RESEARCH
Subunit Structure of Proteins Allows Differentiation Studies Varying protein structures seem to give different enzymic activities, specificities, and functions Varying subunit structures of proteins caused by the different activities of genes during embryonic development have a significant effect on enzymic activity and function during cell differ entiation. This conclusion results from bio chemical studies of the change in en zyme content and structure during cell differentiation—a process by which the embryonic cell ultimately develops into specialized adult cells. Some of the latest progress in this area of research was made public during the Sympo sium on Protein Structure and Func tion During Differentiation, held dur ing the recent 47th Annual Meeting of the Federation of American Socie ties for Experimental Biology in At lantic City, N.J. In the past, numerous laboratories have reported that the enzyme lactate dehydrogenase ( L D H ) exists in more than one form. In fact, five distinct forms of L D H , called isozymes, have been found. Each isozyme of L D H dissociates into four polypeptide subunits of equal size when treated with urea or guanidine, indicating that each isozyme is a tetramer. The isozymes of L D H are of the same molecular weight (about 135,000 ). They differ from each other in net electrical charge; hence, they may be separated electrophoretically. Subunits. Earlier, having found two electrophoretic varieties of subunits, A and B, Dr. Clement Markert of Johns Hopkins University, Balti more, Md., proposed that the five iso zymes of L D H could be formed by five different combinations of these two simple subunits. These would be: LDH-5 = A 4 B°, LDH-4 = A 3 B \ LDH-3 = A 2 B 2 , LDH-2 = A ^ 3 , and LDH-1 = A°B 4 . Dr. Markert and his co-workers were able to obtain all five of these isozymes from equimolar proportions of LDH-1 and LDH-5. The L D H used in the experiments was prepared in crystal 38
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line form from beef tissue contain ing all five isozymes. LDH-1 and LDH-5 were then separated from the mixtures of isozymes by column chromatography on diethylaminoethylcellulose and by electrophoresis through a cellulose column. Each of these two isozymes should consist of only one kind of subunit: A-type poly peptides in LDH-5 and B-type poly peptides in LDH-1. The LDH-5 and LDH-1 isozymes were mixed in equal proportions in 1M sodium chloride, frozen overnight, allowed to incubate at room tempera ture for eight hours, dialyzed overnight against 0.1M phosphate buffer ( p H 7.0), and then resolved by starch gel electrophoresis. Not only did the mixture of LDH-5 and LDH-1 generate all five isozymes, but also in about the expected propor tions of 1:4:6:4:1, which should be obtained at equilibrium if random reassociation of subunits into tetramers occurs, Dr. Markert says. Treatment of LDH-1 or LDH-5 alone in the same way produced no change in these isozymes; each of these dissociated subunits reassembled into its original tetramer only. Proportions Important. The rela tive amounts of the various isozymes produced depend upon the proportions in which LDH-1 and LDH-5 are mixed. The distribution may be skewed according to the input of subunits to generate the various isozyme patterns found in normal tissue homogenates, Dr. Markert says. Since the A and Β subunits have differing amino acid compositions, they are different proteins and prob ably are under the control of separate genes, Dr. Markert says. The isozyme patterns generated in developing tis sues can thus be attributed to the dif ferent relative amounts of A and Β synthesized in each cell as a result of differential gene function. In conflict with Dr. Markert's hy
pothesis is the sixth isozyme which other workers have recently found in human sperm. Synthesis of this sixth isozyme must either involve an addi tional gene or represent a specialized modification of one of the other iso zymes, he says. In other experiments, Dr. Markert and his group have combined subunits from different animal species to form "hybrid" molecules. For exam ple, they have combined subunits from mice and rabbits, mice and horses, mice and cows, and cows and horses to form the hybrid molecules. Study of these hybrid molecules may throw light on the question of which properties of monomers are necessary in forming functional, three-dimen sional protein structures. Two Types. Dr. Nathan O. Kaplan and his co-workers at Brandeis Uni versity, Waltham, Mass., have also found that there are two basic pure types of L D H , each made up of a single kind of subunit. And they also believe that these are under the con trol of separate genes and are dif ferent in their molecular, chemical, Dr. and catalytic characteristics. Kaplan refers to the two types of LDH as H (Dr. Markert's B) and M (Dr. Markert's A ) . He, like Dr. Markert, has also found a total of five isozymic forms of L D H , each con taining four subunits, with three of the isozymes merely being hybrid com binations of H- and M-type LDH. Using the two types of LDH, Dr. Kaplan's group has studied con trol of cell differentiation in embryonic development (as well as the environ mental and chemical factors regulating this control) and the enzyme changes in evolution. Dr. Kaplan finds that enzyme structure is significant in func tion. He also finds that evolutionary changes in enzymes don't occur ran domly and believes that they play an important part in the development of new species.
ISOZYMES. This photograph shows the lactate dehydrogenase isozymes pres ent in each of three preparations after electrophoretic resolution in starch gel. On the top right is LDH-1, on the bottom left LDH-5, and in the middle are the isozymes resulting from a mix ture of equal quantities of these two preparations. All five isozymes were generated in the mixture and in the approximate ratio of 1:4:6:4:1, the ex pected distribution after random reassociation of subunits, Dr. Markert of Johns Hopkins says. The total enzyme activity in the mixture was the sum of the activities of the single-isozyme preparations
The Brandeis group has developed a simple quantitative method for de termining the relative amounts of Hand M-type LDH in different tissues. The relative amounts of these two forms can be used to study the func tional importance of the different types of L D H in metabolism, Dr. Kaplan says. These two different forms of LDH appear to have different func tional roles, as suggested by their catalytic properties, he adds. Physiological. The difference in substrate inhibition between H- and M-type L D H is important physiologi cally, the Brandeis workers believe. Tissues such as most skeletal muscles, where there is usually a sudden need for energy, contain M-type LDH. Tissues such as heart, which require sustained energy and are more aerobic, contain Η-type LDH. The physiological roles played by the M and H LDH's in the breast muscle of different birds have been
studied by Dr. Allen Wilson and Dr. Robert Cahn of Brandeis. Birds that are poor fliers show a low substrate inhibition and hence contain all M-type enzyme. These birds re quire sudden energy in their pectoralis muscles. By contrast, birds who are sus tained fliers have the Η-type enzyme. The breast muscles in these birds are geared for oxidative metabolism. Birds with intermediate flying ac tivities have mixtures of the two forms of LDH. Dr. Kaplan's group has been able to work out a correlation between the composition of LDH and the flying habits of various birds. Studies show that the M form of LDH appears to be more subject to change than the H type is. It also seems to be more sensitive to environ mental and chemical control. Tumors. For example, studies on human tumors show that tumorous tissue has a much higher level of LDH than nontumorous tissue has. The in crease can be almost completely ac counted for by a rise in M-type units, with little change in the number of H units. Normal stomach tissue shows about 10% M-type LDH. But can cerous tissue taken from the stomach shows about 60% M-type L D H units. α-Glycerophosphate dehydrogenase (α-GPDH) appears to be closely linked with the M-type LDH in high vertebrates, according to the Brandeis workers. For example, sustained-fly ing birds containing large amounts of Η-type L D H have little or none of the α-GPDH. By contrast, birds with a large amount of M-type LDH in their pectoralis muscles have a high α-GPDH content. These and other observations indicate that the factors controlling the synthesis of the M-type LDH and the α-GPDH must be quite similar, Dr. Kaplan says. Control. The subunit structure of proteins may control enzymic activity, say Dr. Gordon Tomkins, Dr. K. L. Yielding, and Dr. Norman Talal of the National Institutes of Health, Bethesda, Md. They offer four reasons why pro teins have structures made up of sim ple subunits. These are the conserva tion of genetic information, mecha nism of protein synthesis, repair of genetic damage to protein chains in diploid organisms (complementation), and biological regulation. For example, small virus coat struc tures composed of identical protein subunits may be required to conserve
the information contained in viral nu cleic acids. Or protein synthesis may require an optimum size of messenger ribonucleic acid or ribosomal aggre gate to function properly. But of most interest to the NIH workers is the fact that the subunit structure of proteins may have some thing to do witk the mechanism of biological regulation. For example, biological regulation is indicated by changes in the catalytic properties of the enzyme glutamic dehydrogenase (GDH) with changes in the aggregation-disaggregation structure of the molecule. Hence, a molecular con formational change such as aggregation-disaggregation could serve as a biological control mechanism, Dr. Tomkins says. GDH consists of multiples of a small subunit which has a molecular weight of about 65,000. The largest aggre gate of subunits studied so far has a molecular weight of 1 million. The "monomer" has a molecular weight of about 250,000 (in the past, this was considered the smallest unit). There is also an intermediate aggregate of unknown molecular weight. The largest aggregate catalyzes the GDH reaction. The monomer cata lyzes the alanine dehydrogenase (Ala DH) reaction. And the intermediate aggregate of unknown molecular weight catalyzes both reactions. How ever, it does so less efficiently than does either the large aggregate or the monomer. Components. The GDH system can be resolved into three antigenically different components. One of these has primarily GDH activity, one has both GDH and AlaDH activity, and the third has mainly AlaDH ac tivity. Since it's known that disag gregation stimulates AlaDH activity, the NIH workers believe that the three antigenic components correspond to large aggregate, intermediate, and monomer, respectively. A fourth component, enzymically inactive but antigenically identical to the monomer, has been detected by Dr. Tomkins and his co-workers. This component has the properties of a basic subunit. This observation, to gether with the other evidence, sug gests that the entire GDH structure is built from a single species of subunit, Dr. Tomkins says. Thus the GDH system is an exam ple of biological regulation brought about by molecular conformational change, Dr. Tomkins says. MAY
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of very reactive species, not available at conventional chemical reactor temperatures. • Simple, versatile, equipment requirements.
ARC. The running arc shown here was struck between a graphite anode (right) V 2 inch in diameter and a hollow graphite cathode measuring 2 inches outside diameter and 1 inch inside diameter. The CF4 feed rate was 10 cc. per second; power, 25 kw.; magnetic field, 200 gauss; and reactor pressure, 150 mm. Hg
High-Intensity Electric Arc Shows Promise as Chemical Reactor MIT work shows that fluorocarbons can be synthesized at about 4000° K. A high-intensity electric arc is being put to work as a chemical reactor for synthesizing tetrafluoroethylene ( C 2 F 4 ) , the monomer that's used to make Teflon (C&EN, April 22, page 35). Dr. Raymond F. Baddour, Barry R. Bronfin, and their co-workers at Massachusetts Institute of Technology can produce yields as high as 68 mole % using carbon tetrafluoride (CF 4 ) reactant in their experimental work with an arc reactor. The MIT group uses a high-intensity electric arc as a chemical reactor by introducing reactants into the luminous, high-temperature arc region. One reactant, the carbon tetrafluoride, is introduced as a gas directly into the reaction zone as a convenient source of fluorine, Dr. Baddour told the Electrochemical Society meeting in Pittsburgh, Pa. The other reactant, carbon, is produced as a result of electrode vaporization. A high-intensity electric arc occurs when a large electric current is passed through a gas. Initiation of an arc can be obtained across two electrodes 40
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by applying high-frequency, highvoltage d.c. to ionize the interrupting gas. Once the conducting path is established, an arc can be readily sustained. In the process, a large current of electrons leaves the cathode and travels (through successive collisions) toward the anode. The intense bombardment of a carbon anode with electrons causes it to vaporize; thus it has to be advanced to maintain constant gap and prevent the arc from extinguishing. The luminous reaction zone (plasma) at high temperatures is a region of intense chemical activity. Dr. Baddour and Mr. Bronfin point out the properties that make the arc reactor adaptable to chemical synthesis as: • Extremely high temperatures, ranging from 2000° to 10,000° K. • Very high throughput because of rapid reaction rates and rapid coupling of energy into reactants. • Production of high concentrations
small-scale
Reactor Design. To study the carbon-fluorine system at arc temperatures, the MIT workers constructed an arc reactor consisting of four electrically insulated sections. The central cross-section is 3 inches in diameter and serves as the reactor chamber, which is electrically floating. The anode section is mounted at one port. The section is designed to allow a continuous motor-driven feed of a graphite anode. The anode is centerbored to allow reactant gas to flow directly into the center of the plasma formed between anode and cathode. Other experimental runs have been made with a solid graphite rod, with reactant flowing along the outside of the anode and into the hollow cathode pipe. A stationary hollow graphite cathode is mounted at the opposite port. A water-cooled hypodermic probe is positioned within the hollow cathode. A large viewing window is placed in a port normal to the electrode ports to allow watching the arc. All of the arc reactor's metal parts are watercooled. Electrically insulating gaskets seal the reaction chamber. Power is fed through the electrodes from series-operated motor-generators supplying up to 25 kw. To promote mixing of the cold reactant gas with the plasma formed across the electrode gap, a 200-gauss magnetic field is imposed along the electrode to obtain rapid rotation of the arc. Gas samples are taken through the watercooled probe (estimated quenching rate of about 10 6 ° K. per second) and are analyzed by on-line gas chromatography. Experimental Results. The results of a series of runs at atmospheric pressure show that it's possible to obtain the yield-limiting region (predicted by thermodynamic calculations) of 30 to 40% with the high-power, highfeed-rate condition. The highest conversion rates were obtained at the high power levels—15 to 25 kw.—and fairly high feed rates of carbon tetrafluoride, 25 to 50 cc. per second (standard temperature and pressure), equivalent to 1.5 to 3 liters per minute, the MIT workers say. A power input of 25 kw. vaporizes the carbon anode at the rate of 10
from abstract ideas...fundamental knowledge at Isso Research
Preferential solvation of the neutral ization products is the major factor in determining the effects of solvents on the acidity and basicity of solutes according to studies by Dr. Orest Popovych, an Esso Research scientist. T h e equilibria between an indicator acid, bromophenol blue, and a series of heterocyclic amines in hydrocarbon and hydroxylic media have been de scribed for the first time by a series of equations and constants. These can be evaluated by a combination of visible spectrophotometry (as represented by the above illustration) and electrolytic conductance. T h e ion-pair formation constant Κ Β = (BH+A")/(B)(HA) was
adopted as the measure of basicity of an amine Β relative to a reference acid HA. It was found that while the relative strengths of the bases remained inde pendent of the solvent, the absolute values of the K B ' s were a sensitive function of the medium. The acid-base reactions were most favored by polar and hydroxylic media. For example, adding isopropyl alcohol to toluene caused a rapid initial rise in reactivity which reached a plateau at about 5 % of added alcohol.The addi tion of 0 . 5 % water to a 50-50 isopropyl alcohol-toluene mixture produced a similar effect.
These indications together with other data make it clear that stabilization of ion pairs and ions by solvation seems to be the major factor in determining variation of acidity and basicity as a function of solvent. This new knowledge will permit more meaningful interpretation of acid-base phenomena and a more realistic approach to all research fields where nonaqueous media are involved. . . . adapted from the scientist's notes at Esso Research and EngineeringCompany (P. O. Box 45Β, Linden, New Jersey) scientific affiliate of Humble Oil & Refining Company
Texaco
Petrochemistry on the move
Ammonia—to grow a bumper crop or purify uranium ore Many thousands of tons of Texaco ammonia are produced each year for an almost unlimited number of applications, —as a principal nitrogen carrier in synthetic fertilizers, for making acids and synthetic fibers. Most recently, it has proved its value as an agent for purifying uranium ore. Ammonia is only one of Texaco's growing line of in dustrial chemicals. Others provide the raw materials for manufacturing such products as detergents, paints, plas
tics, explosives and pharmaceuticals. Most likely, one or more Texaco petrochemicals are working for you now. Texaco Inc., Petrochemical Sales Division, 135 E. 42nd Street, New York 17, Ν. Υ.; 332 So. Michigan Ave., Chicago 4, 111.; Texaco Canada Limited, 1425 Mountain St., Montreal 25, Quebec; or Texaco (U.K.) Limited, 29/30 Old Burlington St., London W. 1, England.
AQUA A M M O N I A , ANHYDROUS A M M O N I A , NITROGEN SOLUTIONS, Dl ISOBUTYLENE, CUMENE, ODORLESS MINERAL SPIRITS, NAPHTHENIC ACID, PROPYLENE TETRAMER, BENZENE, TOLUENE, RUST INHIBITORS, AROMATIC SOLVENT, LUBE OIL AND FUEL OIL ADDITIVES
grams per minute. With this power level and a feed rate of 50 cc. of carbon tetrafluoride per second, carbon-to-fluorine ratio is about 2. Species in effluent gas are carbon tetrafluoride, tetrafluoroethylene, hexafluoroethane, and hexafluoropropylene. Tetrafluoroethylene yields increase gradually with decreasing reactor pressure. At 0.1 atmosphere, yields of up to 68 mole% were obtained with high power input. Changing the reactant flow rate has a negligible effect on the yield of tetrafluoroethylene. With fewer collisions at this pressure, equilibrium conditions probably aren't reached. Conversion of carbon tetrafluoride to tetrafluoroethylene falls off as the quench probe is drawn away from the cathode face, defined as zero displacement. This indicates that the plasma is cooling rather slowly, being totally enclosed in the graphite cathode. The cooler plasma regions are apparently unable to support precursors for tetrafluoroethylene production. Normal operation of the reactor results in the immediate coating of the inner wall or the quench probe with a dense layer of carbon after a few seconds of operation, thus providing a cold carbon wall as the quenching device. A carbon surface for quenching had been reported earlier by Du Pont workers to improve tetrafluoroethylene vields in their electric arc process (U.S. Patent 2,852,574). Two Steps. The MIT scientists describe chemical synthesis via the high-intensity arc as a two-step process. The first step is the production of high-temperature-stable species. This occurs in the plasma region, represented as the arc reactor. The second step is the quenching of the highly reactive intermediate species to products . This step, represented as the quench reactor, is essential to obtaining significant degrees of conver* sion. According to Dr. Baddour and Mr. Bronfin, each step has problems which complicate the use of the arc for high conversion rates to desired products. The mixing of an externally supplied reactant wth .the small plasma zone, which has steep radial temperature gradients, is essential and often difficult. The quench reactor must be able to provide cooling rates in the range of 10 6 ° to 10 7 ° K. per second. Consideration must also be given to the proper location of the quenching zone in relation to the plasma. And
the quench surface can affect the product compostion. While the kinetics for the production of high-temperature fluorocarbon intermediates was not known, Dr. Baddour and Mr. Bronfin calculated gas compositions from previously estimated thermochemical properties of the carbon-fluorine system at high temperatures. The composition distribution as a function of temperature for the carbon-fluorine system was calculated by using a free-energy minimization technique. The calculations were made for various carbon-to-fluorine ratios and various pressures up to 1 atmosphere. Below the sublimation temperature (4000° to 4500° K.), the composition is independent of the carbon-to-fluorine ratio, because this is the region where the multicomponent gas phase exists in equlibrium with solid carbon. The sublimation temperature defines the region of the arc reactor temperature, Dr. Baddour and Mr. Bronfin say. Reaction. They describe the arc reacton as:
Viscosity measurements taken while you ^ a ^
CF 4 + C (gas) -> C 2 F 2 (not detected in quench sample) + 2F. C 2 F 2 + 2F -> C 2 F 4 . Calculations indicate that difluoroacetylene (C 2 F 2 ) is stable over a wide temperature range between 2000° and 4000° K. Above 4500° K., all species are reduced to the simple atomic structures. If it were possible to trap species at 4000° K., they would be expected to contain primarily difluoroacetylene. However, difluoroacetylene and free fluorine atoms are violently reactive, and most likely combine to form primarily tetrafluoroethylene, a stable gas at ordinary temperatures and pressures. An additional contribution to the formation of tetrafluoroethylene, though only to about 3 to 5%, is the dimerization of CF 2 . At successively lower temperatures, it might be expected that up to 90% C 2 F 4 formation could be achieved. However, the reaction C 2 F 2 -f 2F -» C 2 F 4 isn't possible here, since the region is poor in fluorine. Dr. Baddour has also used the electric arc device to produce acetylene. Using hydrogen as a feed gas, acetylene concentration up to 25.5% was produced. With methane feed, Dr. Baddour obtained 52% acetylene. He also points out that the unit could be feasible for converting coal into chemicals and fluid fuels.
A c t u a l l y , t h e Lab-Vis m e a s u r e s viscosity i n s t a n t l y . It is a direct r e a d i n g v i s c o m e t e r t h a t f e a t u r e s a p a t e n t e d s e n s i n g device, c a l l e d a p r o b e . This p r o b e is i m m e r s e d in t h e s a m p l e f l u i d w h e r e it v i b r a t e s at ultrasonic f r e q u e n c i e s . Viscosity is electronically p r e s e n t e d on a c e n t i p o i s e m e t e r as a f u n c t i o n of t h e "viscous d r a g " on t h e p r o b e . T h e Lab-Vis m a y be used f o r r o u t i n e s a m p l i n g or f o r c o n t i n u o u s m e a s u r e m e n t s u c h as in l i q u i d phase k i n e t i c s , r a p i d e x p e r i m e n t a l b l e n d i n g a n d pilot p l a n t m o n i t o r i n g . Why has t h e Lab-Vis been so s u c c e s s f u l in i n d u s t r y as well as m e d i c a l research? Read t h e s e s p e c i f i c a t i o n s : Speed: .5 sec. response Accuracy: =±=2% of full scale Range: 0-50,000 centipoises (8 ranges) Operation: •! t e m p e r a t u '; e s t o 6 5 0 ° F · r (pressures from vacuum to 1000 psi Sample Volume: from 2 ml to continuous flow Readout: meter, strip chart recorder (optional) Probes: laboratory, pipeline, and medical For m o r e i n f o r m a t i o n , w r i t e T h e B e n d i x C o r p o r a t i o n , D e p a r t m e n t K-5, 3 6 2 5 H a u c k R d . , C i n c i n n a t i 4 1 , Ohio.
Cincinnati Division « (fcncfpS MAY 13, 1963 C & E N
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