I have two enzyme, one of which is full enzyme and the other is truncated ( small domain containing 85 aa is deleted from full enzyme containing 537 aa). I need concentration of purified enzymes for determining kinetic parameters. I use Bradford assay for measuring concentration, but I think due to lack of small domain, we cannot rely this method. in this method, concentration of enzyme without domain is much lower than that of intact enzyme. lower concentration can be related to deleted small domain? how can I determine the concentration of two enzyme so that deletion of domain doesn't affect concentration determining?
How do you obtain the enzymes and why do you think their concentrations would be similar in the first place? What if the concentration of your truncated protein is actually lower?
You can measure the A280 of your proteins as long as they have tryptophan and tyrosine. Make sure nothing in your buffer interferes with the absorbance though.
Or you can try BCA assay.
If you have the two proteins in very pure form, you may consider to measure directly the absorption of the peptide bond at 205 nm. This is, however, a wavelength where essentially everything absorbs light. Therefore, you must be very careful with buffer components and other components of your solutions.
The technique is very sensitive, and you can find proteins at concentrations from 1 µg up to maybe 50 µg/ml.
For details, please look at the original publication in Analytical Biochemistry 59 (1974), 277-282. MEASUREMENT OF PROTEIN BY SPECTROPHOTOMETRY AT 205 NM.
Good luck - Joh. W.
Dear Arthur, coomassie blue in Bradford test react with Arg and Lys. Arg and lys decreased when we deleted this domain. a decrease in Arg and lys can affect our result in Bradford assay. that's why I'm not certain about that.
thank you for guide
Dear Fereshteh,
From my past experience in analyzing protein samples with varying composition I know that specific methods can give misleading, difficult to interpret or even wrong results. But another factor not to be forgotten is how do you define concentration? Millimoles/liter? Gram/liter? The first is independent of molar weight (or removal of parts of the protein) the second is bound to be lower in case the molar mass is decreased by specific removal of part of the protein. I've used the method quoted below, much to my satisfaction, next to the proposed method the authors quote a number of other really simple methods which may be used in your case.
Good Luck !! Kind regards, Leo
Whitaker, John R., and Per Einar Granum. "An absolute method for protein determination based on difference in absorbance at 235 and 280 nm." Analytical biochemistry 109.1 (1980): 156-159.
Dear Leo, thank you for your answer. it can help me.
Regards
Of the relatively simple colorimetric protein assays (BCA, Bradford, Lowry), I think the Lowry is the least sensitive to sequence differences between proteins.
Quantitative amino acid analysis is another way to measure protein concentration. This can be outsourced to a specialist lab.
If you know the molar attenuation coefficient of you protein you can also determine your concentration at 280 nm (or other wavelengths) very exactly, otherwise there are a lot of colorimetric protein assays. All of this have one big error and this is the standardization with a reference protein (like BSA).
An option is to precipitate you protein (chloroform/methanol), when your protein sample is very pure, and then to determine the weigth of your sample.
If you know exactly the sequence of your proteins and the proteins are pure enough (e.g. 95%). Then, the most accurate way is to measure the A280 and calculate the concentration based on extinction coefficient. Otherwise, you might need to use methods like BCA.
The problem with 280 nm measurement is, of course, that the extinction coefficient depends on the content of aromatic amino acids and cysteine and might be different for your intact protein and the truncated form. But the good news is that, so long as you know the sequences you can calculate an appropriate extinction coefficient for each protein. This is based on Gill and von Hippel, Anal. Biochem. 182, 319-326 (1989) and is available through Swiss-Prot. The program is called ProtParam.
However, being a suspicious person, I like solid back-up and I agree with Adam and Leon about quantitative amino acid analysis. In the old days when most biochemistry departments ran an amino acid analyser the usual procedure was to look for the relative amounts of each amino acid and rely on the fact that when you look at CMCys, Trp the numbers will be small but must be integral - the protein will have 2 Cys or 3 Cys but not 2.5! So usually people did not worry too much about being absolutely quantitative. But if you take an accurate volume of your protein with an accurately measured A280, you need to know if you are seeing 100% of your sample on the amino aicd analyser. So the trick is to add a known amount of norleucine - pure and not found in proteins - to your sample. If you only see 98.5% of the norleucine in the analyser in spite of your best efforts to get every last drop transferred then you know that also there is only 98.5% of your hydrolysed protein and can adjust accordingly. So then you know how many nmoles of Glu, Ala, Asp, Gly etc. are in the hydrolysate and, knowing the sequence, each of those peaks will give you an estimate of how many nmoles of total protein you hydrolysed! If it is carefuly done there can be no argument.
You might find the hospital labs. e.g. paediatrics still running an amino acid analyser.
Once you have done this, you will know for ever how similar or dissimilar the extinction coefficients for your two proteins are and also whether Gill and von Hippel's formula works!
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