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9.9 Forensic Chemistry 3. Protein, amino acid and enzyme analysis

Syllabus reference (October 2002 version)
3. Because proteins are a major structural and metabolic component of all living organisms, the analysis of protein samples can be useful in forensic chemistry	Students learn to: distinguish between protein used for structural purposes and the uses of proteins as enzymes identify the major functional groups in an amino acid describe the composition and general formula for amino acids and explain that proteins are chains of amino acids describe the nature of the peptide bond and explain that proteins can be broken at different lengths in the chain by choice of enzyme compare the processes of chromatography and electrophoresis and identify the properties of mixtures that allow them to be separated by either of these processes discuss the role of electrophoresis in identifying the origins of protein and explain how this could assist the forensic chemist	Students: perform first-hand investigations using molecular model kits, computer simulations or other multimedia resources to present information which describes the composition and generalised structure of proteins perform a first-hand investigation and gather first-hand information about a distinguishing test for proteins perform a first-hand investigation to carry out chromatography to separate a mixture of organic materials such as the pigments in plants perform a first-hand investigation and gather first-hand information to identify the range of solvents that may be used for chromatography and suggest mixtures that may be separated and identified by the use of these solvents perform a first-hand investigation to carry out the electrophoresis of an appropriate mixture and use available evidence to identify the characteristics of the mixture which allow it to be separated by this process

Extract from Chemistry Stage 6 Syllabus (Amended October 2002). © Board of Studies, NSW.
[Edit: 9 Jul 09]

Prior learning: Preliminary module 8.5.5, HSC module 9.2.1

perform first-hand investigations using molecular model kits, computer simulations or other multimedia resources to present information which describes the composition and generalised structure of proteins

• Perform a first-hand investigation using molecular model kits to form 3D models of a range of amino acids (e.g. alanine, cysteine and phenylalanine). Work as a class group to form as many amino acid units as possible and correctly join them by condensation reaction (elimination of water) to form proteins.

  Protein structure , Max Planck Institute for Molecular Genetics, Germany.

perform a first-hand investigation and gather first-hand information about a distinguishing test for proteins

• For a range of organic compounds from both the laboratory and common household products, including proteins (e.g. egg white, aspartame (artificial sweetener)) amino acids (e.g. glycine, aspartic acid, glutamine) perform a first-hand investigation using the distinguishing tests outlined below.
  
  
• While performing the first-hand investigation, safe work practices, such as those outlined in the “Chemical safety in schools” package should be considered and applied. According to the package ninhydrin is toxic by all routes of exposure and should be used in a fume cupboard if the quantity is greater than 25mL. Biuret solution is considered as a hazardous substance that is toxic if ingested and corrosive to the skin. Eye and skin protection should be used.
  
  
• Gather first-hand information by observation and tabulation of results to determine which of the compounds are proteins and which are amino acids.
  
  

  Test	Method
  ninhydrin	Add colourless ninhydrin solution to sample solution. Purple colour indicates presence of proteins or amino acids
  Biuret	Add a few drops of copper sulfate solution to an alkaline sample solution. Purple colour indicates the presence of proteins or peptides, but not amino acids

distinguish between protein used for structural purposes and the uses of proteins as enzymes

• Proteins are a necessary constituent of all living cells.
  
  
• There are two general classes of protein – fibrous and globular. Fibrous proteins are tough, stringy in appearance and are insoluble in most solvents. Globular proteins are predominately spherical in shape and are soluble in water.
  
  
• Fibrous proteins form the major structural component of animal tissue. They are found in skin, hair, muscles, tendons and supporting tissue.
  
  
• Globular proteins have specialised functions as:
  • oxygen carriers (in haemoglobin)
  • communication agents (in nerve cells)
  • defence agents (in antibodies)
  • biochemical catalysts (in enzymes).
    
    
• An enzyme is an organic catalyst produced by living cells that act by lowering the energy of activation required for a reaction. Enzymes are usually protein molecules that have another substance, such as a metallic ion attached to them.

identify the major functional groups in an amino acid

• The major functional groups present in an amino acid are:
  • amino group (-NH₂). At least one amino group is required to give the amino acid some basic (alkaline) properties
     
  • carboxylic acid group (-COOH). At least one carboxylic group is required to give the amino acid some acidic properties.

describe the composition and general formula for amino acids and explain that proteins are chains of amino acids

• Proteins are polymeric molecules. The basic units (monomers) in proteins are amino acids. Up to twenty different amino acids can be found in a protein.
  
  
• The general formula for an amino acid is:

  NH₂-CH(R)-COOH

• Amino acids are able to link together by undergoing a condensation reaction, eliminating water. The link is formed between the –NH₂ group of one amino acid and the –COOH of the other. The bond between the two amino acids is called a peptide bond.
  
  
• The linking of two amino acids is called a dipeptide, three amino acids a tripeptide. When this process is repeated many times, a polymer is formed called a polypeptide.
  
  
• Proteins are polypeptides that contain up to thousands of amino acid units joined together.

  The twenty amino acids , Institute of Chemistry, Department of Biology, Chemistry and Pharmacy, Berlin, Germany.

  Primary structure of polypeptides Wikipedia, USA.

describe the nature of the peptide bond and explain that proteins can be broken at different lengths in the chain by choice of enzyme

• Hydrolysis of a protein results in the cleavage (breaking) of the peptide bonds and the formation of the component amino acids. A catalyst (enzyme) is required for this reaction to occur. The catalyst increases the rate of hydrolysis by about 10¹⁴.

  protein + water → amino acids

• In humans and other mammals, hydrolysis occurs in the digestive system. An enzyme targets specific sequences of amino acids and attacks the peptide bond between two particular amino acids. This allows the protein to be broken into small length chains without completely breaking the protein into individual amino acids. The lock and key theory describes that the protein (substrate) fits into a specific place in the enzyme (the active site) like a key in a lock. Chemical change quickly occurs and breaks the peptide bond.
  
  
• This process can also be performed in the laboratory. Proteins can either be separated into individual amino acids to determine the order of amino acid in the protein or separated into smaller length chain proteins.

perform a first-hand investigation to carry out chromatography to separate a mixture of organic materials such as the pigments in plants

perform a first-hand investigation and gather first-hand information to identify the range of solvents that may be used for chromatography and suggest mixtures that may be separated and identified by the use of these solvents

The following information addresses the above two syllabus points at the same time.

• Perform a first-hand investigation to separate the components present in a range of unknown ink samples. Use a range of solvents (vary the proportion of alcohol and water) to gather first-hand information (chromatography strips) and identify the solvent which produces the best separation for all samples analysed. When you have identified the most appropriate solvent compare the unknown ink samples to a standard (known sample) to identify one of the unknown samples as matching the standard.

  The solvent that produces the best separation is dependent on the structure and functional groups present in the mixture.

perform a first-hand investigation to carry out the electrophoresis of an appropriate mixture and use available evidence to identify the characteristics of the mixture, which allow it to be separated by this process

Web sites suggesting simple ways of carrying out electrophoresis are listed below:

Simple methods of electrophoresis and chromatography , Department of Chemical Engineering, Washington State University, Pullman, Washington, USA.

Lab Investigation: Protein Fingerprinting , BIOTECH Project, University of Arizona, USA.

• Perform a first-hand investigation to separate the components present in a range of unknown ink samples. Use a range of solvents (vary the proportion of alcohol and water) to gather first-hand information (chromatography strips) and identify the solvent which produces the best separation for all samples analysed. When you have identified the most appropriate solvent, compare the unknown ink samples to a standard (known sample) providing the evidence needed to identify one of the unknown samples as matching the standard.

The solvent that produces the best separation is dependent on the structure and functional groups present in the mixture.

compare the processes of chromatography and electrophoresis and identify the properties of mixtures that allow them to be separated by either of these processes

• Chromatography is a method of separation that can be based on a range of chemical and physical properties.
  The most common type of chromatography uses the different solubilities of different substances in a solvent. The degree of solubility is determined by the polarity of the substance and the solvent. The solvent can be altered to increase the degree of separation. Good separation can not be achieved if the different substances have similar solubilities.

  Types of chromatography , Rensselaer Polytechnic Institute, Troy, New York, USA.

• Electrophoresis is a method of separation based on the charge and size of particles of a substance. The pH of the electrophoresis solution (buffer) can be altered to change the charge of the substance particles to increase the degree of separation. Separation of components is usually successful.

  Apparatus for paper electrophoresis , Giorgio Carboni, Italy.
  Gel electrophoresis , University of Utah, Utah, USA.

discuss the role of electrophoresis in identifying the origins of protein and explain how this could assist the forensic chemist

• Electrophoresis allows the separation of individual amino acids present within a protein to be studied and therefore the protein itself to be identified.
  
  
• The proteins present on the surface of a red blood cell determine human blood groups (A, B, AB and O). Electrophoresis of a blood sample identifies the amino acids, and therefore the protein and thus the blood group of the sample. This process can be used as collaborative evidence by a forensic chemist to link or dismiss a suspect to a crime. This method may also be used to help identify a victim of a crime.

  Electrophoresis of blood Kimball's Biology pages, USA