Ninhydrin and Buiret Tests

Ninhydrin Reaction

  • Tests for amino acids
  • reacts to form a purple coloured imino derivative
  • Most amino acids are colourless
  • not all may give a purple colour
  • Proline and hydroxyproline, secondary amino acids give a yellow colour  due its cyclic structure which results in the reduction of flexibility of polypeptide regions. The ninhydrin reaction as this reagent requires free alpha amino group (-NH2) .  Therefore the amino acids which have a free -NH2 (amino) group, are positive for the ninnydrin test but because proline only has -NH (imino) group it is negative.

Buiret Test

  • Tests for proteins
  • the buiret reagent is light blue in colour as it containd copper(II) ions
  • turns purple in the presence of proteins
  • the purple colour is formed when the copper ions in the buiret reagent reacts with peptide bonds on the polypeptide chains to form a complex

Essential and Non-Essential Amino Acids

Essential and Non-Essential Amino Acids

There are 20 amino acids required for adequate health. Some of these 20 amino acids are esseital amino acids as well a non-essential amino acids.

Essential Amino Acids – these are amino acids that cannot be synthesized by the body and have to be acquired through the diet.

Non- essential Amino Acids can be synthesized by the body.

Complete Protein

  • contain all 10 amino acids
  • proteins are derived from animal sources
  • Bears contain some complete proteins

Incomplete Protein

  • lack one or more of the essential amino acids 
  • most vegetable proteins are incomplete
  • Beans are an exception to this generalization

Some more on Glycolysis

ATP can be made in 3 ways :

  • substrate level phosphorylation (animals- glycolysis)
  • oxidative phosphorylation (animals- mitochondria and electron transport chain)
  • photophosphorylation (plants)

First Phase Enzymes

  1. Hexokinase
  2. Phosphohexose isomerase
  3. Phospho-fructokinase-1 (PFK-1)
  4. Aldose
  5. Triose phosphate isomerase

Hexokinase and PFK-1 are both enzymes for the irreversible reactions of the first phase. Their reactions are energetically favorable due to a large negative change of  free energy(ΔG)

Second Phase Enzymes

       6. Glyceraldehye 3-phosphate dehydrogenase

       7. Phosphoglycerate kinase

       8. Phosphoglycerate mutase

       9. Enolase

     10. Pyruvate kinase

The most energetically favorable reaction in glycolysis is reaction 10. between phosphoenolpyruvate to pyruvate using pyruvate kinase. The reaction is irreversible due to ΔG being very negative.

ΔG is close to zero when the reaction is reversible.

Enzyme Classes – Shhhhh!!! Class is in session. . .

Six major classes of enzymes

enzyme classes wordle

Oxidoreductases – catalyses oxidation- reduction reactions

Transferases – catalyses the transfer of C,Nor Pcontaining groups

Hydrolases – catalyses the cleavage of bonds by the addition of water

Lyases – catalyses the cleavage of certain C-C, C-S and certain C-N bonds

Isomerases – catalyses the racemization of optical and geometrical isomers

Ligases – catalyses the formation of bonds between carbon and O, S, N coupled to hydrolysis of high energy phosphate. 


Enzyme inhibitors are molecules that bind to enzymes and decrease the velocity of enzyme-catalysed reaction. There are irreversible inhibitors that permanently bond to enzymes through covalent bonds, as well as    reversible inhibitors that temporarily bond through non-covalent bonds. Reversible inhibitors comprise of competitive, non-competitive, mixed and uncompetitive inhibitors.

Competitive Inhibitor



  • Similar shape to substrate
  • Binds to active site
  • Vmax (max velocity) remains the same
  • Km increases

Non-competitive Inhibitor



  • Does not resemble substrate
  • Binds either to free enzyme or enzyme substrate complex (ES)
  • Vmax decreases
  • Km remains the same

Mixed Inhibitor

  • Binds the same as a noncompetitive inhibitor, either to the free enzyme or ES cmplex, except the EIS cmplex has residual enzymatic activity.
  • Vmax decreases
  • Km  can either increase or decrease



Uncompetitive Inhibitor

  • Binds ONLY to the EIS complex
  • Both Vmax and Km are reduced by the same amount








                                                                                                                                       FIGURE 4.





6 – type of enzyme with more than one active site

8 – inhibitor that produces residual enzyme activity

9 – inhibitor that binds to the ESI

11-non-protein components that help enzymes work

12-substances that diminishes velocity of an enzyme-catalysed rxn

13-catalyses chemical reactions

14-molecular recognition based on structural complementarity


1 – enzymes are what type of biological molecule

2 – binds with active site to form product

3 – point at which all active sites are occupied with substrate

4 – inhibitors that bind permanently

5 – catalyses cleavage of bonds by addition of water

6 – location where substrate binds

7 – numerically equal to the [s] at which rxn velocity is equal to half vmax

10-number of moles of substrate converted to product per enzyme mol per second