Enzyme as protein

Dr.Samina Haq

Quantitative and qualitative test for protein and amino acids

Qualitative test
1. 2. 3. 4. 5. 6. 1. 2. 3. Ninhydrin test Biuret test Xanthoproteic test Millons test Hopkins-cole test Nitroprusside test

Quantitative test
Spectrophotometric assay Protein shows maximum absorbance at 280nm due to presence of tyrosine and tryptophane. Biuret test shows 540nm Lowry test shows 750nm

Ninhydrin Test
Amino acid containing a free amino group and a free carboxylic acid group that react together with ninhydrin to produce colured product. Protein also contain free amino group on the alpha-carbon and can react with ninhydrin to produce blue purple product. When the amino group is secondary the condensation product is yellow.

Biuret Test
It positively identifies the presence of protein in solution with a deep voilet colour.Biuret H2NCONHCONH2 reacts with
copper(11)ions in a basic solution to form a deep voilet complex.

Xanthoproteic Test
The amino acids that contain benzene ring like tyrosine and tryptophan undergo nitration in this test and gives yellow colour.

Millons Test It is specific for tyrosine, the only amino acid that contain a phenol group on which a hydroxyl group is attached. It gives red precipitate.

Hopkins_cole Test
It is specific for tryptophan, the only amino acid containing an indole group.The indole ring reacts with glyoxylic acid in the presence of a strong acid to form a voilet cyclic product.

Nitroprusside Test
It is specific for Cysteine, the only amino acid containing sulfahydryl group. Forms red complex.

Classification and Nomenclature of Enzymes(IUBMB classification)

Enzymes are commonly named by adding a suffixase to the root name of the substrate molecule it is acting upon. Lipase catalysis hydrolysis of lipid triglyceroids. Sucrase= act on sucrose to form glucose and fructose. The latest systamic nomenclature is known as International Enzyme commission (IEC)system is based on type of reaction catalyzed. Enzymes are classified at 4 levels which developed the EzCatDB data base. Basic reaction(R) reaction type (Class) Ligand group involved in catalysis (L) (Sub Class) Type of catalytic mechanism(C) (sub sub Class) Residue/Cofactors located on Protein (P)

1. 2. 3. 4.

Enzyme Classified as Four digits separated by a point.

Enzyme Classification Biochemical Properties Number Oxidoreductase Act on Many chemical grouping, to add or E.C1 . Systemic name: E.C 2 Transferase

remove hydrogen atoms or electrons ()xidation reduction, may require NAD,FAD

Transfer functional groups to donor and acceptor molecules.Kinases are specialized transferase that transfer ATP to other molecules. DNA methyl transferase.

E.C 3

Hydrolases

Cleavage of bond between C and other group by addition of water.(Hydrolysing it)

E.C 4

Lyases

Non hydrolytic Cleavage of C-C,C-S,C-N bond.Add water.ammonia or carbon dioxide across the double bondor remove these elements to produce double bonds.

E.C 5

Isomerases

Convert one isomer to another by transfering one group to another.within the same molecule

E.C 6

Ligases

Catalyzes two reactions in which two chemical groups are joined or ligated with the use of energy.ATP.

SubClass Name

E.C 1

E.C1.1 E.C 1.2 E.C 1.3

Enzyme File type Oxidoreductase Sub sub cLass upto 50 Acting on the CH-OH group Up to of donors 50 Acting on the aldehyde or Up to oxo group 50 Acting on CH-CH group of Up to donar 50