Biokim Farmasi anak 2012

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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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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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Biokim Farmasi anak 2012