edman degradation

ser gluala phelys met leuglu

Ion Exchange Chromatography, Sulfonated Polystyrene Resin

Elution Profile of Amino Acids

Elution Volume

SE A FK M L E

pH 3.250.2 M citrate



Chemistry 365 BiochemistryIndividual Lab Unit #7Sequence Determination Techniques for Proteins and Nucleic Acids

References: Lehninger, A.L.; Nelson, D.L.; Cox, M.M. Principles of Biochemistry, 2nd ed.; WorthPublishers: New York, 1993.Berg, J.; Tymoczko, J.; Stryer. L. Biochemistry, 5th ed.; W. H. Freeman: New York, 2002.

Introduction:

I. Determining Protein Amino Acid Sequence

Once the protein of interest has been extracted and purified, and its molar mass determined, the nextstep is to completely hydrolyze the protein (6 N HCl at 110oC for 24 hours) and determine its amino acidcomposition. Amino acidsin the hydrolysate areseparated and identified byion exchangechromatography.

In most cases, thenext step is to identify thefirst amino acid in thesequence, or in otherwords, the protein's aminoterminal.

The amino terminalis typically reacted with alabelling reagent, such asdabsyl chloride, that formsa bond stable to hydrolysis.The peptide is hydrolyzedand the labeled amino acididentified.

Peptides up to 50 residues long can besequenced by a cyclic procedure where the aminoterminal is labelled, cleaved, identified and theprocess repeated on the shortened chain. Thisprocedure, the Edman degradation method, hasbeen automated. This machine, called asequenator, performs the reactions, separations andidentifications as well as recording all results.

N

N SO2Cl

N

H3C

H3C

dabsyl chloride

dabsyl-amino acid (fluorescent)

N

N S

N

H3C

H3C

O

ON C

R H

O

OH

Endopeptidases and Sequencing by Fragment Overlap

chymotrypsinhydrolyzes peptide bonds on C side of phe, trp, tyr

trypsinhydrolyzes peptide bonds on C side of lys, arg

SE A FK M L E

resulting peptides

resulting peptides



lysglu ser gluala phemet leu

E K S A FM L E

SE A FK M L E

ser ala phelys metglu gluleu

Deduce sequence by comparing fragments


trypsin

chymotrypsin

must be

lysglu ser gluala phemet leu

ser ala phelys metglu gluleu

4 53

3 4 5

3 4 52

2 3 4 5

2 3 4 51

1 2 3 4 5

1 2 3 4 5

Label

Label

NC

S

O

C C

N

H

C

O

O

H

H

H

O

CC

NH

NH2

leumet pheala gluser

NC

S

H2NC

CN

CC

O

O

H

H


H

NH2

C

O

O

N C

S

H

H

O

O

CC

NC

CN

H

H


H

NH2

C

O

O

Release and Identify

Label

Release

Label

EDMAN DEGRADATION

Repeat

phenylisothiocyanate



Identify

Proteins longer than 50 or so residues must be sequenced in an additional manner. The protein chainis broken into smaller fragments by site specific chemical reagents or endopeptidases such as trypsin andchymotrypsin. The resulting segments are separated and sequenced. The correct ordering of these sequencesis made possible by repeating the procedure with chemical reagents or enzymes that cleave specifically atdifferent sites than previously. This second analysis should yield a set of different fragments, which whencompared to the first set and "overlapped", will allow the deduction of the complete protein amino acidsequence.

Recently, the development of recombinant gene technology and DNA sequencing methods make itpossible to sequence proteins via an alternative strategy. If the gene for a given protein can be isolated, andthe nucleic acid sequence determined, the protein amino acid sequence can be deduced through the geneticcode.

II. Determining Nucleic Acid Nucleotide Sequence

The development of a technique by Frederick Sanger has made it relatively easy to sequence largeDNA molecule fragments. The method depends on the abilities to:

1) find two appropriate DNA primers that border the target DNA fragment2) synthesize the complementary strand to the target utilizing the primers, DNA polymerase and

deoxynucleotides

DNA SEQUENCING by the SANGER (dideoxy) METHOD

T C T T

OH

5'

3'

TEMPLATE

dGTPdATP

DNA Polymerase

G

OH

P

P

P

OH

A

P

P

P

PPPP

AAGA GCTC

HO P P P P P P P P

(Target)

PRIMER

A

P

P

P

ddATP

5'

3) generate four sets of labelled complementary DNA fragments, with each set generated by aspecific reaction including a dideoxynucleotide, and

4) separate labeled complementary DNA fragments, by electrophoresis, that differ in length byonly one nucleotide

The four sets of labelled fragments are electrophoretically separated side-by-side, and the pattern ofbands produced directly yields the nucleotide sequence.

For example above, the labelled complementary DNA fragment, pCTTGAGCTGA, can be producedfrom the target pTCAGCTCAAG by four different dideoxynucleotide reactions, each yielding adideoxynucleotide at the 3' end of fragments that all begin at the same starting point.

The "dideoxynucleotide A" electrophoresis lane will show fragments 5 and 10 nucleotides long; the"dideoxynucleotide C" lane will have fragments 1 and 7 nucleotides long; the "dideoxynucleotide G" lanewill have fragments 4, 6 and 9 nucleotides long; and the "dideoxynucleotide T" lane will have fragments 2, 3and 8 nucleotides long.

The final result is that there will be one labelled complementary DNA fragment for each length up tothe total number of bases, and each of these fragments ends with the base at that number position in thesequence of the labelled complementary DNA fragment. The sequence of the original target DNA fragmentmay now be deduced from complementary fragment using base pairing rules.

ddATP

ddCTP

ddGTP

ddTTP

5'

3'

OH

GAACTCGACT

+ dCTP, dGTP, dTTP, dATP

PRIMER

TEMPLATE

CTTGAGCTGPRIMER

CTTGAGPRIMER

CTTGPRIMER

CTTGAGCTPRIMER

CTTPRIMER

CTPRIMER

CTTGAGCPRIMER

CPRIMER

CTTGAGCTGAPRIMER

CTTGAPRIMER

Cyc l e Sequenc i ngCyc l e Sequenc i ng

A G TC

10987654321

-

+

CTTGAGCTGPRIMER

CTTGAGPRIMER

CTTGPRIMER

CTTGAGCTPRIMER

CTTPRIMER

CTPRIMER

CTTGAGCPRIMER

CPRIMER

CTTGAGCTGAPRIMER

CTTGAPRIMER ELECTROPHORESIS

edman degradation

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