vide for this endo-alkylation (bonded to either the apoenzyme or coenzyme) could adversely affect the fit at the receptor site. Dr. Baker's proposal would permit changing a compound more drastically to give better irreversible bonding, even permit exo-alkylation. In exoalkylation, a part of the inhibitor molecule projects over the surface of the apoenzyme and bonds to it by alkylating some active site. And a careful examination of the literature shows that there is considerable evidence for this hypothesis, Dr. Baker says. For example, glutamic dehydrogenase, the enzyme that catalyzes the dehydrogenation of glut am ate, is inhibited by glutarate. This is hardly surprising, he adds, since the molecules are almost identical in shape and differ only in the presence of the amino group. But the enzyme is also inhibited by isophthalate, which has a considerably different over-all shape. This can be interpreted as indicating that the only important configurations of the apoenzyme that have to be matched are the two active sites where the carboxyls are bonded and the distance between these sites. Here, then, is a case where a large part of the benzene ring may be available for the attachment of groups which will more effectively or more selectively inhibit the reaction, Dr. Baker explains. And it offers an opportunity for the substitution of long chains or other groups which would extend over the surface of the apoenzyme and provide many opportunities for strong bonding by exo-alkylation. Present Work. Dr. Baker's present work is on the first two of three steps in developing antimetabolites according to the new hypothesis. By using a wide variety of inhibitors for a specific reaction, he is exploring the geometry of the receptor site on the apoenzyme and determining just how much of the back side of the inhibitor molecule is available for bonding. As he accomplishes this, he is then attaching groups to this back side to determine which groups will extend most effectively over the apoenzyme surface. The third step will then be to determine the location on the enzyme surface of the most active sites for exoalkylation by varying the location of the alkylating group along the chain attached to the rear of the molecule.
Scientists Unravel TMV Protein Structure Establishing sequence of amino acids in tobacco mosaic virus opens way to new studies on viruses The complete amino acid sequence in the protein portion of tobacco mosaic virus (TMV) has been established by a research team which includes Dr. Wendell M. Stanley, Dr. Heinz L. Fraenkel-Conrat, and Dr. C. Arthur Knight at University of California's virus laboratory in Berkeley (C&EN, Nov. 21, page 4 3 ) . This is only the third (others are insulin and ribonuclease) and the largest protein for which the complete amino acid sequence has been determined. TMV is the first virus protein to be thus characterized. This achievement opens the way for a variety of studies on the chemical structure and function of viruses.
The group at Berkeley, which also includes Dr. Akira Tsugita, Dr. Duane T. Gish, and Dr. Janis D. Young, has already shown differences in the amino acid sequence of certain mutant strains of TMV. Studies of the point-to-point relation between the nucleic acid core and the protein coat of the virus can now be made, too. An understanding of this relationship is a key to many of the fundamental biological mechanisms. Recent Progress. Real progress in unraveling the structure of proteins has come only very recently, largely through the development of new techniques. Dr. Frederick Sanger estab-
TMV Protein Has This Sequence
This train of 158 amino acids form the protein subunit of tobacco mosaic virus, according to workers at University of California. Trypsin cleaves the molecule into the 12 peptides indicated by the red arrows. There are 21 amide groups in the molecule that are not in the chain. The circles above show the location of 19 of these.
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lished the sequence of the 51 amino acids in insulin during the early fifties. A few years later, the sequence of the 124 residues in ribonuclease was determined. Now the California group has found the pattern of the 158 residues in TMV protein. Dr. Stanley points out that these rapid advances in protein chemistry have been greatly aided by these de velopments: • Countercurrent distribution and chromatography to separate and iden tify peptides and individual amino acids. • Isolation of highly specific en zymes; carboxypeptidase, for example, attacks a protein or a peptide molecule at the carboxylate end, removes one amino acid residue at a time. • Chemical degradation techniques which can remove one amino acid at a time from either end. • T h e automatic amino acid ana lyzer which accurately determines most of the amino acids in the hydrolyzate of a peptide or protein in 24 hours. Subunits. Until recently, the TMV protein was thought to be a huge molecule having a molecular weight of about 40 million. A real attack on the protein structure became possible when work in the virus laboratory showed that the protein coat of the virus was actually made up of some 2200 subunits, each having a molecu lar weight of about 18,000. These subunits are held together by second ary forces, probably hydrogen bonds. The protein subunits were degraded into 12 peptides of from two to 41 amino acids by trypsin, an enzyme which splits peptides at the carboxyl ate end of arginine or lysine only. The peptides obtained from this step are then split further, either chemi cally or with other enzymes. Step wise chemical or enzymatic degrada tion gives the sequence in the smaller pieces. Bridging Peptides. Once the amino acid sequences in the 12 peptides pro duced from TMV by trypsin were worked out, the order in which these peptides themselves were arranged had to be discovered. "Bridging" peptides were obtained from TMV protein by using subtilisin and chymotrypsin, enzymes which split the amino acid chain randomly rather than systematically. Peptides were found that bridge the points in the protein that had been broken by tryp-
sin, showing how the 12 known se quences were joined. As the California work progressed and parts of the protein structure were published, a group at Max Planck In stitut fur Virusforschung at Tubingen, Germany, also took up the problem. The German group published an al most complete amino acid sequence earlier this year. Dr. Stanley points out that the surprisingly close agree ment between the nearly complete sequence published by the German workers and the complete sequence which he and his co-workers have established is strong evidence for the validity of the results. The minor differences which remain can probably be resolved within a few months. These differences may be due to analytical technicalities; or to the fact that the two groups have been working with slightly different strains of TMV. Dr. Stanley hopes to settle this point shortly through an exchange of virus samples.
Polymer Resists Heat Polybenzimidazoles can withstand temperatures higher than 500° C. New polymers with a completely con jugated aromatic structure show good high temperature stability. Most promising of the polymers: polybenz imidazoles. Preliminary tests show that the polymers are able to withstand temperatures up to 600° C. in a nitro gen atmosphere, do not decompose completely even at 900° C. The polymers are film and fiber formers, also show promising physical characteristics, Dr. Carl S. Marvel of University of Illinois told a St. Louis, Mo., symposium sponsored by the St. Louis Section of the American Chemi cal Society. The symposium honored Dr. Charles D. Harrington of Mallinckrodt Chemical Works, who re ceived the section's 16th Midwest Award. The new polymer may point the way to a host of new heat resisting poly mers, Dr. Marvel says. There's a pressing need for polymeric materials able, to withstand high temperatures. Among potential uses are in high speed aircraft and missiles. Best performers today are fluorine-containing polymers, Dr. Marvel notes. Some rubbers in this group are able to withstand tem peratures up to 350° C.
The search for polymers with even better high temperature performance is along four main lines: inorganic polymers, organic modified inorganic polymers, polymers with metal che lates, and organic polymers with ther mally stable materials. So far, neither high molecular weight inorganic poly mers nor polymers with metal chelates have been made, Dr. Marvel says. And organic modified inorganic poly mers such as the silicones tend to cyclize at about 250° C. But organic polymers made of heat-stable mate rials look promising, he says. Solid State Polymerization. Ingre dients of Dr. Marvel's best performing polymer are diaminobenzidine and diphenylisophthalate. These mate rials polymerize when heated in their solid states at 400° to 450° C. Heating for four hours yields ma terial with an inherent viscosity of 0.6, which indicates a molecular weight of about 54,000, Dr. Marvel says. Longer heating should give material with a viscosity close to 1, and an estimated molecular weight of about 90,000, he adds. The new polymer is soluble in sul furic acid, formic acid, and dimethylsulfoxide. But if polymerization is carried out at 500° C. or above, the material becomes insoluble despite no evidence of structural change. Dr. Marvel says that cross linking may be responsible. Films cast from dimethylsulfoxide solutions have a tenacity of 0.7 gram per denier at room temperature (ny lon runs 4.5 to 7 ) , and 0.5 gram per denier at 200° C. Elongation is 9% at 200° C , and modulus is 16. Color of the film ranges from yellow to yellow-brown. Melting point of the new polymer is above 770° C , Dr. Marvel says. There is very little weight loss in a nitrogen atmosphere at 500° C. At 900° C , weight loss is about 30%. Heating in air at 600° C. for five hours results in a weight loss of 22%.
BRIEF
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X-radiation boosts power of antitoxin by disrupting the "localizing" effect of normal inflammation, says Dr. Reuben L. Kahn of the University of Michigan Medical Center. Mild rather than severe reactions to staphylococcus germs occurred in rab bits of the injection area was x-irradiated first.
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