Applications of Absorption Spectroscopy
G. R. Penzer Universily of Oxford England
in Biochemistry
W h e n a molecule absorbs infrared, visible, or ultraviolet light there are changes in its vibrational and/or electronic states A chromo~horeis characterized by the positions and intensities of its absorption bands, and shifts in the position of a peak as conditions (solvent, temperature, etc.) are altered measure the sensitivity of a transition to its environment. Spectra can thus be used both to identify unknown species and to learn about the phase in which an ahsorher is located. Biochemistry is the observation of natural systems, or of extracts from them, and the description of their function in terms of the known behavior of molecules and ions. A big obstacle in this type of work is the "simple" problem of chemical analysis. The quantities involved are tiny, and ideally a technique should he applicable to an intact system without disrupting its function. Absorption spectroscopy fits this specification for some systems, and is widely used to follow concentrations and to identify chromophores. The overall chemical changes accomplished by living organisms are very large. Frequently they require a series of coupled reactions and a corresponding number of intermediates each of which needs to be ideltified if the reaction sequence is to be properly understood. The steady-state concentrations of these compounds are usually low and their lifetimes are short, but spectroscopy has been used successfully in some cases both to detect and to identify intermediates. Another of the characteristics of a natural system is that its reactions are often caused or controlled by interactions of small energy compared to normal bond energies. These forces may be either intramolecular (as in protein secondary and tertiary structures) or intermolecular (as in coenzyme binding or the association of different strands of nucleic acid). Spectroscopy has been widely used to observe these phenomena by studying the shifts and distortions that are caused in an unperturbed absorption spectrum. This method is quite Table 1.
-
The Range of Spectroscopy and Some of the Units Used
Wavelength (A) m m~crons 0.200 0.300 0.400 0.800 2.000 4.000 6.000 8.000 10.000 15.000
" 1 kcal/mole
692
/
Frequency in cm-' 50 33.3 25 12.5 5 2.5 1.6 1.25 1 0.6
=
(u)
Energy in kcal/mole*
X 10a X lO"
X 10S X 108 X 108 X 10' X 10" X lo5 X loa X 10"
0 . 0 4 3 eV/molecule.
Journal of Chemical Education
ultrmiolet 14.3
2.9 1.9
sensitive, even in the ultraviolet; an absorption at 250 mp corresponds to an energy of 114 kcal/mole, but a shift of 5 mb, which is a hundred times the minimum detectable, is an energy change of only 2 kcal/mole. The major part of this article illustrates the biochemical applications of absorption spectroscopy listed above-studies of the structure and kinetics of both stable molecules and unstable intermediates, and the investigation of intramolecular and intermolecular forces. First, however, it is necessary to consider the scope and the limitations of spectroscopy in the observation of biochemical systems. The Technique
Readily available spectrophotometers cover the range from 1 5 (about ~ 2 kcal/mole) to 190 mp (about 150 kcal/mole)-see Table 1. The only transitions resolvable in the infrared region are vibrational ones; rotational contributions can be neglected. All vibrational modes involve the complete molecule, but the positions of some bands depend mainly on the presence of a single functional group. The precise nature of the molecule to which the group is hound makes small but measurable changes in the position of the absorption. Although a detailed theoretical assignment of absorption bands has been poe8ible for some simple molecules, this is not yet feasible for most speciesof biochemical interest, and so the empirical location of regions of the spectrum where groups typically absorb is an important operation. In steroids, for example, there is no single definitive absorption, but the combination of several lines in the infrared spectrum is usually quite distinct. Unequivocal assignment of structure from absorption spectra alone, however, is not usually possible. Key-group absorptions lie in the range between 3 and Sp, hut beyoad this (9-13~) is the fingerprint region. It is not possible to assign these bands even empirically, but the spectra are still highly characteristic of a particular molecule. Thus compounds can be distinguished which differ in only the smallest r e s p e c t ~ like having deuterium substituted for hydrogen. If an unknown has a spectrum identical in every detail to that of a known substance, i t is reasonably certain that they are the same compound. Because all the energies involved are small, infrared spectra are potentially very useful for the study of weak interactions like hydrogen bonding. For large molecules this potential is reduced because the spectra are very crowded, the number of vibrational modes being given by 3n - 6, where n is the number of atoms. Thus analysis in terms of key-group frequencies becomes
harder, though the spectrum may still be useful as a fingerprint. If there are also weak interactions (as in a protein) they may broaden the absorption hands, and further confuse the spectrum. A major disadvantage of infrared spectroscopy is that water has broad and strong absorptions in this part of the spectrum. Most natural systems have a very high water content crucial to their normal structure and function. This limits the technique mainly to in uitro experiments, to small glimpses that can be viewed through the water windows, and to measurements in D20 whose absorptions differ from t,hose of wat,er hot.h in magnitude and wavelength. Visible and ultraviolet spectra involve electronic transitions, sometimes with a vibrational fine struct,ure superimposed. The energies involved are typical of n-a* and a-s* excitations so that most of the species absorbing in this range contain an umaturated system. As with vibrational spectra the transition of an elect,ron from one orbital to another formally involves wavefunctions which cover the whole molecule. These, however, can be so completely localized in the unsaturated part of the molecule that some electronic absorptions are insensitive to substituents not conjugated or inductively associated with it. For similar reasons these spectra do not depend on inter or intramolecular interactions unless they directly involve the chromophore. There are differences in the characteristic behavior of n-s* and a-a*transitions. Fieure 1shows in a crude way that there is a bigger difference of dipole moment between ground and excited states (transit,ion dipole) for the n-s* excitation.
-
.. .. -
Figure 1.
Changes in polarity during n-r* and
r-r* excitations.
The Franck-Condon principle states that the time taken by an electronic transition is too short for there to be appreciable simultaneous nuclear rearrangement, and so there can be no change in solvation during excitation. Solvents affect the positions of absorption maxima, however, both by their different abilities to solvate the ground-state and by their polarizabilities. The effects of ground state solvation on the energy of an n-a* transition may be large; polar solvents often stabilize the ground state better than the excited one, thus increasing the energy of the transition. The shifts caused for a-s* absorptions tend to be smaller, and in the opposite direction, because many a* states arc more
polar than t.heir parent a states. A polariza.ble solvent adapts well to alterations in the dipole moment of a solute molecule during an electronic excitation because polarizability measures the mobility of electrons, not nuclei. As n-a* transitions have a higher dipole change t,han a-s* ones their positions are more solvent dependent, moving to lower energies as t,he solvent becomes more polarizable. The magnitude of the polarizability effects is usually small compared to those caused by solvation, and they only become dominant for interact,ions between non-polar solutes and non-polar solvents. When a chromophore is bound to a macromolecule (e.g., a coenzyme bound to a protein) its environment is governed largely by the nature of the binding site. This depends critically on the structure of the macromolecule. Quite large spectral shifts may thus result from interactions between the chromophore and groups adjacent to where it binds. Aqueous solut,ions are readily studied in the ultraviolet region as water is without absorptions here, and this has meant that ultraviolet spectroscopy has bee11 more widely used than the infrared in biochemistry. There are other limitations, however. At wavelengths shorter t,han 210 mp oxygen and some inorganic ions begin to absorb, complicating the spectrum. The ext,ent of light scatt,ering from a given solution varies as X-', and so its importance increases in the ultraviolet region, where care must be taken to use clear solutions. Even so, many biopolymers scatt,er light and require that turbidity corrections be made. The simplest way to do this is to plot log (apparent absorption) against log X in a range where true absorptions are absent, and extrapolat,e the line to where they are not. I t is sometimes also necessary to take account of any fluorescence which is emitted. With modified instruments it is possible to measure the visible and ultraviolet spectra of opaque samples. Either the reflected light from the front face of the cell can be observed, or, by using opalescent filters in both sample and reference beams, the light scattering can be averaged out. Either technique leads to the observation of a true absorption spectrum. With more sophisticated equipment it is possible to capture the diffuse transmitted or reflected light by means of an integrating sphere. This is a more sensitive technique. The intensity of an absorption is given by the expression log (Io/I) = cel, where I. and I are the light intensities before and after passing through the sample, r is the molar extinction coefficient of the chromophore, e is its molar concentration, and 1 is the path length of the sample. c is derived from the overlap between the wavefunctions of the ground and excited states, and is a measure of the probability of absorption. Although numerical values can vary widely for a particular type of transition, they are typically a factor of 100 more for a-a* than for n-s* (see Fig. I), which is of the same order as a vibrational transition. e values are generally in the range 100-20,000, which means that to obtain reliable readings with most standard instruments at least 1014-10'6 molecules of chromophore are required. Primary Structure
Spectroscopy is used in biochemistry, as in chemistry, as a way to detect impurities and to investigate the structure of unknown compounds. The species which Volume 45, Number 1 1 , November 1968
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693
are amenable to this kind of study are comparatively small molecules like monosaccharides and their derivatives, steroids, lipids, nucleotide bases, etc. For these molecules the infrared spectrum remains sufficiently simple to he of real use, and is more sensitive to structural differences than the ultraviolet. Applications of infrared spectroscopy to the solution of primary structures of large molecules are uncommon, but there are instances when it provides useful information. It is possible to measure the carhonyl stretching frequency of carbon monoxide bound in red blood cells, to hemoglobins and to hemes. The position of the hand is a very sensitive function of binding strength, and thus varies with the nature of the sixth ligand attached to the iron of a heme complex. Both human hemoglobin and human red blood cells after equilibration with carbon monoxide give the same single sharp absorption (at 1952 cm-') which corresponds to strong binding of the gas. The hemoglobin molecule contains four hemes, hut the fine resolution of a single hand means that there is identical binding of carbon monoxide to each (1). Some measurements have been made of the infrared spectra of intact biological systems, e.g., myelin nerve sheath, bacteria, and whole tissues. The results can usually be correlated with the known composition of the system, showing the presence of water, protein, lipid, etc., and it has also been possible to distingnishspectroscopically between tissue from different sources (2). Generally, however, these measurements have provided little information not already available from other work. Although electronic spectra are less sensitive to structure than infrared absorptions are, they can still be used to monitor changes in unsaturation and conjugation. The different geometrical isomers of some of the unsaturated fatty acids present in lipids can thus he distinguished by their ultraviolet spectra (Fig. 2) (3).
this varies from protein to protein, especially for phenylalanine (4). Common experience shows that visible spectroscopy offers a method for studying changes in heme comp o u n d e t h e color of blood depends on whether it is oxygenated or not, or whether i t is saturated with carbon monoxide. So spectral measurements have been widely used to observe the cytochromes, a family of redox hemoproteins. At room temperature the spectra of these compounds are hard t o differentiate, and the problems are increased by the fact that preparations are frequently both impure and dilute. I t became possible to record more useful cytochrome spectra on the discovery that when the solvent is a glycerol-water mixture and the solution is cooled to liquid nitrogen temperatures, the intensity of absorption increases by a factor of 5 to 10 times, and the resolution of the hands is improved (6). Band sharpening a t low temperatures is widely observed and is attributed to the reduction in vibrational relaxation by collisions with the solvent (in this sense it is analogous to the improved resolution of the spectrum of a vapor relative to a liquid). The increased extinction a t low temperatures is only observed with solvents which form a microcrystalline mass on freezing. This causes internal reflection of the spectrophotometer light beam and increases the effective cell path length. The same effectcan be induced in aqueous solutions at room temperature by adding a suspension of inert reflecting particles, such as talc, and i t has been suggested that a similar process may explain the presence of microcrystalline deposits in the tapetum of an eye. This is a layer behind the retina whose purpose could he to increase visual sensitivity by reflecting the incident light and thus causing more efficient detection (6). A third cause of spectral changes when a sample is cooled is alterations in chemical equilibria (ionization, etc.) which lead to different concentrations of the chromophores. Many cytochromes can now he identified by using the low temperature technique to observe the characteristic absorptions (known as satellite hands) of reduced cytochromes in the region 54&600 mF. The sensitivity and selectivity of the method makes it possible to- study the complex mixtures of cytochromes present in extracts such as mitochondria1 suspensions, and to distinguish the cytochrome cs extracted from different species (7). Not enough has yet been discovered about the sources of cytochrome spectra to provide evidence for the reasons why all the different compounds are spectrally distinct, though spectroscopy in combination with other chemical and physical studies has helped to identify gross structural differences hetween the various classes of cytochrome. Kinetics and the Study of Intermediates
Figure 2. Spectra of the geometrical isomers of octodeca-9:ll :13trienoic acid in cyclohsxone. A, &eloeortearic m i d (tmnr-Irmns-tmnsli 8, odoeastaoric acid (cis-Irons-trans); C, punicic acid (cis-cis-tronsl; D d o taken from ref. (3).
Of the amino acids only aromatic ones absorb a t wavelengths longer than 230 mp, and their spectra are sufficiently different for the amounts of each acid present in a protein to he estimated from spectral observations. It may also be possible to characterize a molecule by the fine structure of these aromatic absorptions as 694
/
Journal of Chemicol Education
Probably the widest use of spectrophotometry is to measure the concentrations of known chromophores, often during a reaction. It offers a convenicnt technique for the routine assay of, for example, enzymes as often either the substrate or coenzyme undergoes a considerable spectral change during reaction. I n addition to such routine work spectrophotometry has been used to detect reaction intermediates, and to follow the kinetics of their formation and decay. Consider for example the reactions of horse radish per-
oxidase (HRPO), which has been studied in detail (8). It is possible to see directly that on adding a peroxide to highly purified enzyme an olive-gree color forms rapidly (compound I) and then gradually changes to pink (compound 11). In the prePence of excess bydrogen peroxide compound I1 turns red into compound 111. These changes are all reversible, but compound I11 is irreversibly degraded to a green compound IV, probably by attack of the hydrogen peroxide on the porphyrin ring. Under some conditions these changes are so fast that rapid reaction techniques (e.g., stopped flow) are required to observe them, hut it is also possible to choose concentrations where the lifetimes of the intermediates are increased to minutes. This allows more accurate spectral measurements, and facilitates studies of the dependence of reaction on various added compounds. These experiments show that addition of an electron donor such as ascorbic acid increases the rate of formation of compound I1 (Fig. 3) and suggest that it was
lor*
7
4
I-
the presence of a donating impurity in early preparations of the enzyme which caused compound I to be overlooked in the original experiments. The behavior of the various intermediates is consistent with the scheme given in Figure 4; the rate-determining step is often the reconversion of compound I1 to HRPO, but this depends on the conditions. The pigment systems of photosynthesis have been subjected to extensive spectroscopic examination. A solution of chlorophyll a in ether has a maximum a t 662 mp, but the chromophore is present in chloroplast (the natural photosynthetic unit) in several environments with maxima in the region 67&680 mp. Shifts, like this, to the red occur when chlorophyll forms ordered aggregates with the pi system of adjacent aromatic rings interacting, and so this kind of order is implicated in the photosynHRPO 1 thetic pigment sysl,n-mMm , f tem (9). Figure 5 I I , , I shows that t& natI v--sv.r J L ---vnI ural environment of chlorophyll is closer to an ordered I 1 monolaver than to the three-dimensional organization Figvre 4. A simple scheme for the reactions of Horrermdirh Peroxidare. of a C I Y S ~ & ~ . ELEC.Rc.l
"trn,,ON
__-
jELmm
.
microcrystol (ethyl chlorophyllide)
~ crystalline moloywe lethyl chlorophyllide)
-
m o l l microcrystols lethyl chlwophyllide)
i
-E
in vivo Inon-fluorescent, photosystem I of plont chioroplartrl omorphMlr monoloyerl (ethyl chlorophyliidel ' vim ~fluxercant,photosystem I t of plont chloroplasts) chlorophyll-protein complexes
660+acctane
Figvre 5.
Figwe 3. Absorption rhonger during the reaction of Horseradish Peroxidare. Dot. simplified from ref. ( 8 ) .
0
rdution lethyl chlorophyllidel
Absorption morimo of chlorophyll o in different rituationr
Measurements of the quantum yield of photosynthesis make it clear that every quantum absorbed has a high probability of causing reaction, but kinetic studies indicate that the minimum size of unit capable of leading to photosynthetic electron transport contains several hundred chlorophyll molecules. This has led to the idea that most of the pigment is present as a light trap which transfers the absorbed energy to a reactive center where the redox processes occur. If a chlorophyll molecule a t this center had a red-shifted absorption the mechanism of energy transfer could be the resonance type suggested for Forster (10). A pigment absorbing a t about 700 mp (known as P-700) has been detected by observing the light-dark difference spectrum of chloroplasts. Two identical samples are taken and one is kept dark while the other is subjected to a flashing light with dark periods of 0.1 sec. During these breaks in illumination the spectra of the two samples can be compared, and it transpires that a species absorbing around 700 mp has been bleached in the illuminated sample (Fig. 6). This effect is rapidly
m-,DF.
I=
]
Figure 6. Light verrus dock difference spectrum for acetone extracted chloroplart particles. Data token f p m ref. ( 1 11.
Volume 45, Number 1 1 , November 1968
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695
reversed in the dark. Assuming that the bleached pigment has the same extinction coefficient as chlorophyll its concentration is one molecule per photosynthetic unit (11). llodern theories envisage photosynthesis as a biphotonic process, with two pigment systems linked by an electrontransport chain. During normal photosynthesis by white light P-700 will he present in a steady-state concentration, but if, as predicted, it is the re8. Specbol changes during the reaction of active center for only one of the Figvre and C, hypertensin I. Dot. taken from ref. 1121. pigment systems, by exciting the other alone it should be possible to build up the concentration of P-700 above its normal value (Fig. 7). This is achieved with light of low wavelengths which excites the accessory pigments and chlorophyll b, but not chlorophyll a. The suggestion that P-700 is an important redox reaction center gains more support from the difference spectrum between oxidized and reduced chloroplasts, which also shows a maximum a t 700 mp. The assignment of its location to the active center of photosystem I is upheld by the observations that the sensitivity to inhibitors of P-700 bleaching HO parallels that of photosystem I, while that of its production follows the oxygen-evolving photosystem 11.
-
Structure of Intermediates
Sometimes it is possible to identify the structure of an intermediate from spectra. Tyrosinase is an enzyme which oxidizes many tyrosine-containing compounds (including proteins) to a variety of quinones and polymers. Three different sorts of reaction sequence can be distinguished spectroscopically (Fig. 8). Type A is characteristic of Nterminal tyrosine peptides, and shows transient absorptions at 305 and 480 mr. These resemble the spectrum of dopachrome (2-carboky-2,3-dihydroindole5,6-quinone), one of the intermediates in melanin formation. The final spectrum has a maximum at 325 mp which is close to those shown by derivatives of 2carhoxy-5,6-dihydroxylindole. These facts are explained by scheme 1 in Figure 9, but the mechanism is impossible for derivatives with blocked amino groups.
Tyrosinase with A, tyroryl-glycinej 8, glulamyl-tyrosine;
mc,!\c6 T~msinvr
A (faat)
Ho
I H
II O
I
R
0
H
0
C'
I
A(.,
/I
I N\CH,Islow1
I
H O R 305 and 480 mr)
HO HOc
! (A,.
I H
c
II O
6
0,
Idow)
I
h l ~ m
R
325 m r )
(.IoY)Compound 475 mr)
Polymer
(Am,
Figure 7.
696
/
Scheme for the photochernicol steps of photosynthesis.
lournol of Chemical Education
Figvre 9. The mechanisms of Tyrosinore action; scheme 1 i s for N-terminal tyrosine% scheme 2 is for C-terminal and protein tyroriner.
C-terminal tyrosine peptides are thus expected to undergo different spectral changes on reaction with tyrosinase, and they generate a transient absorption a t 390 mp (type B). This is attributed to an orthoquinone which reacts slowly to give a broadly ahsorbing polymer. With many proteins a third pattern is observed in which no transient absorption occurs (type C), but this may be caused by a similar mechanism t o type B (scheme 2 in Fig. 9) if the relative rates of the different steps changes (Id). Kinetic studies of some hydrolytic enzymes have implicated an acyl-enzyme type of intermediate (Fig. 10).
Figure 10. Mechanism of hydrolytic enzymes; papain or flcin.
a, chymotryprin;
b,
Direct observation of this species is difficultbecauve of its low concentration, its short lifetime, and the small spectral changes involved when it is formed. These problems have been overcome, however, for m-chymotrypsin. The half-lives for formation and decomposition of the acyl-enzyme a t neutral pH are very short, but they can be increased by a factor of a thousand at pH 3 to several seconds. The enzyme is stable under these conditions, and its mechanism of action is unaltered (the number of active sites is constant and catalytic rate constants form a continuous set as pH is lowered, consistent with dependence only on a basic group with pI< 7.1.). Even under these conditions where reaction rates are slow it is difficult to observe an acyl-enzyme because there are small differences in ext,inction between ester, acyl-enzyme, and hydrolyzed acid. Nevertheless the hydrolysis of the methyl ester of N-acetyl-L-tryptophan to N-acetyl-L-tryptophan and methanol was observed by the absorption changes at 311 mN. On mixing enzyme and ester the optical density fell for about two minutes before rising to a steady value which was still lower than the initial reading (Fig. 11). This infinity reading could he characteristic of either the free acid anion alone or a mixture of this ion and the acyl-enzyme in equilibrium. The second of these possibilities is correct because when the free acid, N-acetyl-L-tryptophan, is added to chymotrypsin the optical densityfalls over several minutes to give an identical infinity reading to that of the hydrolyzed mixture. The initial drop in optical density during hydrolysis of the ester is the result of intermediate being formed faster than it dissociates-a predicted result under t,he conditions used when enzyme
was in excess over substrate. The direct spectral ohservations were not good enough to derive accurate kinetic parameters, hut the approximate values obtained are consistent with other kinetic data ( I S ) . Studies of the proteolytic enzymes papain and ficin have also implicated acyl-enzyme intermediates, but kinetics do not lead to identification of the chemical nature of the active site. A spectroscopic investigation of the enzymic hydrolysis of methyl thionohippurate (C6H6.CO . NH . CHI. CS .OMe) provides direct evidence that the intermediate involves a thiol group from the enzyme. This substrate was chosen because the predicted intermediate has a clear absorption well shifted to the red from other chromophores, but its lack of solubility in water means that measurements have to be made in acetone/water mixtures. The enzyme remains active under these conditions, with apparently an unchanged mechanism. A transient absorption band is formed when methyl thionohippurate is mixed with enzyme--maximum a t 313 mp with papain and at 315 mp with ficin. Model studies show that whereas the absorptions of -CS. OR and -CS. NHgare in the region 23&270 mp, -CS. SR has its maximum a t 305 mp. These results strongly suggest that the intermediate is an acyl-thiol. Methyl bippurate competitively inhibited the formation of the transient absorption in the presence of methyl thionohippurate, showing that they react at the same site. Denaturation of the enzyme arrests the decay of the acylenzyme absorption, showing that deacylation requires a fully active enzyme (14). conformation of Macromolecules
X-ray crystallography has led to the determination of highly detailed structures for some proteins and nucleic acids. No other technique can compete in either the detail or the certainty of the information gained. There are disadvantages, however, in the Xray method. Crystalline samples are required, and although there is evidence that the conformation of many polymers is the same in this state as in aqueous solution, independent checks, where possible, are desirable. More important, the number of species which can be crystallized is a small fraction of the total number
Figure 11. Absorption changes ot 31 1 m p during reaction of chymotryptin with o, N-ocetyl-1-tryptophan m d b, its methyl ester. Dato token from ref. ( 1 3 ) .
Volume 45, Number 1 1 , November 1968
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of biopolymers. Thus a profitable exercise is to use known structures of some proteins to learn about correlations between structure and various physical measurements (of which spectroscopy is one), in the hope that a system may be evolved whereby structures can be inferred from solution studies alone (including sequence determination). Most infrared investigations of conformation have involved fibrous proteins or polypeptides, or nueleic acids. These have the advantages of being easily observed as films, and of containing a degree of organization which enables polarization measurements to be made with success. The transition moment of an ahsorption is a vector, and this means that a molecule absorbs most readily light polarized parallel to the dipole. When the orientation of molecules in a solution is random their absorption is unpolarized, but if some degree of organization is introduced as in an ordered solid, dichroic spectra are obtained. The relative intensities of different peaks depend on the orientations of the different transition dipoles relative to the electromagnetic vector. The most important peptide absorptions in the infrared are the amide I and I1 bands (around 1600 cm-') which are mainly the C=O stretching and N-H deformation modes. Changes in secondary structure (between random coil, a-helix, fl-p1eat)ed sheet, etc.) cause small, but detectable, shifts, band splittings, and changes of intensity which can be used t o suggest structural assignments. Polarization experiments lead to more definite conclusions. The orient.ation of the molecules is achieved by stroking a solution in one direction as the solvent evaporates to leave a film. For an a-helix (in which the hydrogen bonds are parallel to the long axis of the molecule)-like polyr-benzyl-*glutamate-the N-H and C=O stretch bands are most intense for light polarized parallel to the molecule, while the arnide 11 band is polarized perpendicularly. The reverse polarizations are found for pleated sheet structures (like polyglycine), in agreement with prediction (16). Theoretical work bas led to calculation of the band positions and polarizations for different secondary structures in terms of the possible intramolecular interactions. Thus the observed spectra can now be analyzed in more detail to suggest the importance of mixed structures (such as some a-helix and some flpleated sheet) and of deviations from strict adherence to a standard conformation like the helix (16). It is not possible to study globular proteins usefully by infrared spectroscopy both because their mixed secondary structures lead t o diffuse spectra with overlapping bands and because they need to be studied in aqueous solution. The spectra of D10 solutions can be measured, but deuteration of N-H bonds occurs with a considerable shift in absorption. This can be observed for model compounds, such as poly-L-lysine, and a sui'table correction made in assignments, but it is impossible to predict the extent of deuteration inside a globular protein, and so this kind of structural information cannot be deduced from its infrared spectrum (16). It is still possible, however, to use the extent of deuterium-hydrogen exchange to learn about the nature of the protein's surface (17). The secondary structure of nucleic acids has also been 698 / Journal of Chemical Education
studied by infrared spectroscopy. An absorption at 1700 em-' (1685 em-' for deuterated films) has been correlated with the secondary structure of the polymer. X-ray measurements show that this structure is destroyed under the same conditions-heating, treatment with formamide, etc.,-as cause disappearance of the band. A similar absorption is observed when a mixture of the free bases is precipitated from solution by simultaneous crystallization, but not for mechanical mixtures of the solids. Thus the band has been correlated with the hydrogen bonding between bases which stabilizes secondary structure. Polarization measurements show that the direction of this hydrogen bonding depends on the relative humidity over the sample film. Above SOY0 humidity the bases of sodium DNA are perpendicular to the helix axis but a t 70% they have tilted by 13'. These observations are consistent with X-ray data obtained at similar water vapor pressures (18).
The secondary structure of an aqueous solution of nucleic acid can be studied by means of its absorption in the ultraviolet region a t 260 mp. The extinction a t this wavelength falls when an a-helix is formed from random chains of nucleic acid. This hypochromic effect can lead to a change in absorption of 2040Y0, and is proportional to a-helix concentration providing the chains contain more than 8 residues. Theory can account for hypochromism in terms of dispersion force interactions between adjacent base pairs. The low number of pairs necessary to observe the effect demonstrates that the interactions have a short range. The absence of a spectral shift in addition to hypochromism is because coupling between adjacent chromophores is small compared with intramolecular vibrational coupling (19). The nature of a protein's conformation, and changes in it, are reflected in the ultraviolet spectrum. The absorptions of the aromatic amino acids are easy to observe, but with extra precautions, such as evacuating the spectrophotometer to remove oxygen, it is also possible to observe the spectra of cysteine, methionine, hystidine, and the amide absorption in the range from 190-220 mp. The position, intensity, and fine structure of the amide absorption all depend on a protein's shape. On forming an a-helix from a random coil there is a large hypochromic effect, a small blue shift, and the appearance of a long wavelength shoulder at 205 mp, but on forming the 8-structure there is a small hyperchromic effect, a red shift, and a sharpening of the band (Fig. 12). Polarization of absorption for an orientated film of poly-y-methyl-L-glutamate (which forms an a-helix) shows that the main band and the shoulder are polarized perpendicularly. This agrees with Moffit's theoretical predictions, which although they are now seen to be inapplicable to rotatory dispersion should still apply to absorption (80). Studies of this kind enable the amount of a-helix in a protein to be estimated, and the values obtained are in reasonable agreement with those by other methods. The correlation between optical rotatory dispersion and spectrophotometry suggests the absence of left-handed helices because while the rotation method subtracts their contribution, the spectrophotometric one adds it to that of the right-handed helices.
Then by comparing the spectrum of a mixture of free amino acids in the same concentrations as they are in a protein with the spectrum obtained from the intact protein it is possible to estimate the numbers of buried and exposed residues present. This ratio is generally similar for tyrosine and tryptophan, which is consistent with the fact that t h free energies of transfer of the free amino acid from aqueous to non-aqueous solvents are very similar for the two residues. It is also possible to investigate the topography of a protein surface by observing the effects of solvents of different diameters, providing estimates of crevice sizes (21). Many of the characteristics of solvent perturbation studies are well illustrated in some work on the enzyme L-glutamate dehydrogenase. The results are presented in Table 2. The number of chromophores perturbed increases as the size of the perturbant is decreased, but even with D20 a number of residues are unseen (22). The perturbations by ethylene glycol are smaller than Figure 12. Absorption spectra of the peptide bond in poly-L-lyrine in different conformations. A, random soil; B, @-structure; C, a-helix. Data taken fmm ref. (2001.
Protein conformation can be explored by solvent perturbation difference spectroscopy. To measure difference spectra double cells are placed in both reference and observation beams of a dual beam spectrophotometer. The same species are put in each, but they are unmixed in the reference cell and mixed in the observation one (Fig. 13). The difference spectrum provides a sensitivemethod for detecting EFnmctsmall snectral changes (down to 0.05 m ~ ) .-1n solvent perturbation studies the shifts are recorded when a solvent is altered. The method looks a t the surface of a Figure 1 3 . he experirnento~layout protein by way of the for observing difference spectra. a and b are interacting solutes, S is absorptions of aromatic the ,~,.t. amino acids, prosthetic groups, or covalently bound labels. It rests on three major assumptions. (1) The range of perturbing effects is short so t.hat buried residues cannot feel them. Model studies on chromophores buried in micelles have suggested that they are insensitive to perturbants at distances greater than 5-10 A, but there is no doubt that the ranges of different perturbing solutes vary. Some, like sucrose, have a long range and presumably act by changing the bulk structure of the solvent; others, like dimethylsulphoxide, have a very short range which implies that physical contact with the chromophore is required to produce a spectral change. Thus it becomes possible to define three categories of residueburied, exposed, and partially exposed (being perturbed by long-range but not by short range solutes). (2) The perturbant does not alter protein conformation-which is a reasonable assumption for low concentrations of polyhydroxylic perturbants. (3) The perturbant must not be preferentially absorbed or excluded by the protein surface. Before using solvent perturbation on a protein it is necessary to ascertain its affects on free chromophores.
*.;-
Table 2.
Perturber Sucrose Glucose Glycerol Ethylene glycol DzO -
Solvent Perturbation of L-Glutomate Dehydrogenase
Molecular diameter (A) 9.74 7.12 5.14 4.30 2.24
Ratio of ehromophores "seen" to total chromophores PhenylTyrosine Tryptophan danine 0.24 0.27 0.36 0.32 0.44
0.36 0.44 0.43 0.36 0.78
0.10 0.16 0.20 0.13 0.24
These figures are all taken from referenee (ZS?).
expected on the basis of its sizc and the effects of other molecules, but this could be because the glycol is a shorter range perturber than glycerol (21). Solvent perturbation differcnce spectroscopy is not a suitable technique for observing changes in conformation because any perturbation is likely to alter the free energy of a conformational change. Thus even small perturbations are only negligible where one conformation is considerably more stable than any other. Examples are also known where a conformational change does not alter the numbers of buried and exposed groups (25). To follow changes it is more satisfactory to measure directly difference spectra in the same solvent between conditions which cause the conformational change. I t has been common, for example, to measure spectral changes as a function of pH-particularly trying to correlate the results with ionization of the phenolic hydroxyl of tyrosine. This kind of study can detect changes more easily than it can explain them. It is not always necessary to follow the difference spectrum of the aromatic amino acids. Anthracene has been coupled to a thiol group of bovine plasma albumin through a mercury atom, and has been used as a spectroscopic reporter group. The dependence of its spectrum on pH follows closely the changes observed in the tyrosyl absorption of this enzyme. This suggests that the cause of both effects is a general environmental change rather than specific cleavage of tyrosine hydrogen bonds as has also been postulated (24). The use of spectroscopic reporter groups will become a powerful technique as chromophores are found which react specifically with only one of the twenty amino acids. Already some are known, such as 2-hydroxydVolume 45, Number 1 1 , November 1968
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nitrobenzyl bromide and 2-bromoacetamido-4-nitrophenol which can be specific for tryptophan and methionine respectively. Once covalently bound to the protein the spectrum of the chromophore depends on the environment it finds. If it is affected by the binding of, for example, a substrate then the distance between substrate and reporter sites must be close to the sum of their molecular radii, or else the binding of substrate causes a gross conformational change (25). When a reporter group has signaled (by a spectral change) that it is close to a particular site, partial hydrolysis of the protein and analysis of the peptides with reporter groups attached could lead to a partial amino acid sequence for the region of the site. The aromatic absorption bands usually undergo a blue shift when a protein is denatured. This parallels the shift when the solvent of a free acid is changed from a hydrocarbon to water, and is taken as further evidence that some of the residues of a globular protein are in a substantially non-polar environment (26). intermolecular Interactions
The sensitivity of spectroscopy to small changes in the environment of a chromophore makes it an ideal technique for the detection of weak interactions. I n biochemistry it has been widely used to investigate the binding of prosthetic groups and substrates to enzymes. Usually these effects cause shifts in the spectrum of either the protein or the interacting molecule. The analogy with solvent perturbation studies is obvious and measurements of these effects are often made by difference methods. When a flavin coenzyme binds to a protein the absorption maximum may shift in either direction from that of the free molecule (Fig. 14), though usually it moves to longer wavelengths. There is also an increase in fine structure on either side of the absorption maximum. Flavins alone have been subjected to extensive investigation, both varying the solvents and the substituents in the flavin ring (27). By comparing the measurements on flavoproteins with the studies of free
Figure 14.
700
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The molecular structure and absorption rpectrvm of riboflavin,
Journol of Chemical Education
flavins it seems that long wavelength shifts of the visible absorption are caused either by moving to a less polar environment or by replacing oxygen with less electrophilic species in the 2 and 4 positions. Blue shifts, on the other hand, require polar solvents, and perhaps substituents in the benzenoid ring. Sometimes a completely new absorption band may be formed on binding. This occurs when glyceraldehyde-3-phosphate dehydrogenase (GPD) interacts with nicotinamide adenine dinucleotide (NAD+). The new absorption-a featureless band around 360 mp-is attributed to a charge-transfer complex with donation from the protein to the coenzyme (28). A detailed model study has shown that a similar broad absorption occurs in the difference spectrum between indolylethylnicotinamide (Fig. 15) and a 1: 1 mixture of tryptamine hydrochloride and nicotinamide methochloride in the same molar concentrations. This implies that, the binding of NAD+ to GPD * also involves a charge-trans\*m, ferdonation from an indole ring to nicotinamide. This k is supported by finding that Figure 15. The molecular rtrusthe fluorescence of both inlureof indolylethylnicotinamide. dole and GPD is wenched under the conditions which lead to the appearance of the charge transfer band (29). A charge transfer band also results when zinc or cadmium interacts with thionein to form a metallothionein. Thionein is a protein with an abnormally high cysteine content (about 25% of the total amino acids) almost all of which is present with free thiol groups. As it contains no aromatic amino acids except, perhaps, for one phenylalanine, the metal-free protein has no absorption a t 250 mp. When cadmium is added a band forms a t 250 mp and with zinc there is one at 215 mw. Cadmium forms a similar peak in the presence of excess mercaptoethanol (1:2 or 1:3 Cd2+: ligand) whose extinction coefficient and band shape indicate that it is of a charge transfer type. By analogy a similar situation is implicated in metallothionein (SO). Prosthetic groups may make suitable spectroscopic markers for the study of substrate, inhibitor, and activator binding to an enzyme. The prosthetic group of phosphorylase B is pyridoxal phosphate (h,., at 330 and 388 mp) which marks the active center. The enzyme needs two substrates for reaction, glucose-lphosphate and glycogen: it is activated by adenosine monophosphate (AMP) and inhibited by adenosine triphosphate (ATP). No spectral changes are observed between 310 and 390 mp when the enzyme is mixed with either AMP or glucose-1-phosphate alone. When enzyme, AMP, and glucose-1-phosphate are all mixed however, there is a maximum in the difference spectrum a t 360 mp whose magnitude depends on the concentration of protein. The 360 mw band is decreased when ATP is also added to the mixture, in accord with its action as an inhibitor. The species which causes the new absorption may be an enzyme-substrate complex between the pyridoxal phosphate moiety and glucose-1-phosphate, which is stabilized by AMP but destabilized by ATP. A similar spectrum is obtained if inorganic phosphate is added instead of glucose-l-
phosphate in the presence of Ah'IP, suggesting that the substrate binds through its phosphate group (31). Although the inter and intramolecular interactions of proteins and nucleic acids have been studied in more detail than those of other biological systems it is becoming increasingly clear that membrane interactions which involve both protein and lipids have an important natural function. Spectrophotometric studies of whole membranes have yet to get much beyond the empirical stage, but useful results have been obtained from the investigation of phase changes in greatly simplified models like soaps, detergents, and phospholipids. These classes are characterized by having a long hydrophobic chain with a charged region at one end. Infrared spectroscopy has been used to get a picture of the behavior of the molecules on heating. The resolution of the absorptions of sodium palmitate changes from the sharp lines typical of a solid to the more diffuse spectrum of a liquid at a temperature well below the true melting point of this soap. This is evidence that beforp true melting there is a state with charged groups remaining in a lattice while the non-polar groups gain a measure of mobility similar to a liquid. Other phase changes have been detected-usually from the resolution of the infrared spectrum-though their causes are not always understood. When 2-oleoyl-3-stearoyl-LI-phosphatidylcholine is cooled to liquid nitrogen temperature and then warmed back to room temperature the spectrum has changed. After cooling and rewarming it is considerably sharper than before, showing that the normal liquid type of structure is not immediately regained (52). Most applications of absorption spectroscopy in biochemistry have involved empirical use of the technique to detect and follow chromophores. It will be a major advance when detailed theoretical assignments of the absorption bands can be made. A variety of observations are useful in achieving this end, such as the dependence of an absorption band on solvent and on substituents, and the measurement of the magnitude and direction of transition dipoles (33). Finally it is worth noting that the scope of spectroscopic measurements is greatly increased when they can be compared and correlated with other optical properties, such as rotation and light emission. Acknowledgment
I am extremely grateful for the many helpful suggestions given to me by Dr. G. Ii. Radda and Mr. A. D. B. A'Ialcolm, who both read the manuscript of this article. Literature Cited (1) ALBEN,J. O., A N D CAUGHEY, W. S., "Hemes and Hemopro-
R. W., AND terns" (Edztors CHANCE,B., ESTARROOK, YONET~NI, T.) Academic Press, Inc., New York, 1966, p. 139.
(2) MAY,L., AND GRENFELL, R. G., Ann. N . Y . Acad. Sci., 69,71 11957). (3) PITT, G. A. J., AND MORTON,R. A., "Progrem in the
Chemistry of Fats and other Lipids" (Editors: HOLMAN, R. T., LUNDBERG, W. O., AND MALKIN,T.) Pergamon Press, London, 1957,4, p. 233. (4) WETIAUFER,D. B., Adv. P70t. Chem., 17, 304 (1962). E. F., Nature, 164, 254 (1949). (5) KEILIN,D., A N D HARTREE, T., A N D WALD,G., Natu~e,212, 483 (1966). (6) YOSHIZAWA, R. W., "Hemes and Hemoprateins" (Editors: (7) ESTABROOK, CHANCE,B., ESTABROOK, R. W., A N D YONETANI,T . ) Academic Press, Ine., New York, 1966,p. 40.5; E s ~ n s ~ o o K , R. W., "Haernatin Enzymes" (Editors: FALK, J. E., LEMBERG,R., A N D MORTON,R. K.) Pergamon Press, Nev York, 1961, part 2, p. 436. B., Arch. Biochem. Biophys., 41, 404 (1952). (8) CHANCE, R., AND RABINO(9) J ~ c o n s ,E. E., HOLT,A. S., KROMHOUT, WITCH, E., Arch. B i o e k . Bzophys., 72, 495 (1957). TH.,Disc. Farnday Soc., 27, 1 (1959). (10) F~RSTER, (11) KOK,B., "Proceedings of the Fifth International Congress of Biochemistry, Moscow 1961" (Edilor: SIS~AKIAN, N. M . ) Pergamon Press, London, and PWN, Warsaw, 1963,6, p. 73. , T., PETERSON, E. W., A N D MASON,M. S., (12) Y ~ s u n o s u K. J. Biol. Chem., 234, 3291 (1959). G. E., AND BENDER,M. L., J . (13) K$XDY,F. J., CLEMENT, Amw. Chem. Soc., 86, 3690 (1964). A., Biochem. J., 96,189 (1965). (14) LOWE,G., A N D WILLIAMS, T., A N D BLOUT,E. R., J . A m e ~Chem. . Soc., 83, (15) MIYAZAWA, 712 (1961). . . (16) TIMASHEFF,S. N., AND GORHUNOFF, M. J., Ann. Rev. Bioehem., 36, 13 (1967). W. F., JOSEPHS, R., A N D SEGAL,D. M., Ann. (17) HARRINGTON, Rev. Biochem... 35.. 599 (1966). . . (18) SHIMANOUCHI, T., TGUBOI,M., AND KYOGOKU, Y., Adv. Chem. Phys., 7,435 (1964). N. R.. Ann. Rev. Phvs 119) ~ ~, ZIMM.B. M.. A N D KALLENBACH. Chem;, 13, i i i (1962). K., AND DOTY,P., Proc. Natl. Acad. Sci., (20) (a) ROSENHECK, 47, 1775 (1961); (b) GRATZER, W. B., HOLZWARTH, G. I!., AND DOTY,P., Proe. Natl. Acad. Sei., 47, 1785 (1961). (21) LASKOWSKI, M., Fed. P ~ o c .25, , 20 (1966). (22) CROSS,D. G., AND FISHER, H. F., Biochemistry, 5, 880 (19661. . (23) KRONMAN, M. J., A N D HOLMES,L. G., Biochemistry ,4, 526 (1965). (24) WILLIAMS, E. J., A N D FOSTER,J. F., J . Amer. Chem. Sac., 82, 242 (1960). H. R., AND KOSHLAND, I). E., J. Amer. Chem. Soe., (25) HORTON, 87, 1126 (1965); HILLE, nf. B., A N D KOSHLAND, D. E., J . Amer. Chem. Soc., 89, 5945 (1967). (26) Y.mnnr, S., AND BOYEY,F. A,, J . B i d . Chem., 235, 2818 (1960). (27) PENZER, G. R., AND RADDA, G. K., Quart. Rev. (London),21, 43 (1967). (28) VELICIC, S. F., A N D FURFINE, C., "The Enzymes" (Editors:
.
BOYER,P. D., LARDY,H., A N D MYRAACIC, K.) Academic Press, Iuc., New York 1963,7, p. 253. (29) SHIFRIN,S., Biochim. Biophys. Acta., 81, 205 (1964). (30) Kaor, J. H. R., A N D T'ALLEE, B. L., J . B i d . Chem., 236,2435 (1961). (31) BRESLER,S., FIRSOY,L., A N D GMSUNOV, E., Nature, 211, 1262 (1966). (32) CHAPMAN, D., "The Stnlctore of Lipids," Menthuen, London 1965, ch. 4. (33) LIPPERT,E., J . Elektroehem., 61, 962 (1057).
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