Questions related to Enzymology
i am new to enzymology and want to study the discipline through history based books (i,e, how was the Kreb's cycle discovered, methods used then, etc). what are your suggestions to achieve this?
better to be from wiley or springer.
Can alpha glucosidase and alpha amylase work at room temperature and at 37*C? If so why? The specifications for these enzymes are that they work at 37*C. But number of papers have modified their protocols 25*C. How can it work at both room temperature and at 37*C?
I am working in laccase enzyme production by bacteria. In that i have a problem with quantitative estimation of laccase enzyme. I have confirmed the laccase production with plate assay using guaiacol. But i face the problem with quantitative method of laccase enzyme. So kindly suggest me some tips and methods for the laccase enzyme assay. Thanks in advance:)
I tried docking my protein heterodimer to form a hexamer (trimer of a heterodimer) using symmdock server. The output results has given top 20 models of the hexamer and I opened the top model into PyMOL. I see the the parental template has intact structure while the generated partner structures are all broken in the PyMol (Picture attached). When I opened it in coot all the atoms are present and display very well as atoms. But somehow it is messed up with the cartoon representation in PyMol. Please suggest, how can I fix this issue.
I need to characterize two extra cellular enzyme produced by bacteria . For that process the purification of enzyme step is necessary before the characterization process? otherwise can i use the crude enzyme extract (cell free Supernatant) for the characterization process?
Acetate kinases are proposed to exhibit either a "direct in-line" mechanism or a "triple-displacement" mechanism. However, I was unable to find a schematic for any of these mechanisms, even less their respective kinetic treatments to derive rate equations.
Can anyone suggest a reference containing possible rate equations for this case, or at least a clear description of the proposed mechanism so I can try to derive the equation(s) myself?
I appreciate any help you may provide. 😊
Our primary experiment results indicated that food resource arabinogalactan protein (AGP) could combine with phenolic compounds, such as epigallocatechin gallate (EGCG), thus can mask bitter or astrigent taste and function as a good carrier of these bioactive components in food processing. As a kind of proteoglycan, we are planning to modify its sugar moiety and to test whether the sugar moiety take key function in combination with EGCG. Does anyone have experience in the enzymatic treatment of AGP or proteoglycan based on sugar chains?
Hope you all doing well. I would like to bring my concern during kinetics measurement of an exonuclease enzyme. I am performing an exonuclease assay, by varying substrate concentration (25 nM to 500 nM) and fixing the enzyme concentration (2 nM) and I could see the cleavage products very clearly till 300 nM in a linear range. After 300 nM substrate the enzyme struggles to cleave the product as it is attaining the supersaturation. Now, I have a concern that should I also change the concentration of enzyme during this kinetic measurement ? And if I change the concentration HOW and WHEN I need to divide the enzyme concentration in the reaction mixture, during the kinetic parameter calculation ? Because I am little bit concern about the calculation for adjusting the enzyme concentration. Please suggest.
With kind regards
Attached is the image of expression profile of my protein of interest. Starting from the left after molecular weight marker, are total cell lysate (uninduced), total cell lysate (induced), supernatant (Uninduced) and supernatant (induced). IPTG used for the induction was 1 mM. The expression system used was BL21 DE3. Here, the prominent band is in the right range of expected molecular weight. But I am worried I see almost identical expression in both uninduced and induced. Western blot could answer definitely if it is my protein of interest ( need to be done). But is it possible with normal BL21 DE3 cells ? Please give your insights into it.
With kind regards
Hello, I am intending to screen a library of compounds for an in vitro enzymatic assay. The target enzyme follows a Bi-Bi mechanism. The assay development phase is completed with kinetics parameters for both substrates are determined. I am looking into a rational approach to choose the concentration of both substrates and compounds to satisfy the following criteria:
-Inhibition Mechanism-blind design: the mode of inhibition and the substrate of which the inhibitor compete with is unknown.
-Minimize the concentration of the compounds to a reasonable concentration that still able to pick up any possible inhibitory mechanism.
Successful compounds will be used to identify Hits with dose response and IC50 later on.
Is there any recommendations/guideline followed in pharma to deal with this? is there any upper limit for a Hit to be accepted?
I read this report that 3% (v/v) Ethanol increases recombinant protein expression in Escherichia coli. The SDS PAGE gels seem pretty convincing (1).
However, assuming I get great expression of recombinant protein with 3% (v/v) Ethanol, how am I supposed to decontaminate the biohazardous cell waste from the experiment?
Bleach decontamination isn't a good idea because bleach would react with the ethanol.
Autoclave decontamination isn't a good idea because ethanol is flammable.
Is safety just not a priority?
1) Chhetri G, Kalita P, Tripathi T. An efficient protocol to enhance recombinant protein expression using ethanol in Escherichia coli. MethodsX. 2015;2:385-391. Published 2015 Oct 8. doi:10.1016/j.mex.2015.09.005
How to measure the percentage activity of LDH (0.5 ug/ml) using 2 mM of NADH and 10 mM of pyruvate ( 25 °C, pH 7.00 ) and observing oxidation of NADH at 340 nm? Termination of reaction is necessary while measuring the activity?
I am looking for Methods in Enzymology, Volume 22, 1971, Pages 248-252. Specifically, chapter 23: Crystallization as a purification technique.
Alternatively, the following: "A technique for the crystallization of proteins" by William B. Jakoby.
My institution no longer has access to older articles, and I am really interested in the method of crystallization.
I need to calculate Vo in order to than calculate Vmax and Km, I know you can calculate it from absorvance but I don't have that. Is there another way? Can I calculate it using the attached diagram?
I want to calculate an enzyme's energy of activation, but I don't have the enzyme's molecular weight to calculate the rate constant or the pre-exponential factor A. This is part of an assignement I have for a virtual lab of enzymology, we use this virtual lab https://www.ucl.ac.uk/~ucbcdab/enzass/enzymass.htm by ucl and the process of it is basically you choose one of 5 enzymes which are fictional enzymes from what I can tell, and then you can perform "expirements" to determine the enzyme's behaviour in different pH environements etc.
I've attached screenshots of the diagrams the virtual lab has produced about how the enzyme reacts to different temperatures. I've concluded that at around 50°C it produces the maximum amount of protein so I've calcuted the maximum velocity using that at 5.86μmol/min. I had to calculate this because since I don't know anything about the actual enzyme I have to calculate the energy of activation using this equation: logk = logA - E / 2.303 * R * T (where logk=logVmax), our professor told us that we have to use this to calculate the energy. She also told us to use excel to calculate logk and logA and create a y=ax+b diagram. I honestly am really struggling to come up with how to use excel in order to do that since I'm really unexperienced with it.
Please refer to the photos below if you want to see the data I have.
I am a total newbie in enzymology as I started working on this very field couple of months ago. I am working on a nuclease which degrades oligomers of nucleotide from 3' end. Now, I have a setup for the enzymatic assay where I stop the reaction at different time points using a stopping buffer. The oligo substrate is fluorophore labelled and degradation of which can easily be monitored using phosphor-imager that gives the intensity of the substrate degradation or the product formation. My question here is, what will be my approach to calculate the initial velocity, Km and Vmax of the very enzyme using this assay ? Actually, I am looking for calculating those parameters with substrate change (i.e using the top band from each lane) and not with the degraded products formation as it seems complicated to me. Because each nucleotide degradation forms a product w.r.t time and so several products making it complicated. So my choice will be to see how much substrate degraded with time. I have attached the Urea PAGE profile of the assay, where starting from left, lane 1 to lane 5 is is time t=0 min, 10 min, 15 min, 20 min and 30 min respectively. Please help me in this regard.
Hi to all I immobilized a peroxidase on a new synthesized MOF. The reviewer of a journal paper asked me to consider the inhibitory activity and specific affinity of my immobilized enzyme in the introduction. How I can answer this comment of the reviewer for a journal paper revision? What does it mean? In my opinion, these items are related to biosensors. I reported the influence of immobilization on km in my article but I have no idea about the inhibitory activity.
I want to know normal value of Superoxide dismutase and glutathione peroxidase and reduced glutathione and malinoaldehyde in tissue of rat.
Among various factors, the temperature is one of the most crucial factor for enzyme activity. For most of the enzyme assay, researcher use 37oC or normal room temperature as incubation temperature. If we consider the poikilothermic animals like fish, it's natural enzyme activity depends on habitat temperature and each fish (tropical, temperate, polar or cold water species) has their optimum temperature at which it grows best and we know growth is nothing but a consequence of optimum (good) metabolism.
So, is it right to use the mentioned assay temperature for enzyme activity of fish irrespective of its natural habitat?
Do the researchers need to modify the assay temperature according to optimum natural habitat temperature of fish?
Please provide your suggestions.
It is very much confusing when some papers use this term "apparent Km of an enzyme even if they use single substrate ? Not at all clear ! Please help me understanding this. Thanks in advance.
I apologize if my question is not clear. I have a pure chemistry background and I am very new in a Protein Chemistry and Enzymology science.
Hello fellow scientists,
I wish to determine the Dissociation Constant (KD) of a DNA polymerase binding dsDNA. I won't disclose what the DNA polymerase is because it is unpublished work. I have done some binding assays in Agarose gels, but due to the poor sensitivity of the available dyes I had to visualize the relative binding stoichiometrically, and I could not simply just set the protein or DNA concentration around the expected KD.
Previous work in our lab has determined a KD = 20 nm for our DNA polymerase binding a 33mer locked double stranded DNA hairpin.The purpose of using something so complicated was for kinetics assays.
However, I am using a 13-mer dsDNA construct because my goal is to crystallize the DNA complex and a 33-mer is just way too large! My supervisor has advised that I don't believe that my KD is actually 20 nM for my small dsDNA construct.
I am interested in using Isothermal Titration Calorimetry mainly to calculate the KD of my protein to binding this 13mer dsDNA construct. I would titrate my dsDNA into a fixed concentration of protein. I could guess that the KD is 20 nM, but I actually don't know for sure.
I have heard that when you determine the KD you have to have some estimate of the KD and then scan ligand concentrations above and below the KD, measure the response to get a curve of response vs ligand concentration and the KD is mathematically fit or basically it is just the inflection point of the binding curve.
However that advice doesn't tell me if the KD is say 20 nM, what should fixed concentration of my protein be? (I have appreciable amounts of 100 µM protein because I am a crystallographer so excessive protein isn't an issue.). What is the max and min range that I should scan the ligand concentrations? What if the KD is way worse than we predicted and it is actually 1 µM? What fixed concentration of protein should I use and what min and max concentrations of ligand should I use?
Is there a way that I can measure the KD with a certain fixed concentration of protein, and a huge range of ligand concentrations regardless of if the KD is 20 nM or 1 µM? Is that possible?
I am working with cellulases, I have expressed my cellulase enzyme in pET28a expression vector. The bacterial lysate has activity against cellulose substrate but after purification of protein with his-tag using GE 1ml His-trap column, my enzyme does not have activity. I elute my protein with 250mM imidazole, but I have dialysed my sample and still i could not find activity. I need some valuable suggestion.
I do like Cell, Nature, & Science. I find the discoveries to be amazing. But the experiments can be difficult to understand and difficult to replicate without the state of the art scientific instruments. So I have been frequently reading the volumes of Methods in Molecular Biology and Methods in Enzymology. The reason is because these journals give reproducible experiments with simple explanations.
Apologies for an off topic. I am curious to know if someone wants to pursue a second PhD in order to switch into a closely related field from his/her first PhD, would that be considered as post PhD experience ? The person may want to get into such situation when he/she wants to get trained into that field as most of lab doesn't consider if that background is lacking. It is the matter of interest of the candidate to move into that closely related field. Please give your inputs and suggestions in this regard. Thank you in advance.
I’m working with a rather complicated ping pong, bi-bi double displacement reductase enzyme that oxidizes NADH and then reduces a substrate. Interestingly, the enzyme has intrinsic NADH oxidase activity in the absence of the substrate. Of course, upon addition of substrate, the velocity of the reaction increases and NADH is consumed more rapidly. The intrinsic activity introduces a baseline velocity and is complicating our kinetic analyses a bit. Complicating it even further is a noted double substrate inhibition pattern (apparently not uncommon with ping-pong mechanism enzymes). We’ve run a full course of analyses, varying the NADH as well as the substrate and we're now trying to fit the data to an appropriate equation. My question is: How do I take the ‘intrinsic activity’ of the enzyme into account (if I need to at all)? Can anyone recommend an equation and appropriate software that can do this? We currently have SigmaPlot, GraphPad, and Enzfitter (for the double substrate inhibition model). Thanks in advance for any guidance, ideas, and/or discussion.
Hi dear researchers
We used HRP (Biobasic) in phosphate buffer (pH: 6.5) and after adding TMB, the reaction turning to light blue without adding H2O2. After adding H2O2 to reaction media, the brown color was produced (not expected blue color).
There were no changes after decreasing TMB, HRP and H2O2 concentration.
We expect that after adding TMB to the enzyme without H2O2, the reaction doesn’t start and after adding H2O2, we expect that see blue color.
( I have checked and rechecked all calculations, buffer composition, pH etc. )
I want to compare the amount of an enzyme's expression (laccase for example) or presence in the tissue as a result of the experimental treatment. Is there a protein similar to an antibody that might bind with the enzyme and can be filtered then quantified by spectroscopy? or perhaps by comparison of PCR bands. I am interested to hear any other thoughts or suggestions as well. Thanks very much.
Dear All, I am purifying my protein (theoretical PI = 8.9) in a buffer of pH =7.1. till Ni-elution I kept the DTT concentration (1mM). Usually, after elution people used to keep DTT concentration at 5mM. But in my until Ni-elution the protein look absolutely fine. But the moment I add DTT (5mM) thread like aggregate is formed. when I run those aggregate, it shows up as my protein. What could be the possibilities that DTT is making it precipitated. As I know DTT is used to solubilize protein as reducing disulfide bonds. I will appreciate your suggestions.
With kind regards
I am trying to plan an pulldown assay to find some prey protein for my bait protein.
I am interested in using the flag tag because it is small and doesn't disturb structure and doesn't require additional steps to tag your protein like would be necessary with biotinylation.
Plus apparently, the bait-prey complex can be eluted with Flag peptide.
So that is very interesting to me.
However, I haven't been able to figure out how strong Flag-Tag binding is to Anti-flag antibody?
Also, does anyone have a simple protocol for Flag-tag pulldown?
Flag Tag = DYKDDDDK
I have recently started working on improvement of wheat flour for baking. I have come across a research which involved the usage of xylanase, cellulase, beta-glucanase and carbohydrases for improving baking results. I wanted to experiment the same procedure. Since i don't have any background with enzymology i cannot understand how to calculate the dosage that needs to be added to the wheat. If someone can help me in figuring out how to calculate the dosage. I am attaching the link of the article.
I am going to look PPO and POX enzyme activity in fruits. I am getting confuse to make a standard curve. Which reference substances should be taken while making a calibration curve for PPO and POX activity?
I am using catechol as a substrate for PPO enzyme activity and guaiacol as a substrate for POX enzyme activity. I am following this article for the procedure. Or is there any other protocol ?
I want to calculate enzyme activity in the form of 1umol/min rather than on protein basis.
I am quite new in enzymology
Looking forward to your kind suggestions.
Thanking you in advance
Currently I am working with alpha amylase assay using DNS method but I have a problem with the colour which is not changing even I used high concentration from the phenolic standards (Trolox and Gallic acid) and I don't know what is the problem.
I used different concentrations from enzyme (0.1 - 1 U/ml) with soluble starch 1% (w/v) but the colour of samples very close from the control (orange-red colour). In addition, the absorbance of standards or samples are higher than the control, and the absorbance increases with increasing the concentration of standard whereas it should decrease with increase the concentration of standard or sample. As a blank, I tested starch with DNS, the enzyme with DNS and different concentrations of standard with starch and DNS, but I didn't get changes in the colour.
The protocol that I used is:
100µL of buffer or sample + 100 µL enzyme (procine pancreatic alpha amylase) then mixture incubated for 10 min at 25°C, add 100 µL of starch 1%, then mixture incubated for 10 min at 25°C, after that 200 µL of DNS (1%) was add to terminate the reaction and the mixture was incubated for 5 min at 100 °C, cooled to room temperature and then solution was diluted by 3 ml of H2O. The absorbance was recorded at 540 nm.
Can anyone tell me what the problem is or suggest what I can do to solve this problem.
Thanks in advance
I am using KinTek software to do a kinetic fitting. I have determined kinetics of the formations of several species in the time course of 5min and established a model for the enzyme (just as an example: A = B = C = D). I already know that B is always below the detection limit of the instrument during the reaction. Is there a way to restrict the amount of B to lower than the detection limit (such as 0.01 uM) in the software? I only find restrictions for kinetic constants and the starting concentration for species. If there is not a way, is it appropriate to import a sudo-data set (such as a constant amount of 0.005 uM B at each time point, with a sigma value of 0.005) for fitting?
Hi dear researchers.
I have a mixture of nanoparticles and hemin and buffers, …
It was centrifuged and washed several times.
Is there any way to calculate the residual hemin concentration?
( There are not any biological samples)
I am trying to check the interaction between two of my proteins of interest. However before trying any biophysical technique such as SPR/ITC/MST etc, I used normal superdex-200 gel filtration chromatography. I used protein A (Receptor; ~40 kDa) and protein B (Ligand; ~31kDa) in purified form and mixed together with 1:2 molar ratio at temp. 25 degree for 1 hour. Now when I performed Superdex-200 with three individual runs (Ligand, Receptor and Complex). each time I get a surprising result wherein the complex has more retention volume than the Receptor it self (attached result brown: complex, cyan: receptor and blue: ligand).
When I run the peak fractions from each run through SDS PAGE, I get the two different protein bands in the complex run (attached result; lane 1-3 is the Ligand and Lane 4-6 Receptor and lane7-9 Complex peak fractions). Please suggest what could be the possibility of such result. The complex formation shows about 1 degree (57 to 58 degree C) rise in melting temperature every time as compare to their individual ones. Buffer used for the protein are same 1X TBST pH=7.6.
Hi dear researchers
Is it possible that observed increase of Km for one substrate and decrease for another one (for enzymes with two substrates such as HRP) after immobilization?
I have a doubt in calculating the activity of immobilized cellulase i've used the formula Activity of cellulase (μmol/ml min) = 1000 w/Mvt; where, w is the amount of glucose produced, M is the molecular weight of glucose, v is the volume of
the sample and t is the reaction time. Here in the formula instead of ml (i.e volume) i have used grams (g) of beads taken, then the unit for cellulase would be (μmol/g min). The way i am calculating the activity is correct?
I am searching the pGex or pET plazid of HDAC6, if he is possibly for somebody and would give for me, would be needed for my work.
I would like to test the interaction
between my labor's new protein and HDAC6. If somebody is willing to provide me with this plasmids please send to me at Dr. Tibor Szénási Institute of Enzymology,
Research Centre for Natural Sciences, Hungarian Academy of Sciences
1117, Budapest, Magyar tudósok körútja 2.
Fedex number: 317 080 212
There are many research articles exploring alpha glucosidase, salivary and pancreatic alpha amylase inhibition as a therapeutic target for reducing postprandial hyperglycemia (PPHG) in type 2 diabetes. Out of these three, which one is more important/superior and why?
I determine optimal pH and temperature of immobilized enzyme activity. I want to know how pre-incubated it at different pH for pH stability factor.
I'm doing an enzyme assay, but I'm having trouble stopping the reaction so that I can get proper kinetic data.
I can't heat because the substrates are unstable.
I tried filtering, but it doesn't seem to stop the reaction completely.
I can't use acid/TCA precipitation because it will mess up my analytical equipment.
Are there any other ways of easily, and completely, stopping an enzyme assay?
I used NOBA, a chromogenic substrate for phospholipases, to measure (for 120 min) the absorbance (at 405nm) of PLA2 in crude venoms from different snake species. Later, I plot the absorbance versus time curve. As far as I know, I would need the exact concentration of the PLA2 present in each venom, but I don't have that information. Is there another way to calculate each PLA2 activity?
Technically, the absorbance versus time gives me already a clue, but I tested several venoms and the more I add the less "beautiful" my graph becomes so that's why I was hoping to calculate the PLA2 activity and plot it as a histogram to make it more visible.
When titrating a high affinity ligand I am getting Kd values that are very close to the total protein concentration in the system. My understanding on this matter is that, under such conditions, you cannot ignore ligand depletion and assume that your total ligand concentration is equal to the free concentration. My understanding is that a better way to analyse my data is to either decrease the total protein concentration to a value 5-10X below the Kd and repeat the experiment, but I have two questions: firstly, under these conditions, can I assume that my system has reached equilibrium within my assay time frame and get a reliable Kd? And second, would the Briggs-Haldane quadratic equation be a better way to get a reliable Kd under my current conditions (protein concentration close to Kd) without having to change my experimental set up??
I'm designing an assay to test for release of pyrophosphate due to dihydropteroate synthase-like enzyme activity using malachite green reagents. One of my substrates is enzymatically catalyzed and one of the byproducts of this step is pyrophosphate; how can I eliminate the pyrophosphate background, because up until now I have been encountering a giant phosphate wall and the absorbance readings have been way off scale?
EC 126.96.36.199 is the accession number of CYP1B1 enzyme. I know that the first number '1' refers to the major class of the enzyme catalysis reaction, I want to understand the meaning of the rest of the numbers in this enzyme accession number.
I am running some QMMM jobs for finding the transition state and later on I want to do the IRC calculation if i can find the transition state.For me it is now a big problem to locate the transition state and to do the frequency calculation.The system has two layers.High layer I mean the quantum part contains the Adenosyl cobalamin cofactor and the low layer contains EAL enzyme.So the high layer contains 180 atoms which is treated by QM and the low layer contains 10000 atoms which is treated by MM.I am not sure is it possible to do the IRC calculation using gaussain for this type of big system.If anyone can give me some suggestions it would be a great help.
Reactions are carried out in 50 mM Tris–HCl, pH 7.5, 5 mM MgCl2 and 100 mM NaCl using 2 µM cysteine desulfurase (Nfs) in presence or not of 2 µM SufE1. BSA 2 µM can be used as control. In addition to enzymes, pyridoxal 5′-phosphate (10 μM) and DTT (1 mM) are used for all reactions. Reaction is initiated by adding 500 μM L-cysteine for 30 min at 25°C. Then the reaction is quenched by adding 50 μl of 20 mM N,N-dimethyl-p-phenylenediamine dihydrochloride (prepared in 7.2 M HCl). The addition of 50 μl of 30 mM FeCl3 (prepared in 1.2 M HCl), followed by incubation for 20 min leads to formation of methylene blue, which is then measured at 670 nm. Na2S (1–100 μM) is used for calibration?
I know the substrate amount ( 5 different concentrations). Absorbance taken for 0 to 60 minute, rate of 1 min for total 61 readings. Enzyme amount was constant.
While checking the effect of inhibitors on protease enzyme it was found that iodoacetate instead of inhibiting the protease activity it has enhanced the protease activity. I searched alot but could't find the possible reason for this. Can anyone explain what could be the possible reason for the same. Thank you.
Kynureninase is an interesting enzyme involved in many diseases such as schizophrenia and viral diseases, is these a synthetic ligand that can be used to compare the activity of certain compounds?
As a collaboration request: Where can I perform the test against this enzyme?
I want to interpolate the amount of product formed (as concentration or % of conversion) vs reaction time in a biocatalysis process. The fitting equation should have as (y) the amount of product and as (x) the reaction time. I thought to use as the fitting equation the integrated form of the M&M but I am not able to find the correct mathematical form. Or should I use another equation?
i have the structure of unbound protein, ligand and protein -ligand complex?
Lignocellulose is a renewable biomass which is widely available in nature. It contains cellulose (glucose), hemicellulose (xylose, arabinose, mannose etc.) and lignin. How one can analyse a perticular lignocellulosic (wheatbran) biomass to quantify the above to detect the proportion of each of them. Is there any specific and simple methods are available to analyse. please let me know.
Biofuels, bioenergy, carbohydrates, redusing sugars, biocatalysis, enzymology, sustainable biofuels, renewable bioenergy, bioethanol, biotechnology, pretreatment
I am getting Km value of a mutant which is similar to its wild type. How it is possible ? The residue is quite important for substrate binding. Is it possible ? please suggest !
I want to characterise by immunohistochemistry fibronectin, collagen I and III on a biologic matrix but the heat unmasking scrap my tissue. There for, I am in the need to a gentle and efficient unmasking method to preserve my tissue and reveal these proteins in it.
Any suggestion would be helpfull.
I have data from 2 different substracts (in 0 to 100 mg/mL) for the same enzyme at its optimum pH, temperature and time (all of them had a 20 min reaction).
The fact that this data is not related with time, makes it impossible for me to do kinetics (absence of vo).
Is it possible to compare this data in other way?
I'm a student in enzymology and I would appreciate any kind of help.
I am performing an enzyme-inhibitor kinetics experiment with an incresing concentration of substrate and inhibitor. A few years ago, we used to follow a protocol which uses Lineweaver Burk equation ( a double reciprocal plot). But, recently, I am coming across a few research articles saying such linearization of non-linear data is obsolete and now we can calculate Ki' and Km and Vmax using other computational tools. The point mentioned in papers was the linearization methods were used when the computer-based calculation was yet to be discovered. So now, due to advancement in computation, L-B (double reciprocal) and Bowden plots are un-necessary. On the contrary, I still find recent research articles having L-B plots based calculations
Can anyone guide me about this? If these methods are obsolete, what computational; methods or software can I use and how? Please mention some related documentation too in the answer.
I have a decrease of 1/3 to 1/2 in the gene expression of gluconeogenesis enzymes (Glucose-6-phosphatase, Fructose-1,6-bisphosphatase, Phosphoenolpyruvate carboxykinase and Pyruvate carboxylase) in mice liver between my test condition and my control condition and I would like to know if it can lead to a reduction of the flux of the pathway or not? Also is one of these enzymes more critical than the others in the pathway?
Basically I have now two proteins that catalyze the same reaction, but with different affinity for their substrate. The first protein has a lower Km since is enough to visualize the reaction (using a scintillation counter) if I use a concentration around 10-50 nM. When I use the second protein I have to add at least 400-800 nM to be able to visualize the reaction and calculate the different slopes varying the substrate concentration to finally obtain both Km.
As far as I know the concentration of the protein is not relevant to calculate the Km (you only have use a concentration much lower than the substrate of the reaction in a Michaelis Menten-like case). Since my knowledge about enzymology is not very deep, I would like to hear of you that is perfectly possible to compare both Km even if I had to use different protein concentrations to perform the assay.
Thank you very much!
Please colleagues, I need to know how to convert the measure of enzyme activities in plants (Ascorbate peroxidase and Glutathione reductase) from the SI unit of U/L to umol/L. I found that most literature did not report in the SI unit, so I want to convert to make the results comparable to other studies. Kindly assist.
I have optimised an assay with chymotrypsin and N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide substrate. Flavonoids at concentrations ranging from 0.01-1 mM, were then introduced to observe any affects they may have on enzyme activity. Of those investigated, catechin was found to increase activity. However, upon further reading, all research points to catechins having inhibitory effects on serine proteases, yet my results suggest otherwise. I was wondering if anyone (someone with a better understanding of physiochemistry/enzymology than myself) might have a insight or suggestions as to why this might be the case? Assay was carried out in 0.1M tris buffer pH 8.5.
Thanks in advance!
I've just started helping on a project requiring the extraction of high molecular weight DNA from Chlorella luteoviridis (green algae) for PacBio sequencing. My coworker has tried using a whole bunch of enzymes which she creates a solution of and incubates the algae/agar plugs in overnight for gentle digestion of the cell wall to occur. Unfortunately, the cell walls of algae are extremely variable between algae and thus her methods are not as successful on this new strain.
Currently she has tried 4% concentrations of cellulase, pectinase, hemicellulase, and chitinase all in one enzymatic mixture. Additionally, a subsequent incubation in lysozyme has been attempted overnight yet we are still getting poor yields of DNA.
Is there any advice on other enzymes/methods of cell wall digestion you can suggest? Thank you!
Hi, I am interested in measuring nitrogenase activity in free living bacteria. Specifically i am interested in measuring through the 15N2 method which measures the organic nitrogen through mas spectometry. Nevertheless, I would l would like to know why it does not take into account other forms of nitrogen in the supernatant or intracellular. For example Labelled amonnia.