Denaturation studies on natural and recombinant bovine prochymosin (prorennin).


Abstract

1. Prochymosin in solution in the presence of 8 M-urea is fully unfolded, as indicated by its fluorescence spectrum, fluorescence quenching behaviour and far-u.v.c.d. spectrum. 2. Equilibrium studies on the unfolding of prochymosin and pepsinogen by urea were carried out at pH 7.5 and pH 9.0. The results indicate that the stabilization energies of the two proteins are identical at pH 7.5, but that at pH 9.0 pepsinogen is significantly less stable than prochymosin. 3. Kinetic studies on the unfolding of prochymosin and pepsinogen indicate that the processes can be described by a single first-order rate constant, and that at any given value of denaturant concentration and pH the rate of unfolding of prochymosin is significantly greater than that of pepsinogen. 4. Unfolding of prochymosin by concentrated urea is not fully reversible, unlike that of pepsinogen. Kinetic analysis of the refolding of the proteins suggests the presence of a slow process following unfolding in urea; for pepsinogen this process leads to a slowly refolding form, whereas for prochymosin the slow process in urea leads to a form that cannot refold on dilution of the denaturant. 5. The results provide a rationale for an empirical process for recovery of recombinant prochymosin after solubilization of inclusion bodies in concentrated urea. 6. In all respects studied here, natural and recombinant bovine prochymosin were indistinguishable, indicating that the refolding protocol yields a recombinant product identical with natural prochymosin. Study holds ProTherm entries: 7321, 7322, 7323, 7324, 7325, 7326 Extra Details:

Submission Details

ID: M42T4v4h3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:33 p.m.

Version: 1

Publication Details
Sugrue R;Marston FA;Lowe PA;Freedman RB,Biochem. J. (1990) Denaturation studies on natural and recombinant bovine prochymosin (prorennin). PMID:2241930
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
4AUC 2013-05-29 1.6 Bovine chymosin in complex with Pepstatin A
4AA8 2012-12-12 1.8 Bovine chymosin at 1.8A resolution
3CMS 1992-10-15 2.0 ENGINEERING ENZYME SUB-SITE SPECIFICITY: PREPARATION, KINETIC CHARACTERIZATION AND X-RAY ANALYSIS AT 2.0-ANGSTROMS RESOLUTION OF VAL111PHE SITE-MUTATED CALF CHYMOSIN
4CMS 1991-11-07 2.2 X-RAY ANALYSES OF ASPARTIC PROTEINASES IV. STRUCTURE AND REFINEMENT AT 2.2 ANGSTROMS RESOLUTION OF BOVINE CHYMOSIN
1CZI 1997-04-01 2.3 CHYMOSIN COMPLEX WITH THE INHIBITOR CP-113972
1CMS 1990-01-15 2.3 THE THREE-DIMENSIONAL STRUCTURE OF RECOMBINANT BOVINE CHYMOSIN AT 2.3 ANGSTROMS RESOLUTION

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
94.7 Chymosin P18276 CHYM_SHEEP
100.0 Chymosin P00794 CHYM_BOVIN
100.0 Pepsin A P00792 PEPA_BOVIN