Info

*All reagents except cyanogen bromide ate proteases. All are available from commercial sources.

Residues furnishing the primary recognition point for the protease or reagent; peptide bond cleavage occurs on either the carbonyl (C) or the amino (N) side of the indicated amino acid residues.

*All reagents except cyanogen bromide ate proteases. All are available from commercial sources.

Residues furnishing the primary recognition point for the protease or reagent; peptide bond cleavage occurs on either the carbonyl (C) or the amino (N) side of the indicated amino acid residues.

from the total number of Lys or Arg residues in the original polypeptide, as determined by hydrolysis of an intact sample (Fig. 3-27). A polypeptide with five Lys and/or Arg residues will usually yield six smaller peptides on cleavage with trypsin. Moreover, all except one of these will have a carboxyl-terminal Lys or Arg. The fragments produced by trypsin (or other enzyme or chemical) action are then separated by chromato-graphic or electrophoretic methods.

Sequencing of Peptides Each peptide fragment resulting from the action of trypsin is sequenced separately by the Edman procedure.

Ordering Peptide Fragments The order of the "trypsin fragments" in the original polypeptide chain must now be determined. Another sample of the intact polypep-tide is cleaved into fragments using a different enzyme or reagent, one that cleaves peptide bonds at points other than those cleaved by trypsin. For example, cyanogen bromide cleaves only those peptide bonds in which the carbonyl group is contributed by Met. The fragments resulting from this second procedure are then separated and sequenced as before.

The amino acid sequences of each fragment obtained by the two cleavage procedures are examined, with the objective of finding peptides from the second procedure whose sequences establish continuity, be cause of overlaps, between the fragments obtained by the first cleavage procedure (Fig. 3-27). Overlapping peptides obtained from the second fragmentation yield the correct order of the peptide fragments produced in the first. If the amino-terminal amino acid has been identified before the original cleavage of the protein, this information can be used to establish which fragment is derived from the amino terminus. The two sets of fragments can be compared for possible errors in determining the amino acid sequence of each fragment. If the second cleavage procedure fails to establish continuity between all peptides from the first cleavage, a third or even a fourth cleavage method must be used to obtain a set of peptides that can provide the necessary overlap(s).

Locating Disulfide Bonds If the primary structure includes disulfide bonds, their locations are determined in an additional step after sequencing is completed. A sample of the protein is again cleaved with a reagent such as trypsin, this time without first breaking the disulfide bonds. The resulting peptides are separated by electrophoresis and compared with the original set of peptides generated by trypsin. For each disulfide bond, two of the original peptides will be missing and a new, larger peptide will appear. The two missing peptides represent the regions of the intact polypeptide that are linked by the disulfide bond.

Amino Acid Sequences Can Also Be Deduced by Other Methods

The approach outlined above is not the only way to determine amino acid sequences. New methods based on mass spectrometry permit the sequencing of short polypeptides (20 to 30 amino acid residues) in just a few minutes (Box 3-2). In addition, with the development of rapid DNA sequencing methods (Chapter 8), the elucidation of the genetic code (Chapter 27), and the development of techniques for isolating genes (Chapter 9), researchers can deduce the sequence of a polypeptide by determining the sequence of nucleotides in the gene that codes for it (Fig. 3-28). The techniques used to determine protein and DNA sequences are complementary. When the gene is available, sequencing the DNA can be faster and more accurate than sequencing the protein. Most proteins are now sequenced in this indirect way. If the gene has not been isolated, direct sequencing of peptides is necessary, and this can provide information (the location of disulfide bonds, for example) not available in a DNA sequence. In addition, a knowledge of the amino acid sequence of even a part of a polypeptide can greatly facilitate the isolation of the corresponding gene (Chapter 9).

The array of methods now available to analyze both proteins and nucleic acids is ushering in a new disci-

Quick Permanent Weight Loss

Quick Permanent Weight Loss

A Step By Step Guide To Fast Fat Loss. Do you ever feel like getting rid of the extra weight of your body? If you do, it‟s quite normal because

Get My Free Ebook


Post a comment