Effect of Temperature and pH on the Secondary Structure and Denaturation Process of Jumbo Squid Hepatopancreas Cathepsin D.
Authors: Francisco Cadena-Cadena , Luis Cárdenas-López José , Marina Ezquerra-Brauer Josafat , Javier Cinco-Moroyoqui Francisco , Alexis López-Zavala Alonso , del Carmen Santacruz-Ortega Hisila , Alfredo Rivero-Espejel Ignacio 
Published: April 2019

Background:
Cathepsin D is a lysosomal enzyme that is found in all organisms acting in
protein turnover, in humans it is present in some types of carcinomas, and it has a high activity in
Parkinson's disease and a low activity in Alzheimer disease. In marine organisms, most of the
research has been limited to corroborate the presence of this enzyme. It is known that cathepsin D
of some marine organisms has a low thermostability and that it has the ability to have activity at
very acidic pH. Cathepsin D of the Jumbo squid (Dosidicus gigas) hepatopancreas was purified and
partially characterized. The secondary structure of these enzymes is highly conserved so the role of
temperature and pH in the secondary structure and in protein denaturation is of great importance in
the study of enzymes. The secondary structure of cathepsin D from jumbo squid hepatopancreas
was determined by means of circular dichroism spectroscopy.

Objective:
In this article, our purpose was to determine the secondary structure of the enzyme and
how it is affected by subjecting it to different temperature and pH conditions.

Methods:
Circular dichroism technique was used to measure the modifications of the secondary
structure of cathepsin D when subjected to different treatments. The methodology consisted in
dissecting the hepatopancreas of squid and freeze drying it. Then a crude extract was prepared by
mixing 1: 1 hepatopancreas with assay buffer, the purification was in two steps; the first step
consisted of using an ultrafiltration membrane with a molecular cut of 50 kDa, and the second step,
a pepstatin agarose resin was used to purification the enzyme. Once the enzyme was purified, the
purity was corroborated with SDS PAGE electrophoresis, isoelectric point and zymogram. Circular
dichroism is carried out by placing the sample with a concentration of 0.125 mg / mL in a 3 mL
quartz cell. The results were obtained in mdeg (millidegrees) and transformed to mean ellipticity
per residue, using 111 g/mol molecular weight/residue as average. Secondary-structure estimation
from the far-UV CD spectra was calculated using K2D Dichroweb software.

Results:
It was found that α helix decreases at temperatures above 50 °C and above pH 4. Heating
the enzyme above 70°C maintains a low percentage of α helix and increases β sheet. Far-UV CD
measurements of cathepsin D showed irreversible thermal denaturation. The process was strongly
dependent on the heating rate, accompanied by a process of oligomerization of the protein that
appears when the sample is heated, and maintained a certain time at this temperature. An amount
typically between 3 and 4% α helix of their secondary structure remains unchanged. It is consistent
with an unfolding process kinetically controlled due to the presence of an irreversible reaction. The
secondary structure depends on pH, and a pH above 4 causes α helix structures to be modified.

Conclusion:
In conclusion, cathepsin D from jumbo squid hepatopancreas showed retaining up to
4% α helix at 80°C. The thermal denaturation of cathepsin D at pH 3.5 is under kinetic control and
follows an irreversible model.