California Association of Chemistry Teachers
John Leo Abernethy
Department of Medicine UCLA School of Medicine University of California Los Angeles, 90024
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Franz Hofmeister T h e impact of his life
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a n d research o n chemistry
[he modern scientist must have a broad knowledge of many fields. A study of early journals reveals that the trend toward interdisciplinary scholarship began years ago. Among those who crossed boundaries in making major, multiple scientific contributions was the physiological chemist, Franz Hofmeister (1, Z), who was articulate in chemistry, biology, pharmacology, and medicine. About four hundred papers (3, 4) were published from his own research or that of students in his laboratories. Hofmeister's Personal Life
Franz Hofmeister was born in Prague, Czechoslovakia, in 1830. His father was the first doctor in that city to perform a chloroform anesthesia. After the death of his father, Hofmeister's mother and sisters supported him during his medical training at the German University of Prague. He was married in 1891 and continued to live in the family home, where his life was a quiet and happy one. Even as a medical
F r m z Hofmeirter at Strasburg, reproduced from o photograph in Ergebnise der Phyriokgie, 22, 1 11 9231.
student, he had developed an early fascination with chemistry. When H. Huppert was called to Prague from Leipzig, in 1872, to become Professor of Medicinal Chemistry and to establish a laboratory of physiological chemistry, Hofmeister came under his influence. Contact with this brilliant analyst no doubt helped him acquire his highly methodical habits and also probably prompted his submitting articles on the methodology of physiological chemistry to Fresenins' "Zeitscbrift fur Annlytische Chemie." A large chemical institute had been built in Prague in 1879, the year in which Hofmeister obtained his doctoral degree based on analyses of peptones. The following year his name was proposed for the position of Chief of the Institute of Experimental Pharmacology which was being added to the German Medical School in Prague. A trip was arranged for him to visit Oswald Schmiedeberg, who was director of a renowned institute of this type in Strassburg. On his return t o Prague, three modest rooms at No. 4 Hospital Street. were given over to the new institute. By 1885, he had advanced to the status of full professor, and later he became Dean of the Faculty. He was effervescent with projects and always communicative with the rapidly expanding European scientific world. His influence was most part,icularly felt by the young men doing research under his sophisticated direction and by those who heard his dynamic lectures, which had a series of well-planned demonstrations. A faithful collaborator in the more practical aspects of his work was the mechanic of thc institute, H. Jaroslav John. For seventeen years he maintained his strenuous pace of research in contact with the famed men of the medical faculty. I n 1896 he terminated his work in Prague when he was called to the University of Strassburg. He was chosen to fill a position left open by the death of F. Hoppe-Seyler, who had been editor of the famous Hoppe-Seyler's Zeitschrift fur physiologische Chemie since its inception in 1877. Hofmeister made a significant contribution to the first issue of the journaI (5), "Ueher Lactosurie," followed by other important articles in subsequent issues. He became a member of the editorial board when he moved to Strassburg. Hofmeister was made Professor of Physiological Volume 44, Number 3, Morch 1967
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Chemistry in 1896 and became associated with his acquaintance of many years, Oswald Schmiedeberg. He soon gained recognition as an outstanding teacher and developed a superior laboratory manual. He was twice made Dean of the Faculty, during which time he tactfully adhered t o unyielding commitment in important matters where conflicting goals were at stake. His modest demands taxed the available facilities for maximum productivity. He became somewhat of a recluse and left Strassburg only for scientific meetings or when summoned for special reasons. On several occasions he gave lectures that coordinated large backgrounds of research. He covered a diversity of topics, such as "The Chemical Organization of the Cell," 1901, and "The Chemical-Morphological Boundary Area," 1914. He was offered professorships a t three other universities, Heidelberg, Berlin, and Vienna, but he chose to remain a t Strassburg, where he fitted completely into the cultural life. While his research a t Prague had often dealt with physical chemistry, almost nothing was done in this area at Strassburg. He insisted that his students be able to use scientific methods with finesse in obtaining and interpreting results. He was concerned with establishing those qualities in his students that would serve both the Alsatians and the Europeans well. As a consequence, there was seldom an occasion for political antagonism, which could have arisen, inasmuch as Strassburg was located in Alsace-Lorraine. Following the war of 1870, the university was established in 1872 as a German institution. When the Treaty of Versailles was signed in 1919, putting Alsace-Lorraine under French control, many professors left the University. Hofmeister was one of the last to do so. The only pressure placed on him was his own recognition that the University under a new government would need to reduce to a minimum potential elements of ineffectiveness. I n his closing years, he joined the University of Wiirzburg. Now, a t a slower pace, he again took up the continuity of his research, which was his real enthusiasm in life. To him (g), "Logic was a power that could lead chaos into order and help to fashion the world and its inhabitants.'' He died in Wiirzburg on July 26, 1922, a few weeks short of 72 years of age. Scope and Influence of Hofmeister's Publications
Although Hofmeister used several journals for research publication, the Biochemische Za'tschrilt published the largest percentage of his papers. Botany and chemistry became fused with physiology when nicotine, caffeine, morphine, digitalis, and other alkaloids were used for experimentation. An extensive animal laboratory was required for the cats, dogs, rats, and rabbits necessary for studying aspects of a wide spectrum of diseases including beriberi and diabetes. Humans were participants in his work with leukemia and lactosuria. A striking example of the precise role played by chemistry in Hofmeister's work was shown when he established that the sugar involved in lactosuria of pregnant women is not glucose, as had been assumed, but lactose. He isolated the sugar, hydrolyzed it, and subjected reactant and products to elemental analysis, to osazone formation, and polarimetric analysis. As a consequence, the name of this disorder was changed from glucosuria to lactosuria (6). 178
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Animal fluids involved in his research were urine, blood, stomach fluids, intestinal fluids, and animal oils. The heart, kidney, liver, spleen, and muscle were among the organs and tissues used in viuo and in excised condition. Combinations of procedures often led to lecture demonstrations that were sensational teaching aids. His research revealed much pure and applied chemical information concerning lactic acid, glycogen, lipids, proteins, and porphyrins. I n Strassburg, he became progressively more involved with vitamins and deficiency diseases and published several significant papers (3) on this subject. Hofmeister had the capacity for uncovering experiments with a very novel twist. His research on copper binding with leucine, asparagine, and glutamic acid contributed to coordination chemistry. He also investigated the use of such precipitants as phosphotungstic acid for separating proteins. He showed that platinum complexes with ammonia have a curarelike activity that increases with the number of ammine ligauds in the complex. However, the experiments for which the name Hofmeister is most remembered are comprised of a rather compact segment of his total research from his early work in Prague. He and two of his students, S. Lewith and R. Limbeck, published a connected sequence of six papers under the general title, "On the Mode of Action of Salts" (6). A seventh paper (6) was published in 1898 by E. Miinzer, another of his students, who drew general conclusions from the previous reports shortly after Hofmeister moved to Strassburg. The Hofmeister Series
The papers on the behavior of salts published by Hofmeister and his students proposed correlations between precipitating action, dissolving action, and lyotropic swelling of proteins along with diarrhetic action, diuretic action, water binding, osmotic pressure, and other physical chemical phenomena. These were woven into a general outline of salt behavior. The use of a series of salts with a common anion like Li2S04, NanSOa, (NH&S04, and MgSOd should allow relative cationic effects to he observed. Similarly, a series of salts with a common cation, would theoretically permit relative powers of anions to he evaluated, as with NaSOp, NaCl, NaNOa, and NaC103. It was generally found that the order of precipitating power of cations or anions toward certain proteins was the reverse of the order of dissolving power toward other proteins. A good ionic precipitant would be a poor peptizing agent. I n practice, difficulties were encountered in establishing such a series because of a number of factors. For example, it might be necessary to use rather high concentrations of salts to produce a given result. Some salts might not be sufficiently soluble to fit into the series of salts being used. Various methods had to be devised t o circumvent the difficulty. Lewith (6a) used serum proteins, while Hofmeister (6b) used dissolved egg whites for studies of the precipitating power of cations and anions toward globulin and albumin in the protein mixtures. The usual order of cationic precipitating power was Lif > Na+ > Mg++, while the order of anionic power was SO4- > C1- > NO1- > C103-, based on molar concentrations. Many
other ions were studied, including certain organic ones like bicarbonate, acetate, tartrate, and citrate. Various adaptations of two procedures were used in establishing the precipitating power. I n the first method, the protein concentration was set at a fixed value and the salt was added until precipitation occurred. Sometimes equal quantities of salts were used, followed by comparison of weights of dried, isolated protein that precipitated. I n the second method, the salt concentration was fixed while the protein concentration was increased until precipitation took place. Usefulness of the Hofrneisler Series Concept
I n 1925, von Klobusitzsky (7) extended cationic and anionic power series to the rate of settling of dog blood corpuscles. When potassium salts were used, the anionic powers for clotting were: SO4= > C1- > NOs- > Br- > I- > SCN-, (the time required for clotting increased from S O r t o SCN-). When sodium salts were used, it was found that three of the anions changed their order: Cl- > NOa- > S o h , and when ammonium salts were employed, the order > NOS-. Thus the sulfate changed was CI- > its relative position depending on the cation of the salt. The order of clotting action of cations was NH4+ > K+ > Na+. Osmotic phenomena and hydration of ions were important in this behavior. Thomas and Foster (8) placed 50 g of standard hide powder under one liter of water or one liter of salt solutions of various concentrations and covered the aqueous phase with a small amount of toluene to protect against bacterial action. At the end of specified periods of time, aliquots of solution were removed and analyzed for protein content by the Kjeldahl nitrogen procedure. Nitrogen in solution was due principally to dissolved collagen. After 70 days, when 1.3 M metal chloride solutions were used, the order of cationic dissolving powers was: Ca++ > Mg++ > K + Na+ > HzO. Anionic dissolving powers for 1.3 M sodium salts were: I- > Br- > C1- > HIO > SO4- for the same period of time. It is of interest to notice that sulfates have less dissolving power than water toward collagen. This is in accord with the known high precipitating power of sulfates toward other dissolved proteins. Information concerning precipitating powers has established sulfates as excellent agents for fractional precipitation of proteins. Ammonium sulfate has been particularly useful, because it is inexpensive, very soluble, and often causes little or no permanently disruptive action toward protein conformations. This has led to the very effective use of ammonium sulfate in fractionation, isolation, and preservation of activity of enzymes (9). Disadvantages of ammonium sulfate involve difficulties of pH control because of loss of ammonia and contamination of protein with excess nitrogen. Gustavson (10) studied the effect of neutral salts on increasing the solubility of glycine and a few other amino acids. Anionic and cationic Hofmeister series generally followed these patterns: C104- > NOs-
> I- > Br- > SCN- > C1-
and Lif
> Naf > K+; Ca++ >$r++ =B%++
The amino acids were used at or near the isoelectric point. Mild coordinating action of amino acids and cations was undoubtedly involved. When a fiber of collagen is submerged and held taut in a tube of water, and the water is heated, a temperature is reached at which the fiber suddenly shrinks to less than one-third its original length. This temperature, T,, is called the hydrothermal shrinkage temperature, because water is used as the heat transfer medium. For most mature mammalian collagens, the T , is about 6 3 T , although it can be made considerably higher by increasing the weight used in holding the fiber taut. If salt solutions replace the water, the T, is often reduced markedly. The effect in certain cases can be likened to the observed lowering of melting point when one pure solid compound is mixed intimately with a second. When Katz and Weidinger (11) compared 1M solutions of sodium salts in the effect on T , of mammalian tendon, they discovered the Hofmeister anionic power series for lowering the T , to be SCN- > I- > NOa- > Br- > CIOa- > BrOa-. For all of these salts, an increase in salt concentration for lower concentrations decreased the T.. I n some cases, such as NaF, NazS04, NaCl and NaN03, a continuous increase in salt concentration could ultimately cause the T,to take an upward trend. Sometimes the T,attained an even greater value than when pure water was used. Under controlled conditions, for these experiments, the T , in pure water was about 67°C. I n one M NaSCN, i t was about 49°C and in two M NaSCN, it was 35"C, while i t was 65°C in 0.2 M NazSOa and 75'C in 0.6 M NazS04. Lyotropic swelling of collagen (18) and gelatin (13) is the result of the action of neutral salts on the fibrous components of collagen or the partially uncoiled helices of gelatin. The cationic swelling powers for one M solutions revealed this Hofmeister series: Ca++ > Sr++ > Ba++ > Mg++ > Na+ Z K+. The anionic series was SCN- > I- > Br- > Cl- > S O r > SzOs=. Both water and the ions in solution enter the fiber intersticesand force apart non-covalent bonds of thecontiguous protein chains. Direct action of salts and water at the ionic bonds and polar hydrogen bonds are initially particularly strong and this, then, breaks apart the hydrophobic bonds. Often there is a 4- or 5-fold increase in collagen volume, compared to the volume of lyophilized collagen used at the start, and frequently more than a 10-fold increase in dry gelatin volume, for high salt concentrations. Although quantitative rules are not possible, it is usually true that a substantial lowering of hydrothermal shrinkage temperature is accompanied by swelling of collagen. A report by Carpenter and Lovelace (1.4) in 1935 disclosed a strong halide power series in decreasing to a minimum the absolute value of the specific levorotation of purified gelatin solution. As the concentration of alkali metal iodides was increased, a point was reached where a rapid decrease resulted in specific levorotation within a narrow range of iodide concentration. The curve had an appearance of an end-point titration, and the slope of the curve on each side of the mid-point depended upon the salt that was used. For alkali metal bromides, a broader range of bromide concentrations starting at a higher concentration, was necessary to reach a minimum levorotation, and for Volume 44, Number 3, March 1967
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the chlorides an even broader range beginning at a still higher concentration was needed. The Hofmeister series was I- > Br- > C1-. A much weaker effect was noted for the cations with the power series Li+ > Cs+ > Rb+ > Na+. This effect has more recently been interpreted to he a conformational one, based upon the complex processes involved in the conversion of collagen to gelatin and the uncoiling of residual helical portions of gelatin (19). The effects of salt solut,ions on proteins have been especially important in understanding fibrous and other proteins and their conformation changes. As the primary, secondary, tertiary, and quaternary structures of proteins have become clear, the nature of salt effects has been understood better a t the molecular level. Concurrently, the many facets of lyotropic action (15) have been brought more clearly into focus. Important to these interpretations have been t,he nature of the hydration of ions, solubilities of salts, polariaability of ions, stability constants of liganded ions, ionic charge, ionic size, electron population distribution in occupied orbitals of ions, the capacity of ions to become coordinated, and the geometry of the complexes. Equally important has been an understanding of the helical and other conformations of the protein chains and the nature of ionic bonds, hydrogen bonds, and hydrophobic bonds and their ability to be broken or reformed by the action of salt solutions. Osmosis, dialysis, Donnan equilibria, and Zeta potential often play an important role in salt behavior toward proteins. Many of these effects will become more precisely understood as further experimentation provides appropriate information. Acknowledgments
For information concerning Frana Hofmeister, the author is indebted to Prof. Wieland of the Institute
of Organic Chemistry, the Johann Wolfgang Goethe University, Frankfurt am Main, West Germany and Prof. G. Ourisson of the Institute of Chemistry, the University of Strasbourg, Strasbourg, France. Dr. Karel A. van de Putte of Lier, Belgium, and Mr. and 1 . Thomas Charbonneau of UCLA assisted in translation. Dr. Marshall R. Urist of UCLA first called attention to the remarkable impact of Frans Hofrneister on chemistry and medicine. Literature Cited ( 1 ) P O HJ., ~ ~Vaunyn-Schmiedeberg's;lrchiu f i ~ re z p . Path. zlnd
Pharm., 95, l(1922). ~ AND SPIRO,K., E7.g. d m P h y s d . , 22, 1 (1923). (2) P O HJ., (3) Ibid., p. 32. (4) I b d . , p. 29. ( 5 ) HOFMEISTER, F., Hoppe-Se~ler'sZ.phwiol. Chem., 1, 101
,"'. .,. ilY77,
(6) (a) LEWITH,S., Naunw-Schmiedeberg's Arehiv fur e z p . Path. und Phann., 24, 1 (1888); (b) HOFMEISTER, F., o p . cit., p . 247; ( c ) HOPMEISTER, F., op. eit., 25, 1 (1889); ( d ) LIMBECK, R., op. eit., 25, 69 (188'1); ( e ) HOFMEISTER, F., op. eil., 27, 395 (1890); ( f ) HOFMEISTER, F., op. cit., 28, 210 (1891); ( g ) MUNZER, E., o p . cil., 41, 74 (1889). D., Bioehem. Z., 157, 229 (1925). (7) VON KLOBUSITZSKY, (8) THOMAS, A. W., AND FOSTER, S. B., Ind. and Eng. Chem., 17, 1162 (1025). (9) DIXON,M., AND WEBB,E. C., "Enzyme Fractionation by Salting Out," in "Advances in Protein Chemistry," vol. 16, Academic Press, Inc., New York, 1961, p. 216. K. H., J . Am. Leather Chem. ilssoc., 21, 213 (10) GUSTAVSON, ( 1-92fij - *, . (11) KATZ,J. R., AND WEIDINGER, A,, B i o c h m . Z., 259, 191 (1933). (12) VEIS, A,, "The Mncromolecular Chemistry of Gelatin," Academic Press, Inc., New York, 1964. (13) THEIS,E. R., Trans. Famday Soc., 42b,224 (1946). (14) CARPENTER, D. C., AND LOVELACE, F. E., J . A m . Chem. Soc., 57, 2341 (1935). (15) G u s ~ a v s o ~K. , R., "The Chemistry and Reactivity of Collsgen," Academic Press, h e . , New York, 1956. \
Eighth Annual Summer Conference, California Arsociotion of Chemistry Teachers, Asilomar, California, August 14-20, 1966
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