Enzymes #1

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  • Created by: Ivana
  • Created on: 01-12-13 12:26

Intro

Semen stains: 
presence of prostatic acid phosphotases, spermotozoa or P-30 or DNA typing

Saliva stains:
Enzyme amylase or DNA typing

Urine:
Creatinine + Urea but cannot be typed.

Skin tissue:
Sometime spossible to DNA type tissues.  

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Seminal acid phosphotases

  • Remove phosphate from proteins between pH 5-6.5
    After puberty semen contains high levels of seminal acid phosphotases and the levels decline after 40 years.
  • Enzyme based spot test:
    -A-napthyl phosphate is used as a substrate.
    -Upon hydrolysis, the product reacts with a diazonium salt to produce an intense purple colour.
    -Confirmatory tests for the presence of sperm may include microscopic visualisation, detection of prostate-specific antigen (PSA) and DNA fingerprinting.  
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Saliva

  • High concentration of a-amylose which breaks down complex sugars into glucose monomers. 
  • Iodine forms a black coloured complex with complex sugars
  • So if a-amylose is present the comlex will NOT form so will not go black. 
  • The simple starch iodine test has been used but lacks sensitivity. Add iodine dissolved in potassium iodide solution to test sample. If starch is present the sample changes from browny orange to a dark blue black colour. 
  • More commonly detected using an insoluble starch/dye complex (eg amylopectin/ procion red). The hydrolysis of the insoluble complex liberates a soluble coloured complex that can be measured in a spectrophotometer. 
  • Levels of a-amylase are 50x greater in saliva than other bodily fluids. 
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ELISA (enzyme linked immunosorbent assays)

  • Been linked to monitor the presence of a variety of proteins, agrochemicals (eg fetiliser; pesticide) and pharmaceuticals in biological samples.
  • To confirm death by a suspected insect attack, the array of serum TRYPTASE (a proteolyte enzyme released from mast cells as a result of an allerginic reaction to insect venom) may be coupled with an ELISA to detect a specific immunoglobin E. 
  • ELISA based assays are also used to monitor serum proteins - eg blood globulins and post morten conformation of HIV infections. 
  • APPLICATION:
    1) immobilized antigen A (enzyme) is placed at the bottom of a well plate
    2) It is incubated with a primary antibody (rabbit antibody) that is complementary to antigen A. Then it is wash off.
    3)Secondary antibody is added with a marker on it and it binds to the rabbit antibodies.
    -> Can detect amount of enzyme
    ->Suspected insect attack.  
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Enzyme polymorphism

-> Used as markers of genetic individuality 
EG: adenylate kinase, carbonic anyhdrase.
->The use of isolectric focusing (seperating proteins according to their charge) and in situ activity staining produce characteristic isoenzymes patterns.
-> Several polymophic enzymes can be used to establish an indiviudals indentity within a population.
->Compares unfavourably to the info that we can now get from small fragments of DNA. 

Gene168 -----------------> Protein 168
Gene168(a) --------------> Protein 168(a)  one person
Gene168(b) ----------------> Protein 168(b)   another person.

-> far eats asian people have a different structure of metabollic enzymes which means that they have a low tolerance to alcohol.
->can identify gender, race, different diseases, using polymorphic enzymes.  

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Chemical catalysts

-> Without a catalyst chemical reaction require very high temperatures and pressurs to force reactants to products. 
-> Eg platinum, nickel or zeolites help reduce the temp and pressure requirements

The haber process:
-With iron: Needs 400'C and 200 atmospheres of pressure.
-With nitrogen fixing bacter: Needs 15'C and 1 atmosphere of pressure.  

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Biological catalysts

CATALYTIC POWER: (enzymes catalysed rate to uncatalysed rate.)
enzymes accelerate reaction rates up to 10'16 over uncatalysed rate.

SPECIFICITY: enzymes are evolved to be selective for substrates. Due to the active site with has 3D stereospecifictiy for the substrates.
Enzymes in plants have evolved to produce starch from a-D-glucose and cellulose from B-D-glucose. Majority of life have evolved enymes to metabolise a-D-glucose.  

REGULATION: The cell regulate enzymes biosynthesis and the enzymes structure can be altered by:
temp
pH
enzyme activators
enzyme inhibitors. 

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Factors that affect enzyme activity

pH: 
-effects the enzymes structure because charge is part of the forces which hold the protein together.
-changes in pH can alter amino acids charge density,  the solvation shell (A solvation shell is a shell of any chemical species that acts as a solvent and surrounds a solute species) of the protein, and possibly their overall 3D shape.  
-amino acids at active site of an enzyme may alter their charge due to pH change.

This can remove their ability to be involved in the catalyctic event.

Enzymes may have a pH optima for mainting their structure. 

Enzyme structure:
The surface charge (positive or negative) and hydrophobic pockets are NOT evenly distributed over the surface of a protein.  

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