DNA bases but also for short sequences of certain bases. Dr. James C. Dabrowiak, a chemist at Syracuse University, notes that such specificity is "unusual for anything involving a free radical mechanism, which is generally considered a Violent' way to do chemistry." In a sense, this seeming paradox makes bleomycin all the more interesting, in that it can combine "violence" with "deliberation." There is no argument about existence of the radical, Dabrowiak says. However, no one is sure of "its exact nature." Thus, the two observations—a radical mechanism and DNA-base specificity— are reconciled partly by the two-sidedness of bleomycin's structure. The right side copes with DNA, possibly wedging between bases and also binding electrostatically through the positively charged bithiazole on that end of the molecule. Meanwhile, the left side of bleomycin "produces bullets." Ferrous iron binds to several nitrogen atoms in the molecule, much as iron binds to heme nitrogens in hemoglobin (the oxygen-carrying protein in blood). The metal ion reduces oxygen and that breaks DNA by forming some as yet unknown radical. The intermediate steps also are unknown. For example, bleomycin does not itself cause chain breaks in DNA; instead, it renders DNA alkali labile, according to MIT's Hecht. Moreover, the radical pathway seems jumbled. About 10 to 20% of the time, bleomycin destroys itself in a side reaction, leaving an inactive but uncharacterized residue. Also, "more bonds in DNA are cleaved than molecules of bleomycin are bound," Hecht says. "I'm not saying it works catalytically—the word makes me nervous." But there are hints that the iron recycles, allowing each bleomycin to inflict multiple damages. Also, because the left side of the molecule functions separately from the right, it might be possible to improve the left side without affecting the right. Bleomycin also is used as an experimental tool, when combined with radioactive metals such as tellurium and gallium, for detecting certain cancers. The metals "home" to certain tumors—and "they light them up like light bulbs" when viewed with radioactive scanners, according to Glickson in Birmingham. Because bleomycin forms complexes with metals, it has been administered in combination with such metals to enhance detection. "But," says Glickson, "there's been a great deal of controversy over whether this is more or less effective." Glickson's findings tend to fuel the dispute. The metal-bleomycin complexes come apart readily in solution. However, he notes, "there may be time in one circuit of blood through the body for the complex to be maintained." Thus, bleomycin might take the radioactive metals in "piggyback" fashion to tumors, or it might work in some altogether different way. "The whole question as to whether bleomycin affects delivery is up in the air still," he concludes. Jeffrey L. Fox, C&EN Washington 22
C&EN Dec. 11, I978
Vaccines, interferon within chemists' reach Synthetic vaccines, stimulants of the immune response, and the natural antiviral substance interferon are within reach of chemists, thanks to recent advances in understanding the three-dimensional structure of proteins. This prospect was raised by Dr. Christian Anfinsen in the first J. T. Baker Nobel Laureate Lecture, delivered recently at Yale University. Anfinsen is chief of the laboratory of chemical biology at the National Institute of Arthritis, Metabolism & Digestive Diseases. "We know that large polypeptides [that are] direct, one-dimensional translations of genetic information can fold spontaneously into functional and reproducible geometries," Anfinsen says. "This fortuitous aspect of evolutionary design permits us to undertake the chemical synthesis of enzymes, large hormones, and molecules with receptor or recognition properties." Synthetic vaccines that now seem feasible to protein chemists include those against hepatitis, rabies, hoof and mouth disease, and specific influenza strains. Antigens for vaccines might be fragments of viral coat proteins, produced by cleavage of the viral coat with trypsin, Anfinsen suggests. He cites a lysine-alanine copolymer as one example of synthetic adjuvants to stimulate immune response to antigens while being compatible with administration to humans. Antigens might be attached covalently to such an adjuvant. The same synthetic vaccine could be used to immunize persons or animals against several diseases at once. A synthetic vaccine already has been modeled by immunizing animals against coliphage MS II, a virus that attacks Escherichia coli bacteria. The single protein that comprises the coat of this ribonucleic acid (RNA) virus was attached to lysine-alanine copolymer. Interest in the nature and supply of interferon has accelerated since the American Cancer Society ordered 40 billion units from the Finnish Red Cross at a cost of $2 million for trials against human cancer. Anfinsen's current research includes purification, amino acid sequencing, and synthesis of interferon. The NIAMDD investigator speculates that if the amino acid sequence were known, immunochemical methods might be used to guide synthesis. A synthetic peptide, 10 to 20 amino acids long and comprising part of the interferon sequence, could be attached covalently to a column-packing material. Passage of anti-interferon antiserum through the column might trap pure anti-interferon antibody, which then could be isolated by elution with guanidine hydrochloride at pH 2 to 3. The antibody could be attached to column-packing material, in turn, and used to trap interferon for purification and to verify that indeed interferon had been synthesized.
Q. to
Anfinsen: fortuitous evolutionary design
Anfinsen says that synthetic interferon has many attractions, but he thinks that production by insertion of interferon genetic material into bacterial genomes will occur first. Pure synthetic interferon would lack impurities that might cause adverse reactions when injected into humans. Recent advances in solid-state polypeptide synthesis, high-performance liquid chromatography, strategies of linking polypeptide subunits into completed proteins, and abilities of protein chains to fold naturally into biologically active structures make interferon synthesis feasible, Anfinsen says, once the amino acid sequence is known. Natural interferon has carbohydrate moieties linked to the protein, Anfinsen explains, but known antiviral activity of interferon stripped of carbohydrate means that synthetic interferon may be active without a carbohydrate content. Deoxyribonucleic acid (DNA) containing information for interferon biosynthesis might be inserted into bacterial genomes in association with a gene responsible for copious secretion of some other protein, he theorizes. Penicillinase is such a protein. Immunochemical techniques could be used to isolate relatively pure interferon mRNA. For example, purified antibody against interferon could be added to fractions isolated from disrupted human cells previously stimulated to produce interferon. Much of the interferon mRNA might be caught in the process of translating RNA into protein. Anti-interferon antibody would thus react with and precipitate whole assemblies of mRNA, ribosomes, and unfinished interferon protein chains. The J. T. Baker Chemical Co. Nobel Laureate Lecture will be a yearly event to be delivered by a winner of the Nobel Prize at a major U.S. center of learning. The second lecture will be given by Dr. Glenn T. Seaborg in 1979 at an as yet unspecified institution in California. D
