Enzyme Activity - Science topic

I've tried various 2.5% concentrations of glutaraldehyde, but all of them yield pellets that are somewhat challenging to dissolve. that's why I need an alternative approach. I performed cross-linking for 2 hours initially, and then I tried extending the time to 6 hours as well as reducing it to 1 hour. However, I didn't observe any significant changes.

( It's worth noting that I purified my cellulase protein using ammonium sulfate precipitation before starting the cross-linking process. Is it appropriate to use ammonium sulfate precipitation-purified protein for this crosslinking purpose?:

In my laboratory, two students attempted to work with xylanase and exo-cellulase, and both of them achieved success on their very first try. They didn't encounter any significant difficulties, not even when it came to dissolution.

I'm feeling unsure about what steps to take, and time is slipping away quickly. I seem to be stuck in this situation.

The value k_cat is the number of product molecules formed per molecule of enzyme per second, and is reported in units of s^-1. So I don't think that the description of the stock solution in terms of s^-1 makes sense , nor can it be converted to an enzyme concentration. Perhaps you mis-transcribed the units in your quotation from the published account, or there was an error in the paper.

For example, let's suppose the authors meant 998 µM/s as the activity of the stock solution, since the GOX stock solution was defined in similar terms (3 mM/s).

998 µmole/(L-s) = 59,880 µmole/(L-min) = 59,880 U

(The definition you gave for a unit of catalase activity is 1 µmole/(L-min).)

The specific activity you mentioned was 20,000 U/mg.

Thus the stock solution contained 59,880 U/(20,000 U/mg) = ~3 mg.

We don't have information about the volume of the stock solution, unfortunately, so we can't calculate its concentration.

% (w/v) is grams per 100 mL.

1 mg/mL = 1 g/L = 0.1 g/100 mL = 0.1% (w/v)

you can use both beads designed for animal tissue and plant material, either combine them in a single run, or use them sequentially, following these beads selection guidance: https://www.takarabio.com/learning-centers/nucleic-acid-purification/accessory-selection-guides/sample-homogenization-beads

You need 5 units, to be added in 15 µl, which is 0.333 U/µl for the 20X stock solution.

(0.333 U/µl)/(9.70 U/mg) = 0.0343 mg/µl = 34.3 mg/ml

So the 20X stock solution should have a concentration of 34.3 mg of solid per ml.

0.015 ml x 34.3 mg/ml x 9.7 U/mg = 5 U

Since a L-B plot is of the reciprocals of the rates and substrate concentrations, a decrease in activity caused by the presence of an inhibitor is represented as a line that is higher up in the first quadrant than the uninhibited line. If you are seeing the opposite effect, it means that the enzyme was more active in the presence of the inhibitor than in its absence. In other words, the compound is an activator, not an inhibitor. However, it would be a good idea to share the data, since there may be other interpretations that can only be made based on seeing the data.

are you using the assay that employs the use of 0.65% Casein? Do you establish a Standard Curve using 1 mmol/ml Tyrosine? If that is the case and you have run a set of standards from 0.05 mmol/rxn to 0.5 mmol/rxn, the calculations are relatively straight forward. See attached.

Since you frequently refer to colonies ("conies"), it seems that you are not growing a culture of the cells and purifying the enzyme, just extracting the enzyme from the cells in the colony. In that case, there is not much point in studying the kinetics of the enzyme because it has not been purified. The signal obtained may be due to a mixture of multiple enzymes. The amount of the enzyme obtained from a colony may depend on factors such as how many cells are in the colony, the age of the colony, and how well the enzyme were extracted from each colony.

Generally speaking, an enzyme has a single overall conformation. It will not have different structures in different colonies. It might be expressed to different levels in different colonies. Some of it may be in an insoluble form due to failure to fold properly, and the proportion of insoluble, inactive protein may differ between colonies.

If you want to study this enzyme's kinetic properties, you really should purify it.

Thank you so much.

THERE ARE A SERIES OF VOLUMES IN METHODS OF ENZYMOLOGY

also

TitleMethods in Enzymology Volume 61 of Enzyme Structure, Serge N. Timasheff Enzyme Structure: Part H, Serge N Timasheff Methods in Enzymology, ISSN 0076-6879 Volume 61 of Methods in enzymology: Enzyme structureEditorsC. H.W. Hirs, Serge N. TimasheffContributorsC. H.W. Hirs, Serge N. TimasheffEditionillustratedPublisherElsevier Science, 1979 and

Rossomando EF (1990) Measurement of enzyme activity. Methods in Enzymology 182: 38–49

Thank you very much!

COPASI: Biochemical System Simulator.

free download from copasi.org

If you are measuring the conversion of NAD(P)H to NAD(P)+ at 340 nm, the absorbance decreases as the reaction progresses. You should perform your calculation using the absolute value (i.e. positive value) of the negative absorbance change.

Use Beer's Law to calculate the change in concentration of the analyte based on the absorbance change, where the absolute value of the absorbance decrease divided by the path length (0.6 cm) and divided by the extinction coefficient (6.22 mM^-1cm^-1), give the concentration change of the analyte in mM units.

The calculation of specific activity from this result requires knowing the volume of the sample that was measured and the concentration of protein that was present during the reaction.

specific activity = nanomolar concentration of product formed or substrate consumed divided by concentration of protein in mg/liter and divided by time of reaction.

You have 13000 U/g. Is that dry powder or liquid? If it is powder you need to dissolve it in a known final volume of liquid. As you have 100mg, you have 1300 units of enzyme and the units per ml value is determined by the volume. If you have 100mg/1300 units dissolved in 10ml, i.e., 10mg/ml, that’s 130 units per ml. But if you had 1mg/ml enzyme (100ml), you would have 13units/ml.

Metals when present in proteins are there for an evolved reason; usually structural and/or catalytic function. Both HEW and T4 lysozyme have an extensive history of the impact of various metals in their structure and function, so maybe review that literature. The only "problem" I could see from metals might be partial or mixed residency and this giving a mixed population of enzymatic activities, complicating analyses. Many enzymes have been extensively studied by chelating out one metals and then adding back another, see this paper and refs therin.

Avoid freeze thaw cycles. Make your stock and dispense into 50 or 100μl aliquots (single use is even better) which you keep on ice until you freeze the batch. The colder you store your stock at the longer it will last. At -80°C pepsin suspended in 0.01M HCl lasts for a year. Pepsin is more soluble in HCl than it is in Tris so more concentrated stock solutions can be made.

The answer is that it depends. Most often the N-terminal Met is not needed in the protein, but there are some cases where it is. So I would think that while usually you would not need to include it, but on the other hand there is no strong reason not to include.

Do you have access to an absorbance plate reader? You could use one to make the measurements in 96-well or even 384-well plates. It is highly beneficial to also use a multichannel pipettor (8 or 12 channel) to put reagents into the wells. A plate shaker improves mixing of the samples in the wells. Some plate readers can also perform the plate shaking function. If the absorbance measurements are in the far UV (<340 nm), it would require a plate reader designed for such measurements, and special UV-transparent plates.

It varies depending upon the specific protocol of any author you are following, reagents you are using and the abundance of that specific enzyme in your tissue. However to know the best enzyme volume you can perform serial dilution.

The different kinetic constants for the different substrates reflect the chemical differences between the substrates, including the strengths and types of their interactions with the enzyme active site and differences in the activation energy of the reaction. The differences in the kinetic constants between the substrates in this case are actually quite minor, and could easily be within the range expected for experimental variation.

10/20/22

Dear Emre,

I can't tell you which filter is the best one to use. However, this is something that you can easily test for yourself. You can try running small batches of the pepsin & pancreatin separately through each type of filter and measure the enzymatic activity of filtered vs. unfiltered enzymes. One thing to keep in mind is that membrane filters are not always "benign". Sometimes they bind some of the material (in your case, protein) that you are trying to filter. If you look in the catalogs of companies that sell membrane filters, they often describe a particular membrane as exhibiting "Low protein binding". If you follow the above suggestion and find less enzyme activity in the filtrate of one or more of the membranes, it could be that some of the enzyme protein was bound by the filter. To check this, it would be a good idea to measure not only the enzyme activity in the filtered- and unfiltered preparation, but also the protein concentration in the filtered- and unfiltered sample.

Something you mentioned in the first part of your question: Exactly what are you going to autoclave? Autoclaving pepsin, pancreatin, gastric juice and intestinal fluid will denature enzymes and other proteins that they contain.

I hope this information helps you.

Bill Colonna Iowa State University, Ames, Iowa, USA wcolonna@iastate.edu

Here is a paper that may help.

Here is some information about it from Sigma.

V_max depends upon and is, ideally, directly proportional to the concentration of the enzyme. Specific activity divides V_max by the amount of protein present (typically in mg, as in µmol/min/mg). Specific activity can be used to describe the level of activity of any enzyme-containing preparation, without requiring that the enzyme be purified.

k_cat divides V_max (in concentration units) by the enzyme concentration to get s^-1. Purified enzyme is normally used for measuring this parameter.

You can express the enzyme activity in whatever way suits your needs, but you should be explicit about how the calculation is made, the source of the enzyme, what protein concentration was used, and the specific activity of the preparation so that others can repeat the work.

If you keep the enzyme concentration constant throughout all experiments, and its activity remains constant throughout, then, for the sake of your calculations, you can leave V_max in the form of moles of product formed per unit of time or, dividing by the volume of the reaction, as concentration of product formed per unit of time.

Please go through the following reference:

Boudrant J, Woodley JM, Fernandez-Lafuente R. 2020. Parameters necessary to define an immobilized enzyme preparation. Process Biochem. 90:66-80.

Thank you very much for the clarification Annemarie Honegger

You may consider the detection of phosphorylation of LRRK2 substrates via immunoblotting:

1. Autophosphorylation at S1292

2. T73 Rab10 phosphorylation

3. to a lesser extent, T72 Rab8 phosphorylation

You may also consider Phostag assay for Rab10 phosphorylation, as published by the lab of Alessi doi: 10.1042/BCJ20160557

Good luck

10 µL of a 100 g/L (= 100 µg/µL) homogenate contains 1000 µg = 1 mg = 0.001 g.

That will be the number in the denominator when you divide by the number of g to calculate µmol/min/g.

The change in absorbance of 0.1 unit is converted to the concentration of NADPH formed using the extinction coefficient of 6300 M^-1cm^-1 and the Beer-Lambert Law: absorbance = extinction coefficient x pathlength x concentration.

An absorbance change of 0.1 for NADPH with an extinction coefficient of 6300 M^-1cm^-1 in a 1-cm pathlength cuvette corresponds to a NADPH concentration of 0.1/(6300 x 1) = 1.587 x 10^-5 M = 15.87 µM.

This amount of NADPH was formed during 10 minutes, so the rate of NADPH formation was 1.587 µM/min. This occurred on a reaction volume of 2 mL, so it can also be expressed as 1.587 µmoles/(L-min) x 0.002 L = 0.00317 µmoles/min.

This happened when you used 0.001 g of material, so the specific activity was (0.00317 µmoles/min)/(0.001 g) = 3.17 µmoles/min/g.

Dear I am published two paper from the phD thesis to my Student in (2020):

1. Doaa Khalid Mezaal, Essam Fadel Al-Jumaili and Ahmed Majeed Al-Shammari (2020). Production and Purification of Laccase Enzyme by Klebsiellapneumoniae K7 . IISTE.

You can Use Nelson- Somogy Method.

May help you

You may want to Google "simulation of enzymatic catalysis"

Some interesting hits:

Two Sides of Quantum-Based Modeling of Enzyme-Catalyzed Reactions: Mechanistic and Electronic Structure Aspects of the Hydrolysis by Glutamate Carboxypeptidase

If your plasmid really is pPLc245 (and not some derivative of it) then it should not carry the lambda repressor gene and you should get constitutive expression at any temperature. So I can't really explain your result unless RT does not fold properly to have activity at RT or 28.

I'm actually a bit surprised this worked at all for you though, generally plasmids with superstrong promoters like lambda pL are not very stable under conditions where there is no repressor.

Dear Khajezade,

Greetings and have a nice time.

You can remove the pigment easily by using acetone or chloroform. Any one of them is useful to dissolve the pigment well. You can try any one.

With regards.

Hello, I agree with Engelbert Buxbaum, apparently there should be no problems following that methodology, I would also suggest that you review some aspects that may be affecting the analysis, such as:

-preparation of reagents and buffers (verify pH, ionic strength, etc.)

repeat the procedures in the same way for the blank and the sample, eg the incubation time, the same centrifugation time to remove the pellet

check the detection range of your spectrophotometer and the wavelength selected for analysis.

You can also do a quick test and use the reaction blank without enzyme (control -) as the autozero and use the reaction medium plus the enzyme plus the TCA as the blank to inactivate it before the incubation process, after this time centrifuge and then that it would be your positive control to correct any color that could be caused by amino acid residues and then read your sample

I hope I have helped you in some way

regards!!!

This is a case where a non-standard unit definition is being used. According to the Sigma document the unit is:

Unit Definition: One unit will liberate 1.0 mg of maltose from starch in 3 minutes at pH 6.9 at 20 °C.

The 3 minutes is already accounted for by the definition, so you don't have to divide by it.

Look, unless your project is to design methods for expressing protein domains, I'd forget your idea of making it in pieces and hoping they will somehow combine properly. It is 1) unlikely to work and 2) very time-consuming. And 3) you'd need to have the entire protein as a comparison anyway!

Try finding a better way to express the native protein. Have you tried putting it in yeast? E. coli strains that are optimized for large proteins? Under an inducible promotor? Lots of good protocols already exist.

Good luck!

The buffer will have be different or the precipitation will happen again ; ) Some have used cyclodextrins to encapsulate compounds with limited solubility, like chrysin, but you'll need controls to be sure its still accessible to your enzyme. Some nonionic surfactants may help, often bellow their CMC, but controls will need to be done to be sure the assay is still functional. Enzymes that catalyze membrane bound or insoluble substrates are a real challenge, good luck!

And what about TAS??

the so called Pluthero method to purify Taq is quite old (1993) and while simple it can result in low yields and DNA contaminants (see "A simple and efficient method for extraction of Taq DNA polymerase" 2015)

I don't think the ammonium sulphate precipitation is required for activity (please correct me if I' wrong) but it may be worth a try on MMLV RT, as they are very different proteins. Yields are sometimes not very important and Am sulph precipitation can be a very useful method to isolate/purify and concentrate protein preps. I might just use a modern MMLV RT prep like this https://www.protocols.io/view/recombinant-protein-expression-of-mmlv-rt-h-yxmvmxmw9l3p/v1?version_warning=no

iff your constructs are similar, good luck!

If you want to know if the protein is cleaved during its biosynthesis in a cell or following secretion from the cell (either autolytic or by some other protease), you could follow its mass over time following induction of the gene. This could be done using ³⁵S-methionine labeling, immunoprecipitation and SDS-PAGE or by Western blot, both requiring a specific antiserum or antibody.

I think it is difficult to establish whether the cleavage is autolytic or not when the protein is expressed in its native setting. It might help to express it heterologously, so that whatever activating enzyme would normally be present would instead be absent. Then if the protein is expressed in a soluble form but is not cleaved, you would have evidence that cleavage is not autolytic.

Another approach would be to overexpress the protein in bacteria such that it formed inclusion bodies (which often happens but is not predictable). Then, the protein would be unlikely to be cleaved because it would have precipitated before folding. Refolding the protein from inclusion bodies would be necessary to render it in a soluble form. Because proteins in inclusion bodies can be purified before refolding, you could find out whether the pure protein cleaves itself once it refolds by SDS-PAGE or protein mass spectrometry or N-terminal sequencing.

When I want to express proteins with Fe-S clusters and purify cluster loaded protein (holo-protein) I add 0.2mM (final concentration) to my E.coli culture. I induce with lactose and also add L-cysteine as S source. As I grow my cells in LB, I don't think the extra nitrogen I am adding is doing much. I don't think by adding ammonium ferric citrate will impair E.coli growth or protein production, for sure it will not harm the induction experiment under normal conditions. If you could specify a bit more your objective, we might give you more insight.

Did you solve your problem?

Antara Chakravarty

I plotted your data (extracted using webplot, Rohatgi) using graphPad and obtained values very close to what you got, for single y data. Normally I usually plot replicated data, side by side column or enter Mean. SD and N. replicated data usually gives narrower 95%CI data

As described in Cornish-Bowden Fundamentals of Enzyme Kinetics, if the substrates are both mixed together in the reaction with the enzyme, the ratio of the velocity of A to the velocity of B is (kcat/Km*[A])/(kcat/Km*[B]), so if the samples are mixed and the substrate concentrations are equal, A will be converted faster at all concentration, thus the name specificity constant for kcat/Km. However, if the reactions are separate and the concentrations are near saturating, the kcat will determine which rate is faster.

A standard curve must be prepared under the identical conditions as the assay. It is not likely that one that you did not prepare yourself will be accurate.

I'm having the same problem! Any update?

I would suggest you to carry out ELISA or WB analysis of Caspases 3/7 for the detection of apoptosis in flash frogen tissue. If you fix the tissue, the enzyme activity will be lost, hence you can homogenize the tissue and take the sup to anayse capase-3/7 activity using ELISA and level by WB.

As you mentioned in your content, you can go ahead with the TUNEL Assay that is very sensitive ,(For example R & D Systems USA) and you can analyse DNA fragmentation in a single cell of you sectioned tissue.

Best

Not only ligand concentration but pH is also an important factor of chelation .

I've not used Arctic Express directly but have some experience of overnight protein expression systems.

We would grow the starter cultures up to whatever the required OD600 density was (this took some optimisation for each construct so no fixed answer on this point), then turned off the heat and propped the incubator lid open to the ambient temperature (somewhere in the teens). This was using Autoinduction medium containing glucose, glycerol and lactose (there are various recipes for this online), so it only starts induction by metabolising the lactose when really high OD after the glucose is exhausted and derepresses the promoter.

Key things we found improved expression levels were adequate aeration - using 3 litre plastic baffled flasks containing at most 1L of culture would give a very aerated culture, and selecting "good" colonies - I engineered a cleavable fusion GFP reporter upstream of the reading frame so you could identify high expression clones by putting the plate on a blue light box, as well as being able to see where the protein was during purification stages. Sometimes it was necessary to go for colonies that didn't express the highest fluorescence to maximise solubility.

Although it costs more, carbenacillin beats ampicillin hands-down for the same plasmid - it breaks down more slowly and is more stable in acid pH that can arise as cultures are prolonged and denser.

Elimination of chaperonins after culture - there is a protocol to wash some of these off your protein on column by addition of ions and ATP - it is a number of years since I've done this so I can't remember the precise details. Again though, this is published data and can be found by searching for this.

The final answer is 'try try try'. Every protein is different, so asking questions here and doing literature searches, then trialling your findings in your own laboratory is often the only way to optimise the system in your hands - what works for one person may not be completely applicable in another laboratory.

Very best of luck!

Yep, nitrocellulose is a poor choice

Something I would have done differently is to have used a pH-buffered solution during the procedure to maintain the pH at the optimal pH of the enzyme.

What expression system are you using? The typical pET based vectors used in BL21(DE3) strains do not work in MC4100 as it lacks the T7 RNA Pol gene required.

I've edited this answer, hopefully a little clearer now, esp. with an example uploaded.

I think the issue is perhaps that you are not taking into account the fact that S1 displays substrate inhibition. Because S2 does not show this effect you are not comparing like with like. Effectively the enzyme is operating in the presence of an inhibitor with S1, whereas with S2 it is not.

However, even ignoring this effect, the Kcat/Km ratio (or Vmax/Km ratio) does not actually predict which is the better substrate in a real assay situation. You need to consider [S] too.

To see the effect of varying S you would need to plug numbers into the equation v = Kcat [E] S/(Km + S). I think you will find that at low S, S1 has the edge and at higher concentrations S2 has a higher rate. This is actually too simplistic for S1 and Adam B Shapiro has given a more complex equation which is relevant to the situation with an inhibitor in the system.

I have now uploaded data for two hypothetical substrates S1 and S2, neither of which show substrate inhibition, using the standard equation v = Vmax[S]/(Km + S). The tabulated data show that there are conditions in which you can get more product with a substrate with a LOWER Vmax/Km ratio. In fact, you can get almost any rates you like by varying S, thus Vmax/Km (or Kcat/Km) ratios alone do not tell you which is the better substrate.

Hopefully, you can now see some possible explanations why your S2 substrate was better than you expected.

Dear Bohuš Kubala

you can try the pcold vecotrs of takara

i used in some year ago for a protein fusion that was degraded with expression at 20-25-30°C in pet vectors and performing the expression at 17°C, I obtained good results.

you can see an example of it at minute 6' 10'' of the following video

present in my blog: ProteoCool

Sincerely in other cases, with more stable proteins they provided lower expression than vectors based on T7 promoter therefore i suggest to you to test it in parallel with T7 and not replace it.

good luck

Manuele

Dear Bohuš Kubala please have a look at the following original research article in which the addition of osmolytes such as betaine has been studied in detail:

Fortunately this paper is freely available as public full text. I hope it helps.

Good luck with your research!

1. Try adjusting the temperature as you have, even trying at 30 could generate some soluble protein,

2. Adjust the amount of IPTG that you use to induce the expressions

3. Adjust the time you carry out the expression for, if you get soluble proteins at 20 C then leave over night to generate more protein.

4. If there is any information out about you protein of interest, does it require a chaperone protein to assist its folding, if so this will need to be co-expressed with the protein,

5. Check, double check, and get someone else to check that in you sequenced clone your gene of interest is in frame with the His Tag, you will be surprised how often the cloning is done incorrectly.

6.a Change the tag or place it at the c- terminus in the construct, it may be that your tag is affecting the folding of the protein, but less likely here as you have tried to express fragments and they are still stubborn. I would try to express the protein with no tag to see if it is soluble in the cells you are using.

6.b If I have an insoluble protein then MBP (maltose binding protein) is one of the best bets, a bulky soluble protein that can be easily cleaved off in many cases.

7.Change the cells you are using to express, as it is a viral protein, the codon bias may be restricting the accurate synthesis of your protein, therefore it may be worth trying Codonplus BL21, or other derivatives such as Rosetta gami strains

8. Another option is to purify the inclusion bodies of you insoluble proteins, and refold them, this can be done either by refolding on a his-trap or sequential dialysis in decreasing urea concentrations

I hope that offers you some avenues to try out!

9. For the non specific bands contaminating your sample, that is normal - always try to extend the washing step on the purification column to remove non specific binders, the other option there is to identify the concentration of imidazole that your protein of interest elutes at, then create a gradient and collect very small fractions to minimize contaminations. APS cuts before this step also reduce the amount of protein you load onto a column, overloading can generate many non specific interactions!

Good luck!!

Also, Enzymes are used in industrial processes, such as baking, brewing, detergents, fermented products, pharmaceuticals, textiles, leather processing. Here are a range of processes showing how enzymes are used. See the RG link:

It is just a salt form of NE. I haven't done this assay but Googling NE + bitartrate + COMT + assay produces results that show this substrate form has been used by others.

There are several tests available to measure the catalase activity in brain homogenates. Hope you are trying to analyze enzyme activity using colorimetric assay kit. We have used the sensitive kit purchased from BioVision, Inc., CA that is very sensitive reproducible. If you are using the colorimetric assay kit, follow our paper published in Brain Research Bulletin 95 (2013) 54– 60. For more detail, see our research paper. Follow the kit mannual for calculating the catalase activity. All the best.

Please have a look at some enclosed PDFs...

19T-V is better than 19T in targeting the beginning of the polyA site, yes. I believe 19T-VN just gives a slightly tighter clamp.

As to the reaction conditions, yes: heat your RNA + oligodT to 65 for 5 mins, crash cool on ice, then add the buffer/dNTPs/enzyme, mix well, spin down briefly, leave at RT for 10mins, then over to 42 for 25mins.

If you have a solution with 0.5 U/mg is the same of 500 U/g, so if you wish 50 U/g you must dilute 10 times that solution in the appropriate solvent or activity buffer.

You may also have a look for this option;

Thermo M-PER™ Mammalian Protein Extraction Reagent for RIPA like non-denaturing lysis buffer...

Pyruvate carboxylase

Rationale: A biotin-dependent enzyme belonging to the ligase family that catalyzes the addition of CARBON DIOXIDE to pyruvate. It occurs in both plants and animals. Deficiency of this enzyme causes severe psychomotor retardation and ACIDOSIS, LACTIC in infants.

I have been successfully storing at -80 °C, making a separate working solution for other immediate assays, to avoid multiple freeze-thaw.

Thank you for your suggestion Maurice Ekpenyong . Although I’m hoping to look at enzyme activity rather than any cytochemistry. Reading the paper, it sounds like DMSO and snap freezing with LN2 similar to Freezing live cells would be enough. I may have to run a comparison.

best wishes.

Thanks @M Angeles Zorrila Lopez- Perea

Check https://pubmed.ncbi.nlm.nih.gov/22410228/

Hi there,

I think the best example to illustrate your question is the hexokinase/glucokinase which are in charge of the first step of glycolysis. Hexokinase exhibits a low Km compared to glucose concentration because it has the role to incorporate glucose into glycolysis in order to produce ATP at the end. As the cell needs ATP at any time hexokinase exhibit a low Km compared to glucose concentration in cell. Then its activity is more or less constant and near maximal capacity whatever the situation. Glucokinase has the same activity as hexokinase but only at the hepatic level (whereas hexokinase acts in any cell). Its role is to sense glucose level in order to regulate insulin/glucagon balance therefore glucokinase exhibits a higher Km so that glucose concentration variation will induce also a variation in glucokinase activity which will initiate a cellular response in order to optimize glucose metabolism. So basically the same reaction is used in 2 different ways by 2 different enzymes belonging to the same enzyme family: their properties are in accordance with their respective roles in the cell.

For all these reasons (and may be some more) the measured enzymatic activity will be lower by a stationary efficiency factor η_s = turnover of immobilised enzyme / turnover by free enzyme ≤ 1, or by the operational efficiency η_o = τ_f / τ_i ≤ 1 (where τ is the relaxation constant, the time for [S] to drop to 1/e ≈ 37% of the starting value). Note that the proper way to determine τ_f is after destruction of the matrix by ultrasound.

Knowing you are wanting to measure the Enzymatic Activity of a Mannosidase, you should be using a p-Nitrophenyl Mannopyranoside as a substrate, where the product you measure in the reaction is the P-Nitrophenol, which has a specific Extinction Coefficient. You can then simply measure the Activity of your mannosidase in the appropriate buffer system and using the appropriate Stop reagent, to obtain a Delta A405 of a test sample verus a Blank Sample and with Volume of reaction known, dilution of the Mannosidase known, Time of reaction known, and the Extinction Coefficient, obtain your desired umol/min/mg. See example in Attachment.

Juan C Pérez-Casanova - I would not recommend freeze drying the basic liver samples - that's a complex tissue to freeze dry and preserve the enzyme. Solid tissues, even small samples, generally take days to freeze dry, and then the challenge is to reduce the residual moister to <2%. Freeze drying can preserve enzymes well, when properly prepared, most PCR reagents and enzymes are freeze dryed, for example. I suggest that you extract your enzyme, aiquot in buffer and freeze dry the aliquots, then store in the freezer. Do watch out though, even freezing can affect activity as there can be a pH shift in buffer, when you freeze.

@Marco: you are definitly right! I made a slight calculation error... ;)