. Please suggest. Thanks guys.

Rony,

Really, the velocity goes down at higher substrate concentration. But:

1. at low substrate concentration the curve is NOT HYPERBOLIC but sigmoiodal!

2. there can be several reasons for the last part of the curve:

a. nonlinearity of the "calibration curve", a line showing dpendence of the absorbance versus the product concentration that MUST be done BEFORE starting with the experiments at all.

IF that line is not straightt, you MUST find such product concentration range where it is really straight and thus you can and must determine the molar absorption coefficient to be able to use the Lambert Beer law and this coeffcient epsilon must be constant over the concentration range used in teh experiments..

Each method has the concentration range where it can be used, where the line is straight what means that epsilon is constant.

b. substrate inhibition

c. are you not chemist? maybe any can say you what I have told.

Rony,

3. I see one more reason.

I hope on x-axis do you have the initial concentration of the substrate, [S]o.

What do you have on y-axis??????????? Maybe absorbance of something. It is totally unproper! There MUST be the initial reaction rate, vo, in moles/l of product per 1 scond (M s-1) obtained from the tangent(ial) straight line to the kinetic line, calculated mathematically from the soecial formula.

How have you got  tha "number" on the y-axis? 

If you have measured something in one-point way, it is unproper, and it can be reason of such your results.

Ifo so please study how looks the absorbance-time (kinetic) curve for the 1-st order chemical reaction and how to calculate the initial reaction rate, vo!!!!!!!

Mostly people with biological. medical or so on education, but not chemist and physicist, do not know it and they MUST have problems with the kinetic curves, with calculation of vo, with fitting such results to Hill equation.

enzyme-substrate saturation plot and MUST have problems with using the mathematical fitting of the saturation curve by using the Hill rate equation, but never withMichaelsi-Menten equation.

I can say m ore if you inform me wher your probelm is.

elw

Hi Ewa 

the x axis contains substrate concentration which is my/mL because the substrate is chondroitin sulfate A and it doesn't have any proper molecular weight. The y axis is the rate of reaction. So, I plotted absorbance vs time for each concentration and found out the slope. That slope is the value for y axis. Thanks.

1. have you  done calibration curve for the product? please show it.

2. have you found it to be linear in your absorbance range? please show it to me. 

3. have you calculated molar absorption coefficient (epsilon, M -1 cm-1) for the product, in your product concentration range?

4. have you given that epsilon to us? please give me.

5. how have you calculated the initial reaction rate of the1st reaction order?

6. which mathematical formula have you used? please give me.

7. do you have any program to fit to Hill rate equation: ezfit , simfit, sigmaplot or others?

elw

How do i do calibration curve? Can you explain please?

I am looking which reasons are for such a curve:

1. the curve is such, and it is nothing to do, only you must somehow interprete it.

2. there are seveal different mistakes/errors during the experiments.

3. i have given you several questions and you have not answered me carefully point after a point.

4. instead you have answered me giving me question. it is not very polite method to discuss.

5. the calibration cyrve is a straight line: on x axis you have your product concentration and on y-axis you have a measureble quantity as for example absorbance. Or you can say it is a practical LAmbert-Beer law curve. It must be done, but not must be shown, to be sure if you CAN MEASURE what you want to measure.

6. please take several tubes with your reaction product and measure it if it is measurabel or perform the chemical provedure as always and measure. please plot the curve by the mathematical methods and calculaet epsilon (A=epsilon x molar concentration x length of the spectrophotometric cell.

7. if it is straight between "0" and the value "z" it means that the method can be used only in that range. Outside of teh range where the line is not straight the method CAN NOT BE USED.  It could be one of several reasons of your such saturation (vo/[S]o) curve.

8. if you not calculate the initial reaction rate (vo) ,your "y" axis is not proper.

9. you must know a bit of chemistry to use chemical methods in biology. most biologists unfortunately do not know chemistry.

elw

please answer my question. if not I will write nothing more.

the last: you ought perform the same experiment using another enzyme concentration, and maybe that problem will be omitted, when a less amount of enyzme will produce less amount of the product.

elw

Are you sure you measured the initial rate at the higher substrate concentrations? This is a crucial point.

If so, then you appear to have a case of very severe substrate inhibition.

How pure is your chondroitin sulfate. Is it possible that it contains an inhibitor of the reaction? Inhibitory impurities can masquerade as substrate inhibition.

Another potential artifact, if you are doing an absorbance assay, is when the absorbance is too high to measure by the spectrophotometer. If the absorbance starts in this range due to high background from the substrate, there will be no detectable increase afterwards, making it appear that the rate is zero. You must make sure that the absorbance starts and ends in the linear range of detection of the spectrophotometer.

If the apparent substrate inhibition is real, you can try to fit your data to the rate equation for substrate inhibition, which is v = Vmax[S]/{Km + [S](1 + [S]/Ki)} where Ki is the substrate inhibition constant.

This does not take into account the possible sigmoidal kinetics discussed by Ewa luchter-wasylewska.

Hi Adam

what do you mean by measuring initial rate at higher substrate concentration?

do you understand two different phrases: reaction rate and initial reaction rate?

start with the chemistry: definition of the rate  and of the initial rate of 0-order reaction, of 1-st order reaction and so on.

the diagram of the kinetic curve of the 1-st order reaction is not a straight line. therefore you can not read absorbance after for example 1 second but you must calculate mathematically the initial rate using special formulas. if not you are not reading the initial rate of teh reaction and you can have for example 70% of error. 

enzyme kinetics is a matter of chemistry and not of biology and you must learn chemistry, a chapter on teh initial reaction rate calculation of the 1-st order reactions.

the another error may come from the epsilon (above) - chapter spectroscopy in physical chemistry.

elw

Hi Ewa luchter-wasylewska

First of all, I don't have very good knowledge about enzyme kinetics. I am just doing preliminary work. I have taken different concentration of the substrate and took the absorbance readings. Then i found out the slopes which are rates and plotted that which was rates vs concentration. I just wanted to see if that fits the michaelis menten  curve or not. That's the only thing I am doing now. 

The measurement of initial rates is a crucial aspect for steady-state kinetics. If you measure the amount of product formed as a function of time and plot a graph ( a progress curve), you will see the reaction gradually slows down with time. This is because the concentration of the substrate decreases as the enzyme catalyzes its conversion to product. (The accumulating product can also act as an inhibitor in some cases).

The rate of the reaction is related to the substrate concentration according to the Michaelis-Menten equation v = Vmax[S]/(Km + [S]). Unless the substrate concentration [S] is much higher than the constant Km, the rate v decreases noticeably as [S] decreases.

Notice that there is no [P], the concentration of product, in this equation. The theory assumes that there is no product. This can only be approximately true very early in the reaction before much product has accumulated.

Also, the theory assumes that the substrate concentration is equal to the concentration that you put in. This is also only true (approximately) very early in the reaction. For these reasons the rate must be measured early in the reaction. It is often stated, as a rule of thumb, that the rate should be measured before more than 10% of the substrate has been consumed.

So how do you actually measure the initial rate? Collect measurements of product concentration as a function of time. Draw a line through this progress curve that goes through the origin and is tangent to the progress curve. The slope of the line is the initial rate. If the progress curve is rising very steeply, there may not be enough data at early time points to measure the initial rate accurately. In such a case, you should redo the measurement with a lower enzyme concentration.

Suppose you have too much enzyme in the reaction that is monitored by absorbance, and the absorbance exceeds the detection range of the spectrophotometer by the time you make the first measurement. Or, the baseline absorbance may be too high because of high background absorbance from the substrate. For example, that might occur at an absorbance of 3 (It depends on the instrument). All of the measurements will be 3. If you measure the slope of the progress curve, it will be zero, and you will think there was no enzyme activity. This will be incorrect. You simply have not succeeded in measuring the enzyme activity at all.

you can not publish such a curve, before you learn on enzyme kinetics, on reaction steady-state kinetics, on spectrophotometry in the physical chemistry and so on.

why do you ask before being informed on 1-ste level.

elw

Thanks Adam. I appreciate your reply. 

Dear Rony Eshaque

I guess that you are a science student at Wilfrid Laurier University. This means that you should be trained to solve problems. Your question shows that you are studying the activity of the enzyme - chondroitin sulfate A lyase with the EC-number 4.2.2.5 or 4.2.2.20 or 4.2.2.21.

However you don´t state the problem you want to solve. Also the figures you present are not well defined i.e. difficult to understand. What units have the y- and x-axis? Enzyme kinetics is not a problem in itself, but knowledge of enzyme kinetics is required when you study the activity of an enzyme. This is extensively covered in textbooks on enzyme kinetics that are available in your university library (for instance the excellent books by Bisswanger and Cornish-Bowden and others). Before you continue to work with your enzyme you should consult these books, and consult the teachers in biochemistry at your university. Then you could formulate the problem you want to study with this enzyme (determine: the enzyme kinetic parameters turnover number kcat Michaelis Menten constant Km, their pH and temperature dependence, the pH or temperature stability of the enzyme or another property). As this enzyme has been studied by others, you should also inform yourself if the same or similar problem already has been studied for this enzyme. An extensive source of information for the enzyme here is the comprehensive enzyme information system

http://www.brenda-enzymes.org.

I am astonished that none of the previous answers have given you these hints that are essential steps when you start to study a scientific problem.