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Extraction and Quantification ofProteins in Syzygium jambos used as

Diabetic Adjuvants in Puerto RicoYolanda I. Rodrguez-Rivera

BIOL 4990

Mentor: Dr. Jannette Gavilln-Surez

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Proteins

Proteins arebiochemicalcompounds

consisting ofpolypeptides thatfacilitate a biological

function.

Protein Functions

Ramrez Ramrez, G, Heil, M, Orona Tamayo, D, Adame lvarezRM, Ramrez Ramrez, DT, Durn Flores, FD. 2008. ANLISIS DEPROTENAS EN GRNULOS Y HOJAS DE Acacia hindsi iY ENHORMIGAS DE LA REGIN DE IRAPUATO . Guanajuato (MX):Universidad Autnoma de Quertaro (MX). Disponible en:http://www.uaq.mx/investigacion/difusion/veranos/memorias-

2008/10VeranoRegionCentro/17UAQRamirezRamirez.pdf

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Solubility of Proteins

Hydrophilic and Hydrophobic residues

on surface

Charged and polar surfaces will have

higher solubility

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Previous studies

A study shows that a protein named ADMc1 inthe seeds ofMomordica charantia(cundiamor) has hypoglycemic/anti-hyperglycemic properties.

United States Patent Application Publication, Antidiabetic Protein: Pub.No: US2011/0046054

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Previous studies

ADMc1 was found highly effective and neededto be administered only once a day tomaintain normal blood glucose levels.

The protein was detected and purified fromthe seeds of the fruit the plant provides.

Published work states that the polypeptide-pis made of 166 amino acid residues and saidto be of 11 kDa.

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Specific aims of this study

To extract proteins on leaves ofSyszygiumjambos (Pomarrosa del ro).

To quantify the proteins by the Lowrymethod.

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Relevance

Determination of proteins that could serve asbiomarkers of hypoglycemic/anti-hyperglycemic activities for the reduction of

blood glucose in diabetic patients.

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Methodology : Extraction of Proteins

3 replicatesof the plant

Wash pellet withacetone, 10% TCAand 80% acetone.

Centrifuge.

2 g of of drypellet is

resuspended inPhenol

Add dense SDSbuffer andcentrifuge

Separate phases andprecipitate Ph phasewith methanol and

ammonium acetate.

Recoverinsoluble

proteins bycentrifugation

Wash 3 timeswith methanolic

ammoniumacetate.

Wash with 80%acetone

Quantifyproteins inpellet and

supernatant

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Methodology: Protein Quantification

BSAConcentrationand Calibration

Curve

Add BSA [0, 20, 40,60, 80, 100 l] to

these tubes & 100l of unknowns

Add 2 ml of LowrySolution to eachone of the test

tubes

Incubate at roomtemperature for 30

minutes

Add 0.2 ml ofdiluted Folin-

phenol solution toeach tube

Incubate 10minutes at room

temperature

Vortex each tubeimmediately

Determineabsorbance of eachsample at 600 nm

Plot absorbance vsmg BSA to obtain a

calibration curve

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Lowry colorimetric reaction

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Results

y = 7.7027x + 0.0333R = 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

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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)

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Analysis of Results

The amount of protein found in this assay iscomparable to results in Gmez-Vidal, S, et.al.

Given that they were found in the supernatant, theycould be protein inside the cell.

The proteins found on the pellet, which areinsoluble, are probably structural and could pertainto the cell wall.

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Future Work

Analyze protein in leaves of studied plantsand categorize them by their function via 2-dimensional electrophoresis.

Extract and quantify proteins in leaves ofTapeinochious annasae, Costus speciousus and

Tradescantia spathacea.

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References

Gmez-Vidal S, Tena M, Lpez-Llorca LV, Salinas J (2007). Protein Extraction fromPhoenix dactylifera L. leaves, a recalcitrant material, for two dimensionalelectrophoresis. 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. ProteinDetermination - Lowry Procedure; 2011 Sep 16. Available from:http://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.ht

mlRamrez Ramrez, G, Heil, M, Orona Tamayo, D, Adame lvarez RM, RamrezRamrez, DT, Durn Flores, FD [Internet]. 2008. ANLISIS DE PROTENAS ENGRNULOS Y HOJAS DEAcacia hindsiiY EN HORMIGAS DE LA REGIN DEIRAPUATO . Guanajuato (MX): Universidad Autnoma de Quertaro (MX).Disponible en: http://www.uaq.mx/investigacion/difusion/veranos/memorias-

2008/10VeranoRegionCentro/17UAQRamirezRamirez.pdf

http://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.htmlhttp://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.htmlhttp://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.htmlhttp://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.html

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Extraction and Quantification ofProteins in Syzygium jambos used as

Diabetic Adjuvants in Puerto RicoYolanda I. Rodrguez-Rivera

BIOL 4990

Mentor: Dr. Jannette Gavilln-Surez