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Inhibitory Effect of Post-micellar SDS Concentration on Thermal Aggregation and Activity of Papain

A. Qadeer, M. Zaman, and R. H. Khan*

Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh-202002, India; fax: + 91-571-2721776; E-mail: rizwanhkhan@hotmail.com; rizwanhkhan1@gmail.com

* To whom correspondence should be addressed.

Received February 21, 2014
Papain, a cysteine protease isolated from the latex of Carica papaya, is known to undergo irreversible thermal unfolding. In this study, we found that thermal unfolding of papain is accompanied by a simultaneous self-assembly process where this protein is observed to aggregate above 50°C. The extent of aggregation increased with increasing protein concentration from 3-40 µM. The aggregation was confirmed by enhanced turbidity, light scattering intensity, 1-anilino-8-naphthalene sulfonate (ANS) fluorescence intensity and by transmission electron microscopy. Furthermore, we noted that post-micellar concentration of sodium dodecyl sulfate (SDS) remarkably suppresses the thermal aggregation of papain. Far-UV circular dichroism studies revealed that SDS significantly enhances α-helical content of the protein and also tends to prevent its unfolding, and thus inhibits aggregation. Additionally, papain showed maximal activity at 65°C in neutral buffer. However, in the presence of 6 mM SDS (above its critical micellar concentration), the enzyme lost activity by about 10-fold. Thus, promoting the helical propensity of the protein does not appear to be a suitable strategy to overcome the aggregation related problems of industrially important proteins such as papain, which are not only required to be protected against aggregation but also need to remain functionally active in the presence of aggregation inhibitors.
KEY WORDS: papain, thermal aggregation, sodium dodecyl sulfate (SDS), critical micellar concentration (CMC), circular dichroism, 1-anilino-8-naphthalene sulfonate (ANS)

DOI: 10.1134/S0006297914080069