Questions related to Enzyme Activity
The question is: Determining hexokinase activity in rat brain where 10 µl of a 10% (100 g/L) brain homogenate (containing hexokinase) is mixed with 1990 µL of reaction medium (containing the substrate glucose and the coenzymes ATP and NADP+ and the auxiliary enzyme glucose-6-phosphate dehydrogenase). The reaction mixture is placed in a cuvette (light path = 1 cm) in a spectrophotometer at 37°C, where the hexokinase reaction can be seen as an increase in absorbance at 340 nm. The reaction proceeds at a constant rate during the measurement period, and during 10 min a total increase in the light absorption at 340 nm of 0.10 is recorded. The absorption coefficient of NADPH = 6300 x M-1 x cm-1
I need to find the enzyme activity of hexokinase in µmol/min/g. I don't understand how I need to interpret the first line (10 µL of a 10 % (100 g/L) brain homogenate) - and how should I use these numbers?
Thanks in advance!
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 found the dns method to measure the amylase activity, but I cannot get the dns reagent. Therefore, could you suggest me an alternative method or alternative reagent to dns reagent?
Sometimes the enzyme may not be activated or may not function properly unless the it is co-expressed with other protein for post-translational modification or assembly into a functional catalytic complex.
Does a better docking score or stronger binding energy between a mutant enzyme with its substrate always translate into an improved enzymatic activity?
How should RMSD, RMSF, radius of gyration, solvent-accessible surface area (SASA), etc. from molecular dynamics simulation be used to guide enzyme engineering with the aim of improved product synthesis?
Hi, Iinserted gene of Reverse transcriptase into pPLc245 plazmid. If I'm right, this plazmid doesn't code cI857 represor. For expression od reverse transcriptase from this plasmid I used E. coli DH10B. RT was expressed after increase of cultivation temperature to 37 °C. Before this thermo induction I cultivated E. coli DH10B with pPLc245 at 28 °C. But at this temperature RT was not expressed. Why is this possible? Is E. coli DH10B coding cI represor or pPLc245 could contain gene for this represor?
Thank you for all responses.
Hi, every body
I am investigating the enzyme profile of a white-rot basidiomycete fungus. In the enzyme assays, we detect enzymatic activity by color change. However, my fungus produces pigments that do not precipitate with centrifugation, so it causes errors in the results. Does anyone have a solution? Thanks.
I would like to ask you, when I measured the enzyme activity of papain by the national standard method, but the sample tube and the blank tube became zero and negative values when subtracted from each other. The blank tube was added with TCA first, which means that it was caused by the casein itself and showed a light blue color.
How can I adjust the OD value of the diluted papain, which is also light blue, to 0? Is there a problem there?
There is no air bubbles in the measurement, and the instrument operation and personnel operation have been excluded.
Translated with www.DeepL.com/Translator (free version)
I am about to calculate alpha-amylase activity for some fungal samples. I followed the protocol of Sigma-Aldrich (https://www.sigmaaldrich.com/DE/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/enzymatic-assay-of-a-amylase) and incubated my samples in 0.2 % starch solution for 3 minutes.
According to the protocol, the equation for the U/ml value is:
U/ml = (mg of maltose released * dilution factor) / (ml of enzyme applied)
I think I also have to consider the incubation time of 3 minutes, and divide my result from the equation by 3?
Thanks in advance for help.
I have been failing to express a very large trypanosome (160kDa) protein using various expression systems. The protein is a chimera of 3 enzyme activities and I can express truncated forms containing at least one of these enzyme activities. So, I was wondering if I can express and purify these enzyme 'domains' individually and somehow get them to associate, in order to perform enzymatic assays assessing their potentially interactive (regulatory) roles.
For instance can an inducible di-cre recombinase-type system be used? Ideally any recombination domains (or tags) that I may fuse to my recombinant construct would be as small as possible.
I have diluted my chrysin in DMSO, however when run an enzymatic assay in phosphate buffer pH 7.4 (50 mM) and pH 7.6(10mM) it formed precipitation? do you have any suggestion to stop it but still using the same buffer? Thank you
The Vigna radiata plants were subjected to Cd stress ranging from 50-200 ppm. Why does the enzymatic activity (SOD, APX, and CAT) increase under increasing metal stress despite the decrease in protein content?
Hi, I'd like to ask whether it is necessary to precipitate MMLV Reverse transcriptase before affinity chromatography purification (Äkta)? My colleague must do this step with his Taq DNA polymerase. He use (NH4)2SO4 or Na2SO4 + PEG. Without this precipitation is polymerase inactive.
Thank you for your responses.
How to know whether a particular protein is an auto cleaving protein or not (i.e. Self cleaving) ?
Also suppose a particular protein is degrading immediately so How to know whether this is due to Auto cleaving nature of the protein or due to some other proteins (i.e proteases) in the supernatant.
I am trying to assess the activity of PFK enzyme in mESC lines. Recently in the metabolomics analysis, I discovered that although the cells show an increased uptake of glucose 6-P and fructose 6-P, there is a major drop in the levels of downstream metabolites starting from fructose-1,6-bisP all the way up to pyruvate. (except glyceraldehyde-3-P, 1,3-bisPglycerate and 2-phosphoglycerate)
There is also an increase in the levels of glucosamine-6-P, mannose, mannose-6-P and of 6-P gluconate and ribose 5-P. These levels could be increased as G 6-P and F 6-P could be shunted into the pentoseP pathways.
Therefore, I am planning to check if there is a blockage between the F 6-P and fructose 1,6-P by detecting the activity of the PFKinase enzyme.
So far the kits I've found to do this, are based on the calorimetric assay where PFK activity is determined by a coupled enzyme assay, in which fructose-6-phosphate and ATP is converted to fructose1,6-diphosphate and ADP by PFK. The ADP is converted by the enzyme mix to AMP and NADH. The resulting NADH reduces a colorless probe resulting in a colorimetric (450 nm) product proportional to the PFK activity present. One unit of PFK is the amount of enzyme that will generate 1.0 mmole of NADH per minute at pH 7.4 at 37 °C.
I was wondering if there is an in cell method, more precise for cell extracts.. I am not sure how sensitive or specific this calorimetric assay would be.. Any comments/ suggestions would be much appreciated.
Can I say for a given same enzyme, more number of substrate A is changing into the product than substrate B per second ? Please make this this this clear to me. I know Kcat/Km characterized high efficiency. However, I am getting into this situation somehow. Is it possible ? How I would justify or rationalize this in the paper for publication ?
I am a student from a relatively developing country and the commercial enzyme is expensive for us. My graduation thesis is desperately in need of an available standard curve for my AchE assay so that I can calculate my results using it. Please is there anyone who currently doing the AchE assay who can share it with me or where can I find it, are there papers or textbooks that I can find it in?
Your help would be very much appreciated.
I'm trying to run an assay for Caspase 3/7 activity to visualize apoptotic cells in flash frozen, unfixed mouse skin sections. The assay involves a non-fluorescent reagent that is processed into green fluorescence by endogenous, active Caspase 3 or 7 in apoptotic cells.
After thawing the slides for 3 minutes and washing once in PBS to remove OCT, I added the reagent to the unfixed tissue (I've tried 1 hour at room temp, or 30 mins at 37C). After the incubation, I post-fixed the sections with 4% paraformaldehyde, added DAPI mounting medium then visualized. 100% of the cells had green fluorescence, and the nuclei appeared enlarged so it looks like the cells are bursting or autolysing during the incubation. I'm wondering if I should fix the tissue immediately after thawing to stop the damage to the cells.
What would be the best method for post-fixing flash frozen mouse skin so that endogenous enzymatic activity is maintained? Does anyone have experience specifically with using the CellEvent Caspase 3/7 assay from Invitrogen in frozen tissue sections?
PS. I have also tried a TUNEL assay which generated no signal.
Most people say that imidazole doesnt affect the majority of downstream applications for purified proteins but it is a chelator, so you would think that it might chelate the Mg2+ in solution and inhibit Mg2+ dependent reactions. Anyone seen any papers that discuss what concentration Imidazole will chelate magnesium or manganese ions in solution?
Hi everyone, I have been trying to calculate the Km value of a recombinant HCV NS3/4A protease using a FRET substrate, the cleavage of which can be detected at 500 nm using excitation wavelength of 355 nm. I have a RFU vs. time graph of different concentration of the substrate (attached). Next, I am calculating the initial rate which is the slope of the linear initial part of the progress curve, with the ultimate goal to plot the 1/[S] vs. 1/[V] graph and calculate the Km from the Lineweaver-Burk equation. However, if I calculate the slope from this graph for the time range between 0-10 minutes which is the initial linear part, the intercept for the 1/[S] vs. 1/[V] graph is negative but Km value can not be negative. I was wondering if anyone can guide me on how to calculate the initial velocity correctly from the graph I have? Thank you so much!
I'm using (for me new cells) E. coli Arctic express for protein expression (reverse transcriptase). I tried expression at 20°C and 10°C. At 20°C solubility of my proteine was 50% and at 10°C it was about 57%. I'd like to know some your experiences and tips how to use these cells (the best media for them, optimal temperature for night culture or growth up to induction and after induction, how to eliminate chaperonins after expression, how long should lasts expression, some supplements to media, concentration of antibiotics in night culture, concentration of IPTG ... )?
Thank you for all advices!
I'm working in my doctoral thesis in the purification of a lipolytic enzyme from an halophile archea. Currently, I'm trying to purificate the recombinant enzyme using Haloferax volcanii as an expression system. The problem is that suddenly when filtering my crude extract through a 0.45 um nitrocellulose membrane I lose 90 % of the enzymatic activity, when that did not happen before and at most I lost 20 % of the activity.
Has the same thing happened to someone else? Or do you have any ideas that could help me? Thanks!
We are used the protocol for urease extration adaptated (to C. neoformans) from Amin et al.,2013:
Briefly, the broth cultures were subjected to centrifugation (5,000 × g, 4 °C) and the recovered mass was washed twice using phosphate-buffered saline (pH 7.4) and then stored at -80 °C. Subsequently, was thawed to ambient (room) temperature, followed by mixing with 3 mL of distilled water and protease inhibitors (TLCK, Tosyl-L-lysyl-chloromethane hydrochloride) and sonication for 60 s. After centrifugation (15,000 × g, 4 °C), the supernatant was desalted by eluting through SephadexG-25 column. The resultant crude urease solution was mixed with an equal volume of glycerol and then preserved under refrigerator (4 °C) for further uses.
Okay, we ran the suggested protocol, but after 2 days, enzyme activity was no longer observed.
Then, we tried to exclude the TLCK from the protocol, again the activity was observed on the day of extraction, but after it lost the activity.
We also tried to activate urease with sodium bicarbonate and Ni solution, without success.
In another procedure, we repeated the protocol and lyophilized the solution resulting in a white solid. So we tested it on this day, we prepared a urease solution (10 mg / mL) and the activity was good, but after a few days, again the activity was lost.
We always work with refrigeration, and we are careful to keep the solution on ice when we handle it.
Please any suggestions?
Hi, I did heterologous expression of reverse transcriptase in different strains of E. coli at 3 temperatures, 37 °C, 28 °C and 20 °C. As I expected, BL21 provided the best solubility at 20 °C and the worst at 37 °C. On the other hand strain MC4100 had the best solubility at 37°C and the worst at 20 °C. Has anybody similar experience with E. coli MC4100?
Thanks for responses!
As I am characterizing an 3' exonuclease and calculated the apparent Km and Kcat for two different substrates. What I observed is little bizarre. A substrate (S1) which looks like (qualitatively) degraded slowly by the enzyme has Km lower (~60 nM) and lower Kcat (5.1 sec-1) while other substrate (S2) apparently degraded faster, has higher Km (~175 nM) and but slightly higher Kcat (~8.2 sec-1). Now I have difficulty in understanding why is this happening. One more thing I observed is that S1 gets inhibited after 150 nM substrate concentration. While S2 continue to maintain it's saturation plateau even at 8000 nM. I am little perplexed how to rationalize this result. Any help will be appreciated much in this regard. Need an explanation about preferential degradation of the substrate by the enzyme.
Thank you in advance.
Hi, I'd like to try expression of reverse transcriptase with low temperature, 15 °C, due to solubility. Should I use some special plasmids for cold expression? I ordered cells for this purpose - E.coli Arctic express. Is it enough or is it better to combine these cells with plasmids for cold expression? Thank you all!
Is there anybody who adds betaine (trimethylglycine) to media for protein expression? I read that betaine is able to increase solubility of proteins. If you use it for this purpose which form is the best? Is there any proven concentration?
Thank you for all answers!
I am looking for examples of industrial processes which are catalyzed by enzymes (either natural or recombinant). Particularly, I am interested in processes that end up with enzyme removal or inactivation (thermal or chemical), and I am trying to find examples from different areas - the food industry, textiles, pharmaceutics, fuel, etc.
Could you help, please?
First of all, I apologize for my poor English.
I would like to measure the enzymatic activity of COMT, referring to the following reference, but is it theoretically possible to substitute norepinephrine bitartrate hydrate?
Thank you very much.
Anyone know how to measure catalase activity in brain homogenates in terms of μmol/min/g of brain. Reaction was stopped by addition of dichromate and absorbance was measures after 1 min at 570 nm. Controls are without H2O2 for each samples.
Can you plzz provide some references for effect of Mn fertilization on soil enzymatic activities
A special primer has to be used with only 19 bp poly dT (Tm 44.9) instead of 30 bp in original study (Tm 55.2). Will this induce failure of mRNA capture? Will this cause RNA detachment in Reverse transcription using Superscript II (@42 ℃)? If this is a problem, how about start reverse transcription @ 37℃ or 40℃ for 20min, then rise it to 42℃？ Will superscript II be completely block at lower temperature?
I have Laccase from trametes versicolor with 0.5U/mg activity.
I need enzymatic activity of 50U/g in solution. What should be the amount of enzyme I add to obtain enzymatic activity of the above said?
I am working with erythroid progenitor cells in culture and want to assess the activity of an intracellular (cytoplasmic) enzyme. I have a protocol from the literature for the activity assay, but I can't seem to find any detailed info on the lysis buffer and methods used prior to the assay. For westerns I lyse with RIPA on ice and then sonicate, but I know I can't use RIPA for this, so my two questions are:
1. What lysis buffer recipe do you recommend to extract cytoplasmic proteins for an activity assay?
2. What lysis method do you recommend in conjunction with this buffer? Details would be appreciated!
I know there are many lysis buffer options and I'm just not sure how to figure out which one is both easy to make and will work for this purpose, and if one lysis method (freeze/thaw vs sonication, etc.) is more/less suitable for retaining enzyme function for downstream testing?
A full-term female infant failed to gain weight and showed metabolic acidosis in the neonatal period. A physical examination at 6 months showed failure to thrive, hypotonia, small muscle mass, severe head lag, and a persistent acidosis (pH 7.0 to 7.2). Blood lactate, pyruvate, and alanine were greatly elevated. Treatment with thiamine did not alleviate the lactic acidosis. Which of the following enzymes is most likely deficient in this patient?
a) Alanine amino transferase
b) Phosphoenolpyruvate carboxy kinase
c) Pyruvate carboxylase
d) Pyruvate dehydrogenase
e) Pyruvate kinase
Hi every one,
I am extracting enzymatic extract from plant (50 mM Sodium phosphate buffer (pH 7.8), 0.1 mM EDTA, 0.1 % (v/v) Triton; 1 mM PMSF). I mesure the protein content and enzymatic activity fom this extract.
So I want and I need to know if the enzymes extract can be conserved for further enzymatic activity measurement (SOD, CAT, APX, ...).
I really need your help, thank you very much
If I isolate/enrich lysosomes from mammalian cells through either differential centrifugation or using commercial kits, what is the best method of storage to preserve enzymatic activity? Is there a specific buffer that would be the most appropriate? Organelles should be intact at this point so would slow freezing as with cells be more appropriate than snap freezing with liquid nitrogen?
NB: structural integrity of lysosomes is less important than enzymatic activity.
I'm studying digestive enzyme supplements and a large proportion of them contain a wide variety of proteases (Serratiopeptidase, DPPIV-peptidase, pepsin and others) as well as some other, mainly carbohydrate-digesting, enzymes. These are all often contained within the same non-enterically-coated capsule so liquid can leach inside once the capsule is consumed. Would it be expected that the proteases start digesting each other as well as the other enzymes, and would this then reduce the effectiveness of the supplement?
Thank you for any answers
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?
For some reason, we need to have sodium chloride (NaCl, 0.05-0.1M) in our system. Will this affect the reverse transcriptase and block the SMART reverse transcript?
1. Km much, much lower than the prevailing substrate concentration ([S] >> Km)?
2. Km around the prevailing substrate concentration ([S] ≅Km)?
3. Km much, much higher than the prevailing substrate concentration ([S] <<Km)?
Please give examples too. Thank you!
I was wondering if the methods of enzyme immobilisation reduce enzymatic activity. I am aware that everything dependes on enzyme kinetics and the type of immobilisation, ¿but do the enzymes immobilised via covalent or ionic bonding experience a reduction in their catalytic activity?
I want to study the activity of a mannosidase against a substrate using a 96-well plate.
Following the protocol, the reaction lasts for 1h and I will end up with a absorvance value at 450nm.
How can I convert that to enzymatic activity (umol/min/mg)?
I know I have to create a standard curve but, should I create it using known concentrations of the product formed (mannose) or would be okay to do it using known concentrations of the enzyme?
I have a batch of fish liver samples than I would like to preserve for internal controls during EROD assays. Would lyophilizing the fish liver samples prolong the length of time that I can keep them stored -80C without affecting EROD activity?
I'm preparing enzyme-responsive polymeric nanosystems and according to the manufacturer's information, the enzyme is in
-» "50 mM sodium acetate buffer, 1 mM EDTA, pH 5.0.";
-»"≥10 units/mg protein" and
-» unit definition "One unit is defined as the amount of enzyme that will hydrolyze 1.0 µmol of compound x per min at 40°C, using 100 mM Na+/K+ pH 6.0, with 1.33 mM EDTA and 2 mM DTT as the activation buffer."
The flask has 50 micrograms of enzyme.
Concentration: 0.451 mg/mL.
I wanted to prepare 0.5 UN/ mL solution.
Thanks very much
I would like to know how one can decide when to double the volume of total reaction and that of the individual components of an enzymatic reaction such as restriction digestion, PCR, in vitro transcription etc. rather than keeping the same reaction volume while increasing the concentration of the individual components?
For instance, if I want to double the units of T7 RNA polymerase than usual (100 U to 200 U) for in vitro transcription, as I am increasing the template concentration (from 1ug to 10ug), should I keep the total reaction volume constant (50 ul) or should I increase it too (100 ul)?
The example I stated is more of an experimental specific. It would be really helpful to get both, a general reasoning fit for all the enzymatic reactions, and also for a specific experimental setup such as the above.
Thanks in advance !!
Hello. I wanted to relate N mineralization with enzymatic activity in the soil after wheat residue incorporation. Urease is the most widely used enzyme in these kind of studies. However in my experiment, the chemical fertilizer treatment is not ureic. I see studies where they do not specify the type of fertilizer. Is urease suitable to assess N release from plant residues? I have read that nucleic acid can break down into uric acid, but not urea.
I have Started Working on Diabetic Rats and I need to assess the activity of below mentioned Enzymatic activity
I'm new to enzymes and I would like to receive all possible help for this. I have a product that initially contains 57.9g of starch, which I would like to hydrolyze with alpha amylase, that has an activity of 1,11,793 U/G. How much grams of this enzyme should I add to the product so that all of the starch (57.9g) breaks down?
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.
I’ve read that soil samples for soil enzyme assays should be kept cool and at or near field moisture levels. But I cannot find information on how long these samples are viable for under these conditions. Is a month or 2 in the fridge too long to wait to perform the assay? Also, is freezing the samples until they are all ready to be analyzed acceptable, or would this compromise the sample? Reason being I plan on having samples collected from different sites over a 2-3 month time period and need to coordinate sample collection, storage, and shipment. And this leads to my next question…
I’ve also read that comparing soil enzyme assay data from different sites and times is not recommended due to differences in site soil conditions (pH, temp, moisture, etc.) as well as changing conditions over time (rain events, etc.). Is there a way to overcome this, maybe by controlling moisture content? I am essentially trying to compare a treatment effect (+ vs -) on soils under 2 plant types, grown in 3 different climates, and over time following treatment. Any advice here would be appreciated.
The experiment has total of 16 days and its collect sample in 0 hours, 24 hours and 48 hours (7 days) and in 14 days the same. Doing the analyses in the spectrophotometer, I checked that in 7 days did not show any enzymatic activity and in 14 days it present a good enzymatic activity of 144 U/l in 48 hours.
I have an experiment with two levels of two different enzyme and a control treatment (without enzyme) in a completely randomized design. Therefore, a 2 x 2 + 1 factorial design. Can I modeling like a nested design or exist a different model for it?
GDP is the product of GTPase reaction. In the biochemical setup containing enzyme GTP and GDP, GDP acts as an inhibitor as the enzymatic activity was found to decreased. Km increases and vmax and kcat decreases.
The kinetic parameters of the above reaction are same as in mixed inhibition. Some of the previous reports stated that Product inhibition is a part of mixed inhibition where product of the reaction act as an inhibitor.
What can be a major difference between product and mixed inhibition?
If the above reaction is not a mixed inhibition then what else it could be?
Kindly help me in this please.
I have 4 conditions to be investigated:
- Enzyme concentration (3 or more levels)
- Substrate concentration (3 or more levels)
- Solvent: taurocholate (6 mM), taurocholate (12 mM), Triton X-100 (2%), Triton X-100 (5%)
- Incubation time (3 or more levels)
1. What's method to optimize those variables before I can conduct the cholesterol esterase inhibition assay? (not the one-factor-at-a-time one)
2. or because my final goal is the inhibition assay, can I optimize the cholesterol esterase inhibition assay? (I do not know what type of inhibition of my extracts)
Any significant progress in the design or innovation of solid catalyst for biomass hydrolysis and sugar fermentation into alcohols in a biorefinery? If yes, I would like to have some recent updates regarding that subject. Thank you all
I'm measuring fluorescence intensity of the product in an enzyme reaction in 37 celsius degree. I set a kinetic measurement in 1 hour. The fluorescence intensity of the BLANK ( contains only buffer and substrate ) decreases by time and its graph is not a straight base line. I'm thinking if other samples I'm measuring has the same change as the Blank solution, so should I normalise the data by dividing the blank's fluorescence intensity ?
Based on this paper
,TEV protease can cleave between the Gln and several amino-acids (besides Gly/Ser) with acceptable efficiency in its recognition site.
Therefore, it's practically possible to purify many proteins (without an extra residue at the N-terminal end), by using affinity chromatography.
I was wondering if anyone could share their experience/knowledge using TEV protease to cleave between Gln and Met?
I am trying to measure GUS expression, and was wondering if anyone knew what concentration hydrogen peroxide inhibits the GUS protein???
Thanks ! Any help is appreciated!
Generally, the 0.25M sucrose solution is the most common organic solution which is used for tissue homogenate preparation. There are many specific buffer solutions which are used for tissue homogenate preparation for specific enzyme assay. Please provide some references about the use of 0.25M sucrose solution and its advantages as well as disadvantages associated with its use over other buffers. Thank you.
I have been trying to test PPO activity in vegetable juice. I extracted the enzymes using a buffer solution containing potassium phosphate buffer, NaCl, Triton X-100 and PVPP. I tested for PPO activity using catechol as substrate and measured the change in absorbance using uv-vis spectrophotometer. However, after the addition of catechol, the reaction mixture turned turbid, which prevented me from obtaining the initial velocity gradient. Has anyone faced this problem? Any help is appreciated.
I used Kakade method with by adding 0 to 2 millilitre (i.e., 0, 0.5, 1.0, 1.5 and 2 ml) of extract into duplicate sets of test tubes and each tube extract and adjusted to 2ml with distilled water. After that, 2 ml of trypsin solution [4 mg trypsin (Sigma Chemical) was dissolved in 200 ml 0.001 M HCl] was added to each test tube and kept in water bath at 37°C. To each tube, 5ml BAPNA solution.
For the calculation, I first took the average of the absorbance values of each tube and divided by 0.01. Then calculated the differences between each tube and divided by the volume of extract to TIU/ml. Then I plotted the TIU/ml against volume of extract and extrapolated to zero. But I dont know how to calculate TIU/g using the extrapolated value. What dilution factor should I use? Im confused. Can someone please help me?
Recently I added catechol to distilled water and incubated it overnight in a shaking incubator. By morning the solution was all brown. What may be the reason? Does the brown coloring signify production of melanin from oxidation of benzoquinone which may be due to non-enzymatic oxidation of catechol to benzoquinone ?
Please note: in this above experiment there was no enzyme added like catechol oxidase.
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.
for enzyme assay i am using crude enzyme source instead of pure protein. is there chances that i would be able to get maximum result or enzymatic activity.
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 have stored the tissues in -80 degree. I need to check the activity of mitochondrial enzymes and Calcium accumulation. I want to clear, how long we can store the tissues, whether mitochondria is stable in stored tissues.
Hi! I've been studying a particular enzyme that in in vitro experiments exhibits two optimal pH's. All other parameters are the same (incubation time, substrate, temperature, salt concentration, substrate and enzyme concentration) except for the pH.
I'm wondering what the possible mechanistic/molecular physical explanation for this is? I've been looking all over the internet but it's hard to find any discussion on multiple optimal pH's. If anyone could point me in the direction of some literature I'd appreciate it.
Thanks in advance!
I have been searching the literature about this and I have not found an answer. If the atom that accepts a hydride is made more electrophilic, can this speed up the rate of hydride transfer?