Banca de QUALIFICAÇÃO: PRICILA GOMES DA SILVA

Uma banca de QUALIFICAÇÃO de MESTRADO foi cadastrada pelo programa.
STUDENT : PRICILA GOMES DA SILVA
DATE: 23/05/2024
TIME: 14:30
LOCAL: Sala Virtual Remota
TITLE:

EVALUATION OF THE ENZYMATIC ACTIVITY OF A COMPLEX
BETWEEN BOVINE CATHEPSIN D AND BthTX-II FROM THE VENOM OF Bothrops
jararacussu.

KEY WORDS:

Cathepsin D, Snake Venoms, Phospholipases A2, Enzyme Complex

PAGES: 48
BIG AREA: Ciências Biológicas
AREA: Biologia Geral
SUMMARY:

Phospholipase A2 (PLA2) and Cathepsin D are important enzymes in various human
diseases, including neurodegenerative disorders and cancer. Aspartic proteases similar
to Cathepsin D are also essential for the life cycle of various parasites and can mediate
pathological processes in different cellular environments. Recently, Moraes and
colleagues (2022) showed that human CatD and phospholipase A2 from the venom of B.
jararacussu are able to bind and form a stable and functional complex. The objective of
the present work was to identify and characterize the enzymatic activity of the bovine
Cathepsin D and BthTX-II complex against different substrates and physicochemical
conditions. The methodology used to obtain Cathepsin D consisted of grinding 500g of
the bovine liver with 20% acetone in a blender for approximately 3 minutes. Next, the
sample was applied to immobilized affinity chromatography with Sepharose + Pepstatin
A previously equilibrated with 0.1M sodium acetate pH 5.0 and eluted in a step gradient
of 0-100% sodium acetate pH 5.0 + 0.5M NaCl (buffer B) and buffer C containing 0.1M
Tris-HCl (pH 8.5) under a flow rate of 1 mL/min for 40 minutes. Subsequently, the sample
of interest was subjected to a second chromatographic step on a C18 reverse-phase
column (25 cm x 0.45 cm-Discovery) previously equilibrated with 0.1% trifluoroacetic acid
and eluted under a gradient of 0-70% solution B [ACN 99.9% (v/v) and TFA 0.1% (v/v)]
at a flow rate of 1 mL/min. The evaluation of relative mass, purity degree, and the
isoelectric point was observed by SDS-PAGE 12.5% and two-dimensional
electrophoresis, as well as the determination of the enzyme's ideal pH and temperature
activity on the substrate casein, with readings on a spectrophotometer at 660 nm. The
activity of BthTX-II was evaluated on the substrates casein and 4N3OBA. The protein
isolated from the bovine liver showed a relative molecular mass of approximately 32 kDa,
a pI of 5.9, and ideal activity on the casein substrate at pH 3 and temperature 50°C,
similar to data observed in the literature for CatD from other organisms. In addition,
BthTX-II showed significant activity on the specific synthetic substrate 4N3OBA and no
activity on casein, as expected. In summary, the results obtained so far confirm the
effectiveness of the new protocol developed for obtaining, purifying, and physicochemical
characterization of Cathepsin D from the bovine liver. These results provide a solid
foundation for future research and applications in the fields of biotechnology and health.

COMMITTEE MEMBERS:
Presidente - ***.161.386-** - ANDREIMAR MARTINS SOARES - FIOCRUZ
Externo à Instituição - LUIS ANDRE MORAIS MARIÚBA - FIOCRUZ
Externa à Instituição - NIDIANE DANTAS REIS PRADO - São Lucas