Protein dan EnzimKIMIA KLINIK

Rochmy Istikharah

Clinical Significance

Clinical Significance

?

Peptide bond

α-helix

β-sheet

pHIonic strengthTemperature

Solvent (dielectric constant)

Antibodies Coenzymes

Hormone receptors

Denaturation

Electrophoresis Isoelectric focusing

Ion-exchange chromatography

Clinical SignificanceCONCENTRATION

♮ Total protein concentration6-8 g/dL

♮ Selected protein conct.PATTERN

Depend on protein properties

Selected Protein Analysis

Hyperglycemia(e.g. Diabetes Mellitus)

up to 25% albumin glycosylated

nonenzymatically

Related with HbA1c in DM

Normal condition8% albumin glycosylated

nonenzymatically

Useful for assessing diabetic control in Px with hemolytic

anemia

Total Protein Analysis

Total Protein Analysis

≥ 2 peptide bonds

Cu2+ + Protein Cu2+-Protein complex–OH

Less sensitive than Lowry

!

Protein Analysis• Skrining

• Total protein 6-8 g/dL dengan 3,5-5 g/dL albumin• Pattern of serum protein in agarose electrophoresis

(Serum protein electrophoresis: SPE)

Able to diagnose

Unable to diagnoseSelected Protein

Analysis

Methods: Protein Elektroforesis

Prinsip: Pemisahan protein berdasarkan perbedaan muatan &/atau bobot molekul (metode standar elektroforesis)

Protein Serum Elektroforesis

pI: isoelectric point (ampholyte & net charge = 0)

Horizontal electrophoresisCth. Agarose electrophoresis

Vertical electrophoresisCth. SDS-PAGE Automated electrophoresis

Plasma Protein Elektroforesis

SPE Patterns/Serum PEP

Modified fromVithoulkas H. HomeoNews2008;9:9-10

SPE PatternsModified fromVithoulkas H. HomeoNews2008;9:9-10

SPE PatternsModified fromVithoulkas H. HomeoNews2008;9:9-10

IgA

SPE PatternsModified fromVithoulkas H. HomeoNews2008;9:9-10

Large spike in γ regionHomogenous Ig

O’Connel et al. Am Fam Physician 2005;71(1):105-112

immunofixation electrophoresis

McPherson & PincusHenry’s Clinical Diagnosis21st Ed. 2006

Enzyme

Kimia Klinik

Rochmy Istikharah

ENZIMGlossary

• Proenzim (Zymogen)

• Enzim

• Holoenzim

• Apoenzim

• Koenzim/kofaktor

• Isoenzim

Plasma & Non-plasma Specific Enzymes

Plasma • Normal ada di plasma

Plasma adalah tempat aksinya

• Konsentrasinya tinggi dibanding organ

• Cth. Enzim pd koagulasi

Non-plasma• Plasma bukan tempat

aksinya• Konsentrasinya di

plasma rendah (normal krn sel apoptosis)

• Konsentrasi meningkat???• Apoptosis, nekrosis,

obat, etc

Harper’s Biochemistry26th Ed

• Regenerasi & penyimpanan ATP

• CK-BB: (CK-1)brain type

• CK-MB: (CK-2)hybrid type

• CK-MM: (CK-3)Muscle type

Kreatin + ATP

Kreatin fosfat + ADP

CK Mg2+

Copyright ©2007 American College of Cardiology Foundation. Restrictions may apply.

Anderson, J. L. et al. J Am Coll Cardiol 2007;50:652-726

Faktor yg mempengaruhi nilai rentang enzim

• Waktu sampling• MI: CK-MB: 6 jam

• Usia• Gender:

massa otot, olahraga, konsentrasi hormon

• Ras• Olahraga

Alkaline phosphatase (U/L)

[AMP buffer, p-nitrophenyl phosphate substrate,

30oC] <10 th 157 – 270 10 – 18 th 90 – 320 >18 th 40 – 100

CK serum

30 – 50 U/L500 - 1000 U/L

Pengukuran Konsentrasi Enzim

Pengukuran Tidak Langsung:

aktivitas enzim

1.Single reaction

Pengukuran produk atau substrat yg dikatalisis oleh enzim tsb jarang

2.Coupled reaction

Pemeriksaan Langsung:

Immunoassay

• Monoclonal/polyclonal antibodies• Mengukur

konsentrasi enzim itu sendiri

• Contoh teknik: ELISA

• Suhu• Suhu dijaga dlm rentang ±0,1o

• Perbedaan 10o aktivitas enzim meningkat 2xnya• pH: pH optimum utk enzim aktif• Garam

• Terlalu tinggi aktivitas enzim turun drastis• Konsentrasi protein:

• Plasma manusia, protein: 70 g/L; Urin hampir tidak ada• Penambahan albumin pd pemeriksaan amilase di urin (spike)

• Inhibitor & pengganggu lain