1. Extraction and Quantification of
Proteins in Syzygium jambos used as
Diabetic Adjuvants in Puerto Rico
Yolanda I. Rodríguez-Rivera
BIOL 4990
Mentor: Dr. Jannette Gavillán-Suárez

2. Proteins
• Proteins are
biochemical
compounds
consisting of
polypeptides that
facilitate a biological
function.
Protein Functions
Ramírez Ramírez, G, Heil, M, Orona Tamayo, D, Adame Álvarez
RM, Ramírez Ramírez, DT, Durán Flores, FD. 2008. ANÁLISIS DE
PROTEÍNAS EN GRÁNULOS Y HOJAS DE Acacia hindsii Y EN
HORMIGAS DE LA REGIÓN DE IRAPUATO . Guanajuato (MX):
Universidad Autónoma de Querétaro (MX). Disponible en:
http://www.uaq.mx/investigacion/difusion/veranos/memorias-
2008/10VeranoRegionCentro/17UAQRamirezRamirez.pdf

3. Solubility of Proteins
 Hydrophilic and Hydrophobic residues
on surface
Charged and polar surfaces will have
higher solubility

4. Previous studies
A study shows that a protein named ADMc1 in
the seeds of Momordica charantia
(cundiamor) has hypoglycemic/anti-
hyperglycemic properties.
United States Patent Application Publication, Antidiabetic Protein: Pub.No: US
2011/0046054

5. Previous studies
• ADMc1 was found highly effective and needed
to be administered only once a day to
maintain normal blood glucose levels.
• The protein was detected and purified from
the seeds of the fruit the plant provides.
• Published work states that the polypeptide-p
is made of 166 amino acid residues and said
to be of 11 kDa.

6. Specific aims of this study
To extract proteins on leaves of Syszygium
jambos (Pomarrosa del río).
 To quantify the proteins by the Lowry
method.

7. Relevance
Determination of proteins that could serve as
biomarkers of hypoglycemic/anti-
hyperglycemic activities for the reduction of
blood glucose in diabetic patients.

8. Methodology : Extraction of Proteins
3 replicates
of the plant
Wash pellet with
acetone, 10% TCA
and 80% acetone.
Centrifuge.
2 g of of dry
pellet is
resuspended in
Phenol
Add dense SDS
buffer and
centrifuge
Separate phases and
precipitate Ph phase
with methanol and
ammonium acetate.
Recover
insoluble
proteins by
centrifugation
Wash 3 times
with methanolic
ammonium
acetate.
Wash with 80%
acetone
Quantify
proteins in
pellet and
supernatant

9. Methodology: Protein Quantification
BSA
Concentration
and Calibration
Curve
Add BSA [0, 20, 40,
60, 80, 100 µl] to
these tubes & 100
µl of unknowns
Add 2 ml of Lowry
Solution to each
one of the test
tubes
Incubate at room
temperature for 30
minutes
Add 0.2 ml of
diluted Folin-
phenol solution to
each tube
Incubate 10
minutes at room
temperature
Vortex each tube
immediately
Determine
absorbance of each
sample at 600 nm
Plot absorbance vs
mg BSA to obtain a
calibration curve

10. Lowry colorimetric reaction

11. Results
y = 7.7027x + 0.0333
R² = 0.9939
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04
Absorbance(600nm)
BSA Concentration (mg/ml)
BSA Calibration Curve

12. Results for Syszygium jambos supernatant
• Concentrations of unknowns:
– X1 = 0.33 mg/ml
– X2 = 0.57 mg/ml
– X3 = 0.44 mg/ml
[Protein] = 0.45 mg/ml of supernatant
[Protein] = 0.23 mg/g dry weight
In comparison with:
[Protein] = 0.25 mg/g dry weight
(Gómez-Vidal, et.al)

13. Analysis of Results
• The amount of protein found in this assay is
comparable to results in Gómez-Vidal, S, et.al.
• Given that they were found in the supernatant, they
could be protein inside the cell.
• The proteins found on the pellet, which are
insoluble, are probably structural and could pertain
to the cell wall.

14. Future Work
• Analyze protein in leaves of studied plants
and categorize them by their function via 2-
dimensional electrophoresis.
• Extract and quantify proteins in leaves of
Tapeinochious annasae, Costus speciousus and
Tradescantia spathacea.

15. References
Gómez-Vidal S, Tena M, López-Llorca LV, Salinas J (2007). Protein Extraction from
Phoenix dactylifera L. leaves, a recalcitrant material, for two dimensional
electrophoresis. Electrophoresis, 29: 448-456.
Hurkman WJ, Tanaka CK (1986). Solubilization of Plant Membrane Proteins for
Analysis by Two-Dimensional Gel Electrophoresis. Plant Physiol., 81: 802-806.
Biology at Davidson [Internet]. 2000. Biology at Davidson in North Carolina. Protein
Determination - Lowry Procedure; 2011 Sep 16. Available from:
http://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.ht
ml
Ramírez Ramírez, G, Heil, M, Orona Tamayo, D, Adame Álvarez RM, Ramírez
Ramírez, DT, Durán Flores, FD [Internet]. 2008. ANÁLISIS DE PROTEÍNAS EN
GRÁNULOS Y HOJAS DE Acacia hindsii Y EN HORMIGAS DE LA REGIÓN DE
IRAPUATO . Guanajuato (MX): Universidad Autónoma de Querétaro (MX).
Disponible en: http://www.uaq.mx/investigacion/difusion/veranos/memorias-
2008/10VeranoRegionCentro/17UAQRamirezRamirez.pdf

16. Extraction and Quantification of
Proteins in Syzygium jambos used as
Diabetic Adjuvants in Puerto Rico
Yolanda I. Rodríguez-Rivera
BIOL 4990
Mentor: Dr. Jannette Gavillán-Suárez