Extraction and Quantification of Proteins in Syzygium jambos used as Diabetic Adjuvants in Puerto Rico

Yolanda I. Rodrguez-Rivera BIOL 4990 Mentor: Dr. Jannette Gavilln-Surez

Proteins
Proteins are biochemical compounds consisting of polypeptides that facilitate a biological function.
Ramrez Ramrez, G, Heil, M, Orona Tamayo, D, Adame lvarez RM, Ramrez Ramrez, DT, Durn Flores, FD. 2008. ANLISIS DE PROTENAS EN GRNULOS Y HOJAS DE Acacia hindsii Y EN HORMIGAS DE LA REGIN DE IRAPUATO . Guanajuato (MX): Universidad Autnoma de Quertaro (MX). Disponible en: http://www.uaq.mx/investigacion/difusion/veranos/memorias2008/10VeranoRegionCentro/17UAQRamirezRamirez.pdf

Protein Functions

Solubility of Proteins
Hydrophilic and Hydrophobic residues

on surface Charged and polar surfaces will have higher solubility

Previous studies
A study shows that a protein named ADMc1 in the seeds of Momordica charantia (cundiamor) has hypoglycemic/antihyperglycemic properties.

United States Patent Application Publication, Antidiabetic Protein: Pub.No: US 2011/0046054

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.

Specific aims of this study

To extract proteins on leaves of Syszygium jambos (Pomarrosa del ro).
To quantify the proteins by the Lowry method.

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

Methodology : Extraction of Proteins

3 replicates of the plant
Wash pellet with acetone, 10% TCA and 80% acetone. Centrifuge.

Recover insoluble proteins by centrifugation

Separate phases and precipitate Ph phase with methanol and ammonium acetate.

Wash 3 times with methanolic ammonium acetate.

Wash with 80% acetone

2 g of of dry pellet is resuspended in Phenol

Add dense SDS buffer and centrifuge

Quantify proteins in pellet and supernatant

Methodology: Protein Quantification

BSA Concentration and Calibration Curve
Add BSA [0, 20, 40, 60, 80, 100 l] to these tubes & 100 l of unknowns Add 2 ml of Lowry Solution to each one of the test tubes Incubate 10 minutes at room temperature Vortex each tube immediately

Add 0.2 ml of diluted Folinphenol solution to each tube

Determine absorbance of each sample at 600 nm

Incubate at room temperature for 30 minutes

Plot absorbance vs mg BSA to obtain a calibration curve

Lowry colorimetric reaction

Results
BSA Calibration Curve
0.35 0.3

0.25
Absorbance (600 nm)

0.2

0.15

y = 7.7027x + 0.0333 R = 0.9939

0.1

0.05

0 0 0.005 0.01 0.015 0.02 BSA Concentration (mg/ml) 0.025 0.03 0.035 0.04

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
(Gmez-Vidal, et.al)

Analysis of Results
The amount of protein found in this assay is comparable to results in Gmez-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.

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

References
Gmez-Vidal S, Tena M, Lpez-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 Ramrez Ramrez, G, Heil, M, Orona Tamayo, D, Adame lvarez RM, Ramrez Ramrez, DT, Durn Flores, FD [Internet]. 2008. ANLISIS DE PROTENAS EN GRNULOS Y HOJAS DE Acacia hindsii Y EN HORMIGAS DE LA REGIN DE IRAPUATO . Guanajuato (MX): Universidad Autnoma de Quertaro (MX). Disponible en: http://www.uaq.mx/investigacion/difusion/veranos/memorias2008/10VeranoRegionCentro/17UAQRamirezRamirez.pdf

Extraction and Quantification of Proteins in Syzygium jambos used as Diabetic Adjuvants in Puerto Rico
Yolanda I. Rodrguez-Rivera BIOL 4990 Mentor: Dr. Jannette Gavilln-Surez