Individual proteins of known subunit molecular weight and the pyrophosphatase wi
ID: 189076 • Letter: I
Question
Individual proteins of known subunit molecular weight and the pyrophosphatase with a native molecular weight of electrophoretically separated by SDS-polyacrylamide gel electrophoresis. The marker dye migra 3900 dals were denatured in a buffer containing both SDS and -mercaptoethanol. A sample of each protein was gel for protein revealed the following: ayt Protein Serum albumin catalase ovalbumin carbonic anhydrase myoglobirn MW 69,000 60,000 43,000 29,000 17,000 Distance Migrated (cm) 1.6 2.2 3.5 5.2 7.4 o, 13 6.43 The pyrophosphatase migrated 6.7 cm. However, when the B-mercaptoethanol was omitted, the pyrophosphatase migrated 3.8 cm, Determine the subunits molecular weight and comment on the quaternary structure of the enzyme. Bmertae to emnol c (5 pts) A peptide was isolated and subjected to sequence analysis. The experimental data are as follows: a. Amino acid composition: Ala (2), Gly, His, Glu, Lys (2), Pro, Ile, Tyr,Met, Val. b. Brief carboxypeptidase incubation: free Tyr released. c. Dansyl chloride reaction:N-dansyl-Glu. d. Trypsin digestion (three fragments) Fragment A composition: Ala (2), Gly, Ile, Lys, Met, Val. Fragment A dansyl chloride reaction: N-dansyl Ala Fragment B composition: Pro, Tyr. Fragment B dansyl chloride reaction: N-dansyl Pro. Fragment C composition: Glu, His, Lys e. CNBr (two fragments) Fragment D composition: Ala (2), Glu, His, Lys, Met. Fragment E composition: Gly, Ile, Lys, Pro,Tyr, Val. Fragment E dansyl chloride reaction: N-dansyl Gly. Using this information and what you know about amino acid structure and protease specificity, propose a sequence that is consistent with the data. If in doubt about the order of the amino acids, place them in parentheses.Explanation / Answer
5. Dansyl chloride gives a N-terminal amino acids = N-Glu
Trypsin cleaves at C-terminal of Lys
Cynogen bromide cleaves at C-terminal of Met
Carboxypeptidase cleaves at tyr
From Cynogen bromide we get 2 fragments,
Glu-Ala-Ala--His-Lys-met / Gly-Ile-Lys-pro-try-val
In trypsin 3 fragments,
Ala-Ala-Gly-Ile-met-Val-Lys / Glu-His-Lys / Pro-Tyr Tyr -C terminal
Rearrane this N-ter is Glu and C-ter Tyr
Glu-His-Lys-Ala-Ala-Gly-Ile-met-Val-Lys-pro-Tyr
But if we use CyBr the order is not coming. So change the methione place.
Glu-His-Lys-(Ala-Ala)-Met-(Gly-Ile-Val)-Lys-pro-Tyr This the order of the sequence.