Question

Determine the approximate pI for each protein (A-E) and (b) draw the 2D proteinIEF/SDS gel diagram showing the approximate positions of the proteins.

A.78 kD, 4Asp, 2Lys, 1Arg

B.57 kD, 2Glu, 2Asp, 3Arg, 2Lys

C.97 kD, 2Asp, 1His, 2Lys

D.45 kD, 3Glu, 1Cys, 2Lys, 1Arg

E.50 kD, 2Asp, 1His, 2Cys, 2Lys

Answer 1

In this case the question does not provide any info regarding the pka values so I m assuming some general values the pka of the - carboxyl group is around 2.0 the pka of the - amino group is around 9.5 pka of R chain amino group of lysine is around 10,5, the pka of the R chain carboxyl group of Aspartate is around 3.5, the pka of R cahin amino group of arginine is around 12.5, pka of R chain carboxy group of glutamate is around 4.2, the pka value of the R chain of Cysteine is around 8.33 and the pka of R chain of histidine is 6.0..

Now to calculate the following pi of the given proteins we have to take the avg of every ionisable group present in it along with the N terminal group and the C terminal group.

For Protein A the amino acid seq is DDDDKKR so the no of ionisable group present here is 9 so taking there respective pka and doing avg we get the pi = (3.5+3.5+3.5+3.5+10.5+10.5+12.5+9.5+2.0)/9 = 59/9 = 6.55

For Protein B the sequence is RRDDRRKK so the number of ionisable group present here is 10 so the pi will be

(9.5+4.2+4.2+3.5+3.5+12.5+12.5+10.5+10.5+2.0)/10 = 70.9/10= 7.09

For protein C we have the sequence DDHKK so the number of ionisable groups become 7. pi of the protein is

= (9.5+3.5+3.5+6.0+10.5+10.5+2.0)/7= 45.5/7= 6.5

For protein D we have the sequence EEECKKR  so the number of ionisable group becomes 09. so the pi of the protein will be = (9.5+4.2+4.2+4.2+8.3+10.5+10.5+12.5+2.0)/9= 63.9/9 = 7.1

For protein D we have the sequence DDHCCKK so the number of inonisable group becomes 09 the pi of the protein will be

= (9.5+3.5+3.5+6.0+8.3+8.3+10.5+10.5+2.0)/9= 62.1/9= 6.9

The graph of the SDS electrophoresis is as follows.

The protein C has the maximum weight in the question so it will run the least distance when place in SDS PAGE gel and the protein D having the minimum moleculer weight ran the maximum distance in the gel.