Pepsin is a member of the aspartate proteases family and it shows maximum activity at  acidic pH (http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.4.23.1).

My suggestion is to keep pepsin at neutral (7-7.5 pH at -80°C to limit autolysis. The activity can be restored upon re-acidification using proper buffer (see http://www.ncbi.nlm.nih.gov/pubmed/11106168).

As you suggested, pepsin activity is lower  over time because the concentration of the substrate  will be lower and lower over time. The enzyme kinetic obeys to a first-order rate law ( http://www.ncbi.nlm.nih.gov/pubmed/11106168).

Furthermore pepsin appears to be sensitive to substrate structure, with a measurable effect on the kcat reported by several authors (http://pubs.acs.org/doi/abs/10.1021/bi00825a001). This effect on mcat will have, in turn, an effect on the velocity of the reaction.

http://pubs.acs.org/doi/abs/10.1021/bi00825a001

http://www.ncbi.nlm.nih.gov/pubmed/11106168

http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.4.23.1

Hi Alberto, proteinases like pepsin do not cleave peptide bonds at random.

Pepsin prefers to cleave peptides that fit well into the active site. Therefore it preferentially cleaves some peptides better than others. I quote from this paper:

 http://peds.oxfordjournals.org/content/21/12/699.full.pdf+html

"Pepsin has the highest preference for Phe and Leu in the P1 position, followed by Met, but also tolerates Tyr, Trp, Cys, and Glu in the P1 position. The P1’ position does not have as strong an influence on pepsin specificity as the P1 position, although aromatic residues (Tyr, Phe, and Trp) and, to a lesser extent, Ile and Val are preferred. Conversely, the presence of His, Lys, Arg, and Pro in the P1 position strongly disfavors pepsin activity. Pepsin also discriminates against Pro residues in the P2 position and, to a lesser extent, in the P2’ and P3’ positions. In addition, Arg, Lys, or His residues are disfavored in the P3 position"

(To explain, the P1-P1' is the peptide bond that is cleaved. P2, P3, etc. residues are N-terminal to the cleavage site. P2', P3', etc are C-terminal to the cleavage site.) 

my guess  that 2 factors could contribute to slowing down:

1) Proteases have preferred sites both structure and sequence wise,

once preferred sites  are depleted, reaction slows down.

2) Pepsin kills it self over time 

Thank you Steingrimur and Misha for your answers! In your opinion, how quickly does the pepsin "kills itself"? Does it dies out linearly over time, or faster?

Hi Alberto, I don't know of any systematic study on the autolysis (i.e. self digestion) of pepsin.

But I do know that researchers working with proteinases keep them at -80oC, or lower, to ensure that there is no free H2O around to promote hydrolysis.

Pepsin is a member of the aspartate proteases family and it shows maximum activity at  acidic pH (http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.4.23.1).

My suggestion is to keep pepsin at neutral (7-7.5 pH at -80°C to limit autolysis. The activity can be restored upon re-acidification using proper buffer (see http://www.ncbi.nlm.nih.gov/pubmed/11106168).

As you suggested, pepsin activity is lower  over time because the concentration of the substrate  will be lower and lower over time. The enzyme kinetic obeys to a first-order rate law ( http://www.ncbi.nlm.nih.gov/pubmed/11106168).

Furthermore pepsin appears to be sensitive to substrate structure, with a measurable effect on the kcat reported by several authors (http://pubs.acs.org/doi/abs/10.1021/bi00825a001). This effect on mcat will have, in turn, an effect on the velocity of the reaction.

http://pubs.acs.org/doi/abs/10.1021/bi00825a001

http://www.ncbi.nlm.nih.gov/pubmed/11106168

http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.4.23.1

Concerning autocatalysis - you can try minimizing the effect, by having many fold more substrate than pepsin - given that the process is statistic in nature, the probability that pepsin finds a genuine substrate molecule is much higher than it finds another molecule of pepsin. Over time your substrate concentration will diminish, and the autocatalysis will gain in importance.

I think you are mixing up certain things. For example Pepsin does not easily undergo hydrolysis. The thiol group in its active site (since it is a cysteine protease) has to be deprotonaned prior to hydrolysis of the substrate by a basic amino acid like histidine (in its active site) which will be reprotonated after the hydrolysis is over and it is by the amine group of an amino acid from one end of the small peptides released by it.  However, by repeated use, no doubt the low substrate concentration might show its lowered activity, but the most important part is that when more and more free amine ends are released from the substrate, it tried to buffer the low pH required for the activity of the enzyme which affects the enzyme activity.

Jai, I'm afraid you are mistaken. Pepsin is an aspartic proteinase.

No question is naive Alberto..Pepsin, an aspartic protease,  is most efficient in cleaving peptide bonds between hydrophobic and preferably aromatic amino acids such as phenylalanine, tryptophan, and tyrosine. So initially when pepsin comes in contact with a protein it shows logarithmic pattern of function in cleaving the peptide bond and as the substrate (here peptide bonds) start reducing with time the activity reduces. There may also be feedback inhibition also due to the aminoacids that are formed as a biproduct which would reduce the activity of the protein. Fianally when the substrate decreases and feedback inhibition starts working due to the accumulation of product, autolysis also starts action thus leading the a combined effect of reducing pepsin activity. Hope this helps ...

All the best

Pepson is an aspartic protease. In a study conducted by Melissa H. Palashof , where 9 proteins (ranging from 68 KD to 8 KD) including the E.coli cell lysate is  digested with pepsin, it was inferred that  at all pH points pepsin will often cleave  after most bulky hydrophobic residues such as leucine and phenylalanine. Additionally, the

residues that most often occur immediately following the cleaved peptide bond are

tryptophan and tyrosine. The data also show that pepsin has a low probability of cleaving after the residues arginine, cysteine, proline and histidine.

It may happen that the smaller peptides may be rich in arginine, cysteine, proline and hisitidine and so after a while the cutting stops completely. Inorder to confirm you may for MALDI -TOF or LCMS to know the amino acid composition of these smaller peptides... Good luck

About study conducted by Melissa H. Palashof. I can't found this study. Can you help me find this article?

After three years (!) I have some sort of an answer myself: with my colleagues, we were able to develop a probabilistic computer model of pepsin activity, based on frequency of cutting reported in literature (mainly from Hamuro et al, 2008 and Powers et al, 1977). Here is a link to the paper, I hope it will be useful to further the discussion :-) Article In silico modeling of protein hydrolysis by endoproteases: a...