VOL. 18, No. 1, 1964

Infrared Spectroscopy of Biological Materials*' Leopold May

Department of Chemistry The Catholic University of America

Washington, D.C.

Abstract The apphcatmn of infrared spectroscopy to varmus problems m

the blolog~cal field ~s dlustrated by considering ~ts use m the structural determination of proteins and a chmcal s~tuatton. Samphng problems with blolog~cal materials are &scussed W~th the advent of the :far- infrared spectrophotometer the study of b~olog~cal materials m the :far- infrared region ~s now possible Typical results with various materlai such as tissue and macromolecular components of the t~ssues are shown. The far-infrared spectra o£ amino acids are d~scussed

Introduction

Infrared biological spectroscopy has great value m the study of biological systems. It can be used to study the structural aspects of the various macromolecular compo- nents within the tissue. In addition, it can be used to sup- plement other chemical or physical methods of analysis for the qualitative or quantitative determination of these components within tissue. In speciahzed cases, infrared spectroscopy affords a rapid qualitative and quantitative identification of constituents, for example, the analysis of gall stones.

Sampling

Since biological materials are generally soluble m water and rarely m organic solvents, the techniques of sampling generally used are those concerned with solid samples. However, it is sometimes quite desirable to study the bio- logical materials in water solution. The usual techniques for using water solution of this nature have been described previously (1, 2). It is most desirable to study the macro- molecular components m solution since tissue components exist m a md~eu of water (the amount of water in tissue is generally between 80 and 95 % ).

The usual techniques used in solid samples are appli- cable to biological materials: mineral oll mulls; KBr disks; and polyethylene disks. The mineral off mull has the dis- advantage of having bands that would interfere with the bands appearing in the spectra of substances. Potassium bromide disks are used because KBr has no strong bands with the exception of one at about three microns due to the hydroxyl stretching frequency. These disks may also be saved for future use in a desiccator. Polyethylene disks (3) can be used for solid sampling, although they have bands similar to that found with mineral oil mull. There is no band near three microns, and this is very useful in determining hydroxyl groups in a molecule. It has the disadvantage that the sample and polyethylene must be heated to about 100°C so that many biological materials such as protein would probably become denatured.

One finds that when different preparative schemes for tissues are examined, i.e., section, homogenate, lyophihzed film, and KBr disks, the spectra m each case is identical

Presented at the First National ISA B~omedlcal Soences Instrumenta- tion Symposmm, June 1963, Los Angeles, Cahf

~Some o£ the work reported here was supported by Research Grants NB02927-03, Natmnal Institute of Neurological D~seases and Bhnd- hess, and GM10574-01, Dlws~on of General Medical Sciences, U S Pubhc Health Service.

(4). Although the tissue prepared m these ways may have the same spectrum, there is no assurance that individual macromolecular components such as proteins will not be- come denatured using various preparative schemes. To test the possibdlty that this may or may not have occurred, an experiment was run using two proteins, rxbonuclease and alcohol dehydrogenase. These were prepared as films in KBr disks as well as m agar films (5), and the biological actiwties of these proteins were measured by determining their enzymatic activities. The proteins were denatured in all cases with the exception of the film of ribonuclease. The structure of the protein was found from the position of the amide bands around 1660 and 1530 cm -1. Although denatured to a great extent m agar and not m the other preparations, nbonuclease appears to have the a helical structure in all preparations. On the other hand, alcohol dehydrogenase is denatured in all preparations. It appears then that infrared structural determination should not be made without some independent measure of the amount of denaturation of the protein. Hence it would be best to use water solutions for studying these materials since this is the form in which it does appear m the cell.

Measurement of Protein Structure

Elliott and Ambrose (6) found that spectral difference could be observed m folded and extended polypeptide chain and fibrous proteins. They concluded that for the folded a form the amide I band occurs at about 1655 cm -1 and the am~de II band occurs at about 1545 cm q. With the extended form (/3), the bands occur at about 1630 and 1520 cm -1. It was later shown that these correlations may not be applicable to proteins. Mlyazawa and Blout (7) and Krlmm (8) later showed that these correlations were not completely correct. They mterpreted the positions of the amide I and II bands in terms of the folded, extended, and random coil as well as pleated sheet chain type o( structures. These types of correlations are shown in Table I, which is assembled from these works. Recently the amide V band has been examined by Mlyazawa (9) using syn- thetic polypeptides and found to vary with the structure of the polypeptide (Table I) . We have observed that ribonuclease has a band appearing at about 700 cm "1 which would indicate that the protein exists in the /3 form, but the positions of the amide I and II bands suggest that it is in the c~ form. Mlyazawa (I0) has found that the amlde VII band is sensitive to the conformation of the poly- peptide chain. I t varies from 217 cm -1 in polyglycine I (antiparallel chain extended form) to 365 cm -1 in poly- ¢lycine II (helical form). This spectral re3eion requires further study before these results can be applied directly to the determination of the structure of proteins.

Clinical Application

An example of the usefulness of infrared spectroscopy in clinical chemistry is the application to the analysis of gallstones and urinary calculi. For a complete analysis of urinary calculi including trace elements, it is necessary to

6 A P P L I E D SPECTROSCOPY

TABLE I. A M I D E BANDS AND S T R U C T U R E OF P R O T E I N S a

Absorptmn Bands, cm -*

Random Vtbratton cr (hehcal) fl (extended) Cod

Parallel Anh-Parallel Cham, Chain, Pleated Pleated Sheet Sheet

S N - H 3280 ~ 3280

1650s~7" 1 6 4 0 s 1660s Am*de I(S C = O ) 1 6 5 2 m ~

Am*de II (S C-N, 8 N - H ) 1 5 1 6 w ~ , 1540 s 1535 s 1546s

Am~de III (8 N-H) 1250

Amide V ( S N - H ) 610-620 700 650

1 6 4 5 w ~ , 1 6 8 5 w ~ , 1630s~ 1 6 3 2 s ~

1 5 3 0 s ~ , 1 5 3 0 s ~ 1550 w a

~r--paral le l d~chrotsn, ~r- -perpendmular d~chro~sm, s - - s t r ong , m - - m e d m m , w - - w e a k , S - - s t r e t c h

do infrared, x-ray diffraction, and emission spectroscopic measurements (11). However, it has been found with gall- stones that one could analyze for the type of stones using infrared spectroscopy solely (12). The analysis can be completed within 20 to 30 min after removal from the patient whereas chemical analysis usually takes a day. Either Nujol mulls or KBr disks are used, and one can quite easily distinguish between inorganic and organic stones. Generally, the inorganic samples have fewer bands than the organic stones. Analysis of gallstones by this method has led to a reclassification of the types of gall- stones (12). It was found that xt was better to use Nujol mulls with gallstones but KBr when analyzing urinary calculi. Efforts have been made to use infrared spectroscopy in the study of blood serum and has been used successfully to analyze for lipldes in blood and other tissues (13, 14).

Far-Infrared Spectroscopy With the advent of commercial far-infrared spectro-

photometers, it is possible to examine this spectral region to determine what useful information could be found for biological materials. Typical proteins, ribonuclease and bovine serum albumin, appear to have a strong band near 700 cm -I and weaker bands near 590, 513, and 360 cm -1. The band at 590 cm -a may be related to the band (570- ¢;40 cm "1) found in monosubstituted amides (Table II) . The 513 cm -1 band r~ay bo rebted to absorption found with the ma;or constituent of the nrotoins, namely the amino acids (Table III~ Generally, the absorption in this soectral region is considered to be due to skeletal wbra- tions. The results of a number of studies with various amino acids show these bands can be assigned to the wbra- tions shown. The skeletal vlbrat~ons include the func- tional ~roups as well as the hydrocarbon chain of the amino acids. It is interestin~ ro note that the number of hydrocarbons on tho alr~ha carbon seems to effect the posi- tion of the bands. As the m~mber of carbons increase, the band moves towards lower frequency (Table III) .

T A B L E I I . F A R - I N F R A R E D A B S O R P T I O N BANDS OF

P R O T E I N S AND AMIDES~ c m -1

Protein 700 588 513

Polypept~des Helical form 610-620 365 a Extended form 700 217 b Random cod 650

Amides e 600-650 700-730 570-640 350-440

8 O C N ¢z N H ~" C = O 8 0 C N

"Polyglycme II (12) bpolyglycme I (12) ~From (15)

TABLE III. F A R - I N F R A R E D A B S O R P T I O N BANDS OF A M I N O

AC1DS~ c m "1

Functzonal Groups

C O O - 640-660 480-495 C O O H 667 --C (NH. +) CO0-

Hydrocarbon Chatn

430-450 335-350

C H 3 - - & C 600-610 One carbon 520-560 420-435 T w o carbon 459 300-320 Branched Chain 390-400 285

Correlation of the bands in the spectra of rat tissues can be made with the absorption of proteins and other constituents (Table IV). There appears to be a &fference in the spectra of two types of tissue. In the second set, there appears to be a shoulder at 676 cm "1, which does not appear in these other tissues. The 700 cm -x band in the spectrum of human cerebrospinal fluid can be related to the protein at about 700 cm -1. The intensity of this band is correlated with the concentration of protein in the samples, which was measured by determining the ultra- violet spectrum of a solution of the spinal fluid. Since these spinal flmds were simply prepared by lyophihzing spinal fired, the presence of phosphate which is shown at 600 cm -1 is to be expected since this is the major salt constituent of the spinal fluid. There are bands that still remained to be explained, and the origin of these will be clarified after additional biological materials have been studied in this region.

T A B L E I V . F A R - I N F R A R E D A B S O R P T I O N BANDS

OF TISSUES, c m -1 a

Ra t Cerebral Tissues b 700 535 Rat Spleen, Spinal Cord, Blood 700 676 sh 535 Human Cerebrospmal Fired 700 600 Assignment Protein DNA POC 3 DNA

465

~CsBr Disks; DNA-desoxyrtbonucleic aod, sh--shoulder bCortex, Medulla, white matter

Summary This paper has reviewed some of the problems of infra-

red biological spectroscopy. The problem of sampling is always present when one is deahng with these complicated molecules. One of the greatest uses of infrared spectroscopy is the structural determination of macromolecular compo- nents, such as protein. The characterization and relation- ship of the infrared spectrum to structure of these compo- nents have stdl not been completed. Extension of the infrared spectroscopy into the far infrared has just begun. It appears that it may yield additional auxiliary mforma-

VOL. 18, No. 1, 1964

tion that may be of value to those studying blologlcal materials. As an analytical tool, infrared spectroscopy has been shown to be superior to the ordinary chemical :ech- niques in certain clinical situations, such as the analysis of gallstones and urinary calculi.

Literature Cited (1) E. R. Blout, ANN. N. Y. ACAD. ScI. 69, 84 (1957) (2) D. M. Klrschenbaum, APPLIED SPECTROSCOPY

17, 149 (1963) (3) L. May and K. J. Schwing, ImP. 17, 166 (1963) (4) L. May and R. Boccalatte, Unpublished observations (5) J. Androsme and C. Krupa, BIOCHEM. Z. 338, 212

(1961) (6) A. Elliott and E. J. Ambrose, NATURE 165, 291

(1950); A. Elhott, PROC. INTEIN. CONGV,. Bzo- C H E M . , 3RD 1955, 106

A T

(7) T. Miyazawa and E. R. Blout, J. AM. CHEM. Soc. 83, 712 (1961)

(8) S. Krimm, J. MoL. B~OL. 4, 528 (1962) (9) T. Miyazawa, Y. Masuda, and K. Fukushlma, J.

POLYMER ScI. 62, 562 (1962) (10) T. Miyazawa, BULL. CHEM. Soc. JAVAN 34, 691

(1961) (11) G. Chlhara, N. Kurosawa, and E. Takasaki, CHEM.

PHARM. BULL. 7, 622 (1959) (12) G. Chlhara, el al., ImD. 8, 771 (1960) (13) G. J. Nelson and N. K. Freeman, J. BIOL. CHEM.

234, 1375 (1959) (14) H. P. Schwarz, ADV. CHN. CHEM. 3, 1 (1960) (15) W. Nowak, Ph.D. Dissertation, The Cathohc Uni-

versity of America, 1963 (16) Sr. M. St. Lawrence Eaton, S.N.D., M.S. Disserta-

tion, The Catholic University of America, 1963

Infrared Spectroscopy By

Attenuated Total Reflection*

Bennett Sherman

Instrument Division, Barnes Engineering Company, Stamford, Connecticut

Abstract

A review ~s given of the attenuated total reflecuon method m infrared spectroscopy Some spectra of biological material are included to dlustrate ~ts value m the b~omedmal field

Introduction

Infrared spectroscopy as applied to the study and an- alysis of organic and biological materials is well estab- hshed. In general, the substances of interest are separated by some physical-chemical means such as centrifuge, or column chromatography. However, these substances must be obtained in a form that is relatively transparent to the infrared over the spectral range in which the characteristic absorption bands are to be observed or recorded. In some cases ~t is possible to imbed the substance in a pressed alkali-halide plate, usually KBr. In any case, the important physical characteristic is the relatively good transmission to the infrared of the final preparation. Turbidity and the presence of significant amounts of hght-scatterlng solids in the test substance likewise interfere with the quality and usefulness of the infrared absorption spectrum ob- tained. In spite of these limitations, infrared absorption spectroscopy has been used extensively and successfully in studies of molecular structure of materials and substance~ of biological interest. An excellent review of various tech- niques in this field can be found in the publication of a conference held by the New York Academy of Sciences Jn 1957 devoted to the subject of infrared analytic spectro- scopy of materials and substances of biological interest ( l ) .

~Presented" at the Flrs~ Nat iona l ISA Bxomedlcal Scxences I n s t r u m e n t a - tmn Symposium, June 1963, Los Angeles. Cahf

Attenuated Total Reflectance (ATR)

In 1961, J. Fahrenfort (2) described a new technique that he devised for obtaining useful and intense infrared absorption spectra from materials otherwise unammenable to transmission methods. Basically, the technique employs the phenomenon of total internal reflection.

When radiation is totally internally reflected from a suitable face of a dielectric prism surrounded by a medmm of lower index of refraction, there is a penetration of the radiant energy into the rarer medium beyond the totally reflecting face for a short d~stance. If there is no absorp- tion in the second medium, the time average of the trans- fer of energy into the rarer medmm is zero and the radia- tion is totally reflected. A close analysis of the situation, when the rarer medium contains materials which absorb, shows that part of the incident radiation on the internally reflecting prism face is absorbed and in those spectral regions that are characteristic for the absorbing material. Thus, spectra quite similar to transmission spectra can be obtained. Harrick (3) discusses the observation of ab- sorption in the rarer medmm and derives a formula for the depth of penetration of the radiation into the rarer medium, discounting absorption effects. This can be con- strued as a guide to the effective sample thickness for a spectrum as if it were obtained by transmission methods. Thus, as Harrick and Fahrenfort point out, absorption spectra could be obtained that are characteristic of sample thicknesses up to several microns.

From the formula for "depth of penetration" it can be seen that the appearance of an infrared absorption band will depend upon the difference of the indices of refraction