1

Short notes on topics where Iceland spar (mostly in polarimeters)

aided in research on amino acids, peptides and proteins, 1840s-1920s

Definition, discovery and characterization of amino acids

Amino acids are the basic structural units of proteins. An amino acid consists of

a central carbon atom to which are bonded an amino group -NH2, a carboxyl

group -COOH, a hydrogen atom, and a group called the side chain. In proteins,

the carboxyl group (minus OH) of one acid is joined to the amino group (minus

H) of another one. The term for this is a peptide bond, and one speaks of

dipeptides composed of two acids, tripeptides etc.

The first amino acids to be discovered (isolated by hydrolysis, ~1820) and

named were cystine in urinary calculi, leucine from muscle and wool, and

glycine from gelatin. These were followed by aspartic acid (and the related

asparagine) in the 1820s, tyrosine in 1846, and alanine in 1850. Then came

valine in 1856, serine and glutamic acid (and glutamine) in the 1860s,

phenylalanine and lysine in the 1870s, arginine in 1886, histidine in 1896,

proline, isoleucine and tryptophane around 1900, followed by thyroxine,

methionine, and threonine early in the 20th century (Fischer 1907a, Sayhun

1944). All known proteins are composed of a selection from these 21 amino

acids or certain modifications of some of them (cysteine, hydroxyproline, ...).

Polarimeters aided in the identification of amino acids obtained from

various sources. For instance, Schulze and Likiernik (1893) established that

leucine from animal horns and from three quite different materials had the same

optical activity. Fischer and Suzuki (1905) and Abderhalden (1907) similarly

found that the properties of cystine obtained by hydrolysis of serum albumin,

bladder stones, hair and feathers were identical.

Early work on proteins

Bouchardat (1842) confirmed a conclusion by J.B. Biot that albumin and other

proteins which occur widely in organisms, are optically active. A few examples

will be cited here on further research in that field through the 19th century.

Becquerel (1849) and Hoppe (1857) describe how they made use of polarimeters

in measurements of albumin in serum, milk, urine and other bodily fluids when

diagnosing illness. The former designed a polarimeter dedicated to this purpose

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which was available commercially for a while. A. Béchamp completed a

doctoral thesis in medicine in 1856 on albumin and related proteins, and

according to the Dictionary of Scientific Biography (1980-90) he employed a

polarimeter to discover various properties of these that had not been resolved by

older methods. He later published valuable papers on these proteins, again with

polarimetric observations playing an important role (e.g. Béchamp 1884).

Fig. 1. Left mirror-image pair: lactic acid. Right pair: the amino acid alanine.

Optical activity of amino acids

All the amino acids except one turned out to fulfill the condition (of at least one

carbon atom being bonded to four different groups) necessary for molecules to

exhibit mirror-image symmetry. See the example in Fig.1. Due to this symmetry

property in turn, an amino acid molecule will rotate a polarized light beam either

to the right (d-) or left (l-).

L. Pasteur discovered optical activity in aspartic acid and asparagine in

1851, and found that it varied greatly with the pH of the aqueous solvent. The

optical properties of the other acids were studied by various investigators, aiding

in attempts to isolate and to synthesize these acids (Fig. 2). Synthesis of the

acids generally produces optically inactive mixtures of the d- and l- components

in equal amounts. These are called racemic mixtures, the name originating in

Pasteur's research on tartaric acid in the late 1840s.

Resolution of racemic mixtures

Amino acids obtained by the decomposition of proteins are indeed optically

active, whose glycine whose side chain consists of a single hydrogen atom.

Racemization may take place during the decomposition or later, depending on

solvent conditions such as its pH (Kossel and Weiss 1909) or temperature.

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Fig. 2. List of optically active or inactive (dl-) amino acids already synthesized,

from Fischer (1907a).

Pasteur suggested in the 1850s three methods to resolve racemic mixtures

of organic compounds. One involved precipitation and manual separation of

left- and right-handed crystals from solution, and another utilized selective

consumption of one antipode by microbes. In the third method, a racemic acid is

made to form a compound with say a pure d-antipode of a base. The resulting d-

acid/d-base and l-acid/d-base salts will then have different physical properties

such as solubility.

Pasteur's methods were not used much until the 1880s. The manual

separation method was applied to asparagine by Piutti (1886) and to glutamic

acid in 1893. Around 1890 the microbe method resolved glutamic acid, leucine

and aspartic acid. The third method was applied (Fischer 1899) to tyrosine and

aspartic acid, and later to other amino acids. Piutti noted that his d-asparagine

had a sweeter taste than the naturally occurring l-asparagine. This was the first

instance of different physiological effects by antipodes on higher organisms;

such differences have subsequently been of great importance in pharmacology.

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Fig. 3. An example of the application of Walden inversion to produce antipodal

versions of lactic acid, the amino acid alanine (see Fig. 1) and propionic acid

from one another (Walden 1919).

The Walden inversion

P. Walden (1896; see Fig. 3) discovered an unexpected class of chemical

reactions, by means of which it was possible to convert in a simple way a left-

handed molecule into the same right-handed molecule, or vice versa. For several

years after Walden's initial series of papers on this phenomenon in 1896-99, it

was regarded as a mere curiosity. E. Fischer (see next section), A. McKenzie

and his collaborators (McKenzie and Clough 1908, etc.) greatly enhanced the

practical scope of Walden's discovery. They related it to other intramolecular

changes such as racemization already known to take place in the wide range of

organic nitrogen compounds. Hence, optical activity considerations were often

taken into account in research on these changes (e.g. Kossel and Weiss 1910).

The Walden inversion has continued to be a valuable tool in various fields of

stereochemical research (Karrer and Kaase 1919, Walden 1919, Kenyon and

Young 1941). It also stimulated theoretical chemistry in general: by 1930 at

least 25 different theories of its mechanism had been proposed and tested.

E. Fischer's research on amino acids and peptides around 1900

Fischer (e.g., 1899, Fischer and Mouneyrat 1900, Fischer and Warburg 1905)

used Pasteur's (1853) third method with alkaloids or benzoyl- and formyl-

compounds to resolve racemic mixtures of amino acids. He wrote in 1902-09

dozens of papers on the synthesis of amino acids (Fischer and Leuchs 1902, etc.)

and demonstrated relations between them, for instance how serine could be

transformed into alanine and cystine (Fischer and Raske 1907) by chemical

treatment. Fischer (1906, 1907a and later) employed the Walden inversion with

success in his studies on amino acids, and others soon followed.

Fischer (e.g. 1900) also found ways to isolate the various amino acids and

to produce their esters which were well suited for conversion to other

derivatives. Fischer and his collaborators went on to synthesize many dipeptides

and related compounds, continuing to polypeptides with at least 18 amino acids

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(Fischer 1905, 1907b, Fischer and Zemplén 1909, and many other papers). This

work sometimes involved polarimetry (Fig. 4).

Fig. 4. Top: A paragraph from Fischer (1900) on properties of ethyl-leucine

ester. Bottom: Beginning of a description by Fischer (1907b) of the synthesis of

a polypeptide consisting of nine units of glycine (Glykokoll) and one of leucine.

Fischer's successors

In the early decades of the 20th century, many scientists followed E. Fischer's

example in applying polarimetry in research on amino acids and proteins. This

research concerned the sources of the proteins in nature, their molecular

structure and relationships, compounds derived from them, their breakdown

products, methods of synthesis, and so on (cf. Edlbacher 1927, Sayhun 1944).

Only a few randomly selected examples will be mentioned here. In particular,

Fischer's former student E. Abderhalden carried out valuable studies on the

hydrolysis of synthetized polypeptides by agents such as enzymes from

digestive fluids (Fig. 5). Measurements of optical activity could for instance

yield information on which of the peptide bonds in a protein an enzyme will

attack, the kinetics of such processes, and the inhibiting effects of admixtures.

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Fig. 5. Introductory statement from Abderhalden and Gigon (1907) on the value

of optical activity observations in the study of protein degradation by enzymes.

Ehrlich (1904) studied the properties of leucine and isoleucine (which he

isolated from molasses) in detail. Locquin (1907) subsequently showed that an

amino acid synthesized by himself and L. Bouveault possessed the same optical

activity and other properties as Ehrlich’s isoleucine. Abderhalden and Weil

(1913) isolated a new amino acid called norleucin from hydrolysis products of

nerve tissue. Leuchs and Brewster (1913) synthetized oxyproline whose

molecule has two asymmetric carbon atoms. Dakin (1918) discovered and

synthesized hydroxyglutamic acid. Gamgee and Hill (1903) and Gamgee and

Jones (1903) measured hemoglobin and some nucleoproteins with a polarimeter.

Asymmetric action of enzymes

Some amino acids (designated by d- or +) rotate light to the right, and others to

the left (l- or -); however, it is also possible to define the handedness of the

molecules themselves, a property designated as D or L. All amino acids in

natural proteins turned out to be of the L-type, as was first suggested by Clough

(1918) on the basis of a detailed study of optical rotations in these acids and

numerous related compounds. This is of fundamental importance in biological

chemistry: for one thing, natural protein-splitting enzymes only act on bonds

between L-acids (see Levene 1925). The action of enzymes on related

asymmetric compounds such as esters (Warburg 1906) was also studied.

More on characterization of amino acids and proteins

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Results on the characteristics of amino acids and proteins must have aided in the

acquisition of further knowledge regarding the role of these compounds in

biochemical processes, as well as in applications to pharmaceutics, agriculture,

food science and other practical fields (Fig. 6). Levene and van Slyke (1909)

concluded from their optical measurements of casein and other substances: “An

exact analysis of the important leucine fraction of protein is thus rendered

possible for the first time.”. Similar work on other milk proteins was reported by

Dudley and Woodman (1915). Osborne and Harris (1903, see also Lindet and

Ammann 1907) measured carefully the optical activity of at least a dozen

proteins such as gliadin and globulin from various plants. This property may

have been used subsequently in research on cereals: in a paper on the optical

activity of gliadin proteins in wheat Dill and Alsberg (1925) claim that “The

properties of flour depend largely upon the proteins it contains”. Dakin (1920)

was the first to estimate the relative proportions of over a dozen amino acids in

gelatin, using a new separation method along with “Innumerable analyses...by

polarimetric observations”.

Fig. 6. Part of large tables of optical activity in animal and vegetable proteins in

different solvents, obtained by various authors (from Robertson 1918).

In a discussion of E. Fischer’s research on the common amino acid

cystine, Toennies and Lavine (1930) state that “The only practical quantitative

criterion for the purity of l-cystine is its optical rotation.”. Freudenberg and

Rhino (1924) demonstrated, using a polarimeter among other techniques, that

the configuration of alanine (Fig. 1) is identical to that of lactic acid, with an

NH2-group replacing OH. Similarly, aspartic acid was found to be analogous to

malic acid (see Freudenberg and Lux 1928). P. Karrer and his co-workers (e.g.,

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Karrer et al. 1926) also made significant discoveries regarding the configuration

of several amino acids and their interrelationships, aided by polarimetry.

Fusel oil

Following a chance discovery by Salkowski (1889) it was realized that yeast

contained enzymes that could cause decomposition of the yeast cells themselves.

This phenomenon came to be called autolysis. Optical activity probably played

only a minor part in research on autolysis, which was eventually found to be of

wide-ranging importance in the living world. However, polarimetry for instance

aided in explaining the common appearance of some unpleasant liquids during

fermentation in the brewing industry. They were collectively known as "fusel

oil", and remained for a long time a major culprit in the health problems caused

by alcohol consumption. These compounds were in fact not derived from the

sugars (or starch) that were being fermented, as people had believed. Rather, the

amino acids present in the brewer's plant material and in the yeast were losing

their NH2-groups (Ehrlich 1907, Neubauer and Fromherz 1910-11), resulting in

the formation of various alcohols, aldehydes and organic acids. These findings

greatly increased interest in both the scientific and the applied aspects of

fermentation. Using polarimeters, chemists soon isolated enzymes (Harden and

Zilva 1914) that catalysed steps in such processes.

On miscellaneous other methods

The above notes have all concerned the use of polarimetry in groundbreaking

research on the chemistry of amino acids and proteins. However, many other

physical and chemical techniques have been applied in order to clarify the

structure and properties of these important compounds. In the late 19th and early

20th centuries, spectrophotometry was among such techniques.

Noorden (1880), Branly (1882) and several others in the next decades

used Nicol-prism spectrophotometers in studies on hemoglobin. Klug (1895)

made a series of experiments on the digestion of proteins with similar

equipment. Branly concluded that these meters (Fig. 7) had great advantages

over other instruments. Soret (1883) used Iceland spar dispersing prisms in his

spectrophotometer when investigating how blood and its various components

absorb ultraviolet light. He found strong absorption at just over 400 nm. This

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was due to porphyrins which are characteristic for so-called hemoproteins, and

the wavelength interval in question was subsequently named “Soret’s band”. In

some proteins he also found an absorption peak around 275 nm; it may for

instance be responsible for some of the damage to animal tissues caused by

ultraviolet light (UVB-rays). Spar prisms had a minor role in the continuing

extensive research on the absorption spectra of porphyrins (e.g. Marchlewski

and Schunck 1900).

Fig. 7. The spectrophotometer designed by Branly (1882) for research on blood.

N and N' are Nicol prisms made from Iceland spar.

From around 1930, various new methods (although already known for

decades) involving polarized light were applied with advantage to the study of

the structure of proteins and other large organic molecules. These methods

included optical rotatory dispersion, flow birefringence (Maxwell effect),

analysis of scattered light (nephelometry, etc.), and microscopy.

Further information may be found in the 4th edition (Sept. 2015) of my

report JH-2015-02: Iceland spar and its influence on the development of science

and technology in the period 1780-1930.

Leó Kristjánsson

10

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