User:Syedal82

Peptide hydrolysis- 1) I took approximately 1 mg peptide sample in a small vial and dissolve it in .05ml of N HCl. 2) I sealed the vial tightly and place it room temperature in 1 weak. 3) After hydrolysis, I transferred the hydrolysate to a watch glass and evaporate the liquid by gently heating under a heat lamp in the hood. 4) Then, I added .1 ml of distilled water, repeated the evaporation, and prepared the residue by dissolving it in .1 ml of distilled water. Paper chromatography- 1)	Using a pipet, I applied 5 uL of the different amino acids ( Glycine, Alanine, Leucine, Phenylalanine and Tyrosine) in the Whatman paper. 2)	I also applied the hydrolysate peptide to the paper in two points. 3)	I placed the sheet in a chromatogram tank containing 1 cm layer of acetonitrile and .1 M ammonium acetate (60:40). 4)	I took out the whatman paper when the solvent reached 1 cm from the top of the chromatogram. 5)	I sprayed the Ninhydrin to the paper and dry in the hood. 6)	Then the spots were appeared and I circle the spot.

NH2-Terminal analysis- Dansyl chloride treatment- Procedure- 1)I weight 1 mg of the unknown peptide and transferred into a conical centrifuge tube. 2) I dissolved the peptide in 0 .5 mL of .2 M sodium bicarbonate and added .2 mL of the solution of dansyl chloride in acetone. 3) I incubated solution in 1 hour at 37 degree Celsius. 4) After completion the reaction, I evaporate the solution to dryness bys using nitrogen gas. 5) Then I dissolved the residue e in acetone .5 ml acetone 6N HCl solution. 6) I evaporate the acetone in the vial with a gentle steam of nitrogen gas. 7) I sealed the vial and kept it in the room temperature for 1 week. 8) I evaporated it under a watch glass under a lamp and dissolved the residue in 50% aqueous pyridine. Thin layer chromatography- 1)	I applied 3 uL of Glycine, Alanine, Leucine, Phenylalanine and Tyrosine in the TLC paper. I also added 5 uL of the unknown. 2)	I developed the plate in chloroform “methanol: aceteic acid (75:5:1) until the solvent reached 1 cm from the top. 3)	I dry the plate and viewed the plate under UV light. I circle the spots with the pencil.

Analysis of results- A)	Total hydrolysis of Unknown peptide-

The colors of the two amino acid are dark purple and light purple. From the calculation of the Rf value, we can see that the Rf value of Alanine and Leucine are similar to the two unknown amino acid.

B)	NH2- terminal analysis-

The Rf value of the unknown and the Rf value of Alanine are similar.

Result- The unknown amino acids are Alanie and Leucine. The dipeptide is Leucine-Ananie.The structure is given below-

H       O   H              /         //    / OOC- C-NH-C-C-NH2 /             /          CH2        H           / CH-CH3 /       CH3