TECHNOLOGY
Analyzer finds peptide primary structure New low-cost sequenator unravels amino acid sequence via Edman degradation of peptides bound to resin At Boston University, cliemists Richard A. Laursen and Alex G. Bonner have built a relatively inexpensive peptide sequencing machine using solidstate methods. Last week at the an nual meeting of the Federation of American Societies for Experimental Biology in Atlantic City, they reported favorable results in work to date un raveling peptide chains with their new sequenator. The analyzer can be built with a few weeks* labor in the laboratory, from commonly available components cost ing about $5000. So far, the Boston chemists have degraded peptides through seven cycles or residue deter minations and believe the machine has promise for sequencing larger pep tides, peptide mixtures, and proteins. "Not all of the bugs have been worked out yet," they state, but yields thus far are about 96 to 97'< per cycle. The machine is designed to sequence peptides of up to 20 residues—the size range of most peptides found in protein enzymic digests. Finding the primary structure or amino acid sequence of proteins has
become one of the more important objectives in biochemistry as research focuses more and more on functions of enzymes, hormones, and other pro teins. As the chemistry of this se quencing has become routine, the frontier has switched to automation of the analysis for rapid, quantitative re sults. In the past two years, several sequenators have come on the market based on the degradation procedure developed by Dr. Pehr Edman. These instruments, from companies including Beckman Instruments, Bio-Cal Instru ment Co., and the Illitron division of Illinois Tool Works, carry price tags in the $30,000 to $45,000 range (C&EN, April 28, 1969, page 51). Now Dr. Laursen and Mr. Bonner believe the time and cost of protein structure determination can be re duced significantly. Their innovation has been attaching subject polypep tides to an insoluble resin, aminopolystyrene. This is essentially the reverse of the solid-state method for synthesizing polypeptides, developed in the past
Sequencing instruments use Edman degradation Ri R2 R3 ι ι ι P h - N = C = S + H2N-CH-CO-NH-CH-CO-NH-CH· I Coupling ρΗ8·9 S
Ri
,
R2
II
l
1
I
R3 I
Ph - N H - C - N H - C H - C O - p N H - C H - C O - N H - C H I Cyclization (Splitting) Ι ι Anhydrous acid Μ
•
N
I ψ
\
Ph-NH-C
R2
R3
, I I CH-Ri + H2N-CH-CO-NH-CH
s-c Thiazolinone
11
Conversion 11 Aqueous acid Phenylthiohydantoin for analysis of one chain residue 52 C&EN APRIL 20, 1970
Repeat degradation and analysis
few years by Dr. R. B. Merrifield and Dr. Bemd Gutte at Rockefeller Uni versity, New York, N.Y. (C&EN, Feb. 10, 1969, page 62). This procedure was later commercialized by Schwarz Bioresearch division of Becton, Dick inson and Co. in an instrument costing $18,500. The Edman degradation has two major steps: coupling of phenyl isothiocyanate with the polypeptide at pH 8-9, and cleavage of the resultant phenylthiocarbamyl peptide by cyclization in anhydrous acid to give a thiazolinone and a peptide shortened by one residue. The thiazolinone is then converted to a phenylthiohydantoin for identifica tion, and the residual peptide is run through further cycles of degrada tion. Altered. The Boston University chemists have altered this procedure by attaching the polypeptide to the resin. This linkage occurs through an amide bond between the carboxyl terminal amino acid of the polypep tide and an amino group of the resin. However, first the side-chain func tional groups of the polypeptide are guarded from side reactions by block ing their amino and carboxyl groups with appropriate protecting groups. The resulting insolubilized poly peptide is packed with glass beads into a thermostated glass column, and the degradation process begun. Each cycle takes two hours and is controlled automatically by a programer. The product phenylthiohydantoins—labeled with a radioactive group from the starting reagent—are identified chromatographically by comparing the labeled compound with unlabeled phenylthio hydantoins corresponding to the com position of the peptide. The heart of the sequenator is the reaction column, which is 3 mm. in inner diameter and 120 mm. in length, with fittings on each end through which reagents are pumped. The in let end is connected to a series of five electrically controlled pneumatic valves, which control flow of five re agents. A valve at the outlet end of the column directs effluent to a waste bottle or the fraction collector. All valves and fittings are of glass, Teflon, or Kel-F. Operation is controlled by a disk-type programer.
