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PROTEIN
Dr.rer.nat. Mardiyanto, MSi, Apt
BIOCHEMISTRY
Kamis, 5 Desember 2013
Lanjutan
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Daftar Pustaka
Mathews, 2004. Biochemistry (3rded)
Berg M J, Tymoczo J L, Stryer L, 2003, Biochemistry (7thed)
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It is the partial double-bond character of the
peptide bond that defines the conformations apolypeptide chain may assume.
It is shorter then a single bond
Rigid & planar
AA = Amino Acid
AA AA AA AA -
Regarding The Secondary and
Tertiary Structure
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Konfigurasi cis dari grup peptida memilikiinteraksi ruang yang hebat, sehingga antar asam
amino akan eksis dengan konformasi bentukan
di i f
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Prediction of Structure
WTAASSTTRP PAASSAATYW QRRPALILAS ILTASSTRAQ
WYDRPILKLD SSAILELLPO KLYHKLQAYT SPLIIKLILT
GFLIPAASDK LE
Mari Berkunjung ke http://web.expasy.org/protscale/
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Pengetahuan yang dalam akan susunan asamamino penyususn protein itu membantu dalamprediksi struktur protein.
. Prediksi struktur protein membantu dalammemilih metode yang tepat dalammempurifikasi
. Metode yang tepat dalam purifikasi dapatmenjaga stabilitas protein yang akandipurifikasi
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Goals of purificationvary with the intended use of theprotein.
Purity is defined by the general level of proteincontaminants and also by the absence of contaminants of
special interest such as endotoxin, viruses etc.
Protein purification can be divided into 5 stages.
a) Preparation of the source
b) Knowledge of protein propertiesc) Development of an Assay
d) Primary Isolation
e) Final Purification
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HIGH RESOLUTION PURIFICATION
Chromatography is the usual method of preparing highly purified activeproteins.
Chromatographic operations are classified as low-pressure, medium-pressure, high-pressure depending on the pressure used to force liquid
through the packed bed.
Feature Low-Pressure Medium Pressure High-Pressure(HPLC)
Particle Size 40-150m 10-75m 2-15m
Flow Driver Gravity, peristaltic Piston or syringe Positive displacement
Run Time 40-100min 15-60min 0.5-30min
Apparatus Cost Low Medium High
Resolving Power Lowest Intermediate Highest
Particulate tolerance Low Very low Lowest
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HIGH RESOLUTION PURIFICATION
Chromatographic operations are broadly classified as
a) Ion exchange Chromatography
b) Hydrophobic Chromatography
c) Affinity Chromatography
d) Size exclusion Chromatography
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Ion exchange Chromatography
In this case, a cation (or alternatively an anion) is attached to the resin beads.Depending upon the electrical properties of the proteins, they may attach tothe column. For example, positively charged proteins will stick to a negativelycharged column. These proteins can then be removed by washing the columnwith either a strong salt solution or changing the pH of the wash buffer.
Anion exchangers such as DEAE ( Diethyl amino ethyl) are used.
Attraction of proteins at a pH above the isolectric point of the protein.
Cation exchangers such as CM ( Carboxy methyl) are used.
Attraction of protein at a pH below the isoelectric point of the protein.
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Ion exchange Chromatography
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Ion exchange Chromatography
Elution
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ELUTION
Done by washing the column with a strong salt solution (NaCl) whichincreases the ionic strength thereby pushing out the proteins.
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AFFINITY CHROMATOGRAPHY
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SDS-PAGE
Apa itu ?
SDS = Sodium Dodecyl Sulphate
PAGE = Poly Acrylamide Gel Electrophoresis
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MALDI-TOF
Matrix-assisted laser desorption/ionization (MALDI)
Time-of-flight mass spectrometer (TOF)
M--------H---------I---------A--------S--------R--------P--------I---------I---------I dst
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Dr.rer.nat. Mardiyanto, MSi, Apt
BIOCHEMISTRY
Pemeriksaan Protein Dari Tubuh Manusia
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Where is Protein located in
human body ?
Two kinds of Protein :fibrous protein
andglobusprotein
Structural
Functional, such Enzyme
When protein has an active site, it becomes Enzyme
in terminology
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Blood Composition
Plasma
Plasma is fluid component of blood.
Comprises ~55% of total volume of
whole blood. Contains proteins, sugars,
vitamins,minerals, lipids, lipoproteins and
clotting factors.
95% of plasma is water
Red Blood cells (RBC)
Whole Blood Whole Blood after centrifugation
Note: clotting has been prevented
White Blood cells (WBC)
& Platelets Cellular
Components
Serum
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Blood Analysis
Sumber
Veins
Arteries
Skin puncture-capillary blood
Faktor-faktor yang mempengaruhi pemilihan sampel
Analyte under investigation
Patient vascular status
ease of collection
Cara Koleksi
Syringe
Evacuated tube
Additives
Separator gel
Intravenous lines
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Significantly affected by hemolysis:
Hemolysis-rupture of red blood cell
Can be due to improper collection
Significant increase inpotassium, magnesium,phosphorous
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Cara Pengkoleksian
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Measuring
Unit dan Lambang hasil pengukuran :
1. Metabolites (glucose, urea etc) areexpressed as mg/dL or mmol/L.
2. Electrolytes (Na+, K+) as mmol/L or
meq/L (popular terminology)
3. Enzymes as Units/L
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The units of measure commonly used to express theconcentrations of electrolytes in plasma
are :
milliequivalents (mEq)/L
= Mass (g) x 1000 x valency
MW
and millimoles(mmol)/L
= .. ?
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Test results
Variations, Errors, Interferences
Variations
Clinical variationswithin an individual and betweenindividuals
Analytical variations-no test is perfect. All tests have somedegree of variations for repeated measurements of the samesample.
The final test result is affected by factors that occur Pre-analytically
At the time of the test
After the test is completed
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