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7/30/2019 Extraction and Quantification of Proteins in Syzygium jambos used as Diabetic Adjuvants in Puerto Rico
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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.html7/30/2019 Extraction and Quantification of Proteins in Syzygium jambos used as Diabetic Adjuvants in Puerto Rico
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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