CLS 331 • Experiment • No.1 The hydrolytic activity of salivary amylase on starch Introduction Amylase is a hydrolytic enzyme which breaks down many polysaccharides such as starch. Starch Is a polymer of D- glucose units linked by α-1, 4 glycosidic bonds. The starch is made up of two polysaccharides: Amylopectin (branched-chain polysaccharide).. Amylose (unbranched-chain polysaccharide The hydrolytic effect of amylase on starch results in yielding maltose (composed of two D-glucose molecules ) as the end product. There are two broad groups of amylases: α and β- amylases. The β- amylases rapidly hydrolyse the amylose portion of starch to maltose by acting on residues at the non reducing terminals. They hydrolyse α-1, 4 glycosidic links in polysaccharides so as to remove successive maltose units from the nonreducing ends of the chains. The α amylases, in contrast to the β- amylases, cause a rapid loss of the capacity of amylase to give a blue colour with iodine, also the rate of appearance of maltose is much slower in the α amylases catalysed reaction than in the βamylases catalysed one. Types of amylase: α - amylases – human amylase β- amylases – bacteria and plant amylase Source of amylase: 1- Pancreas 2- Salivary gland Secretion: Serum or urine Hyperamylasemia Increase the secretion of amylase in blood Some Causes of Hyperamylasemia Acute pancreatitis, Salivary gland lesions, etc The Assay In any enzyme assay, the rate of the reaction can be known by measuring the amount of substrate (s) that is utilized or the amount of product (s) that is formed in unit time, it also called enzyme unit. In the case of amylase, The substrate is starch (colourless) The product is maltose (colourless) They can be converted to coloured products by specific chemical reactions. Starch +Amylase Starch + Amylase + Iodine (qualitative) maltose (colourless) blue colour Principle At a pH of about 6-7 and in the presence of chloride ions, α amylases catalyses the hydrolysis of starch to maltose with the intermediate formation of various dextrins. Dextrins are polysaccharide produced by the hydrolysis of starch Starch + Cl+ + α amylases maltose + intermediate formation of dextrins Steps of the reaction: 1. Starch + α amylase + Higher dextrins 2. Intermediated dextrins 3. Lower dextrins + maltose Iodine Iodine Iodine blue colour reddish brown colour colourless This point known as Achromic point ( time taken to reach the point at which the reaction mixture no longer gives a colour with iodine) Procedure Part1: ml saliva Control 1 Test1 Stock saliva 9.5ml H2O 0.5ml saliva 5ml Test tube1: mix then incubate at 37 C Control tube1: gently boiled for 2 min, cooled and then placed in the water bath at 37 C Part 2 Control 2 Test 2 5ml starch 2ml phosphate buffer 1ml NaCl Mix well, then incubate at 37⁰C X 5-10 min After incubation, C1 T1 1 ml 1 ml C2 In incubator T2 Spotting Plate Add 1 drop of iodine solution in 2 rows C Add 3-4 drops from C2 Add 3-4 drops from C2 after 1 min, mix Add 3-4 drops from C2 after 1 min or half min, mix Add 3-4 drops from C2 after 1 min or half min, mix add 3-4 drops from C2 after 1 min or half min, mix T Add 3-4 drops from T2 Add 3-4 drops from T2 after 1 min min, mix Add 3-4 drops from T2 after 1 min or half min, mix 3-4 drops from T2 After 1 min or half min, mix 3-4 drops from T2 After 1 min or half min, mix Result Test tube -> blue color then reddish brown color then colorless (A chromic point 4-40 minute)? Control tube -> blue color why? Report Principle Results; identify the time to reach the A chromic point for each tube. Reference value. Comment on each result exercises Hazard of chemical used