[hlaurenc]

I recently carried out an investigation into the specificity of chymotrypsin and trypsin which I am in the process of writing up. In short, combined substrate with enzyme, incubated for 15 mins and measured at 430nm. Used trendline equation to determine mmol products formed per minute.

N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and N-succinyl-L-phenylalanine-p-nitroanilide (NSPLN) were the synthetic substrates used to be recognised by trypsin and chymotrypsin, respectively.

Results came back as expected; however, when doing the opposing combinations (chymotrypsin-BAPNA and trypsin-NSLPN), activity levels were recorded. I have been racking my brains and books for hours and hours and can't seem to understand how there would be any activity levels as trypsin doesn't cleave at phenylalanine, nor chymotrypsin at arginine.

I'm a first year 'mature' student undergraduate returning to university with no science background - this part is mind boggling!

Thank you in advance

[AWK]

Check the specificity of the enzymes a little more closely. Even the Worthington manual can help a little.

[hlaurenc]

Thank you. I did actually look through this yesterday evening but it didn't explain anything to me.
I'm an undergrad for Human Nutrition doing an intro to Biochemistry - this may be way above my level of understanding as far as chemistry; however, it's frustrating as there's an explanation out there somewhere.

My knowledge goes as far as knowing chymotrypsin cleaves after residues with aromatic sides chains i.e. tryp, tyr and phenyl and trypsin cleaves at residues after positively charged side chains, i.e. arg and lys.

My results show a reaction between the substrate, that contains arg, and chymotrypsin and also between the substrate, that contains phenyl, and trypsin.

Is it something to do with the N-terminal for either? Is arginine is present when activating chymotrypsin from chymotrypsinogen by trypsin?
Trypsin - proline residue at the end or something to do with the acidic residue?

I'm not looking for the answer to be given to me, just a shove in the right direction:)

[AWK]

The enzyme specificity is determined for peptides and not for amino acid derivatives. Perhaps the substituents on the nitrogen atom mimic some amino acid fragments.

[hlaurenc]

Ok thanks, so in other words, I need to look closer at the N-terminal of the polypeptide as opposed to just researching C-terminal?

[AWK]

Look for the similarity of the substituents on the nitrogen atom to the other structural elements needed for the activity of the enzyme.

[hlaurenc]

Thanks for your help

[Babcock_Hall]

You say that activity levels were recorded, but you don't say how much activity.  That might be a useful piece of information, possibly as a percentage, relative to the expected combination of enzyme and substrate.

[hlaurenc]

The activity levels were measured spectrophotometrically at 430nm to subsequently calculate the umol products formed per minute.

Results as follows (umol p/min):
Trypsin/BAPNA   0.038 (as expected)
Trypsin/NSLPN   0.006 (?)
Chymotrypsin/BAPNA   0.011 (?)
Chymotrypsin/NSLPN   0.028 (as expected)

[hlaurenc]

0.1ml of enzyme, 0.9ml of substrate and 1.0ml pH 8 buffer solution - incubated at 37˚C for 15 mins

[Babcock_Hall]

One of the things that occurred to me was that there could be an impurity of trypsin in the chymotrypsin and vice versa.  However, your rates are too high for this to be a likely explanation.

[hlaurenc]

Thanks anyway @Babcock_Hall
Will get there