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Anal. Chem. 1989, 6 1 , 642-650
PERSPECTIVE: ANALYTICAL BIOTECHNOLOGY
Investigations of Protein Structure with Optical Spectroscopy: Bovine Growth Hormone Henry A. Havel,* Robert S. Chao, Royal J. Haskell, and Thomas J. Thamann
The Upjohn Company, Kalamazoo, Michigan 49001
Optical spectroscopy provldes a wealth of information about proteln structure that is dWficult to obtain from other methods. Investlgaths of changes ln prhnary, secondary, tertlary, and quaternary structure are partlcuiarly well-sulted for optical technlques such as UV absorptlon, clrcular dichrolsm, fluorescence, Raman and Infrared spectroscopy, as well as light scattering methods. Each method ha$ unlque areas of applkablllty and contributes to structure determlnatlon In a dlfferent manner. The appkatlon of these methods Is demonstrated wlth examples of studles performed on bovlne growth hormone. Some of these Include: determlnatlon of soiutlon-state structure, monltorlng dlfferences between solution- and solld-state structure, determlnatlon of molecular size distrlbutlon, and lnvestlgatlons of proteln foldlng mechanisms. I t Is demonstrated that by judicious choke of methods, a reasonably complete descrlptlon of protein structure can be obtalned.
Proteins are large, complex molecules whose manifold biological functions make them targets for commercialization using biotechnology. Recombinant DNA techniques, in particular, provide the synthetic tools for industrial production of proteins. Because the biological activity of a protein is determined by its three-dimensional structure, protein structure elucidation can play an important part in successful applications of biotechnology. Protein structure is traditionally subdivided into primary, secondary, tertiary, and quaternary structure levels (Figure 1). Primary structure is the specific amino acid sequence of the protein and the location of any disulfide bonds between cysteine residues, while secondary structure describes local structural elements of the protein chain such as a-helix, ppleated sheet, and @-turn. Regions with little local order are referred to as “random” or “disordered”. A third level of protein structure (tertiary) involves the orientation of, and nonbonded contacts between, the secondary structure elements to form the folded structure of a protein. The last protein structure level (quaternary) relates the separately folded protein chains to one another; in multisubunit proteins this involves the relative positions of the subunits. A complete description of protein structure requires information about all four structural levels. As outlined in Table I, a variety of spectroscopic methods are available to determine protein structural features. Optical spectroscopy, in particular, provides knowledge of protein structure that is difficult to obtain by other experimental techniques ( I ) . It should be emphasized that combining information from each of the various methods leads to a high 0003-2700/89/0361-0642$01.50/0
level of understanding about protein structure even though no single technique is able to fully describe the system. This “systems approach” is common in the application of molecular spectroscopy to structure elucidation, but due to the complex nature of protein structure determination, its implementation becomes imperative if a useful level of knowledge is to be achieved. This report describes applications of optical spectroscopy to the determination of primary, secondary, tertiary, and quaternary structure. The optical techniques considered are UV absorption, circular dichroism (CD), fluorescence,infrared (IR) and Raman spectroscopy, as well as light scattering methods. The examples used are those where optical methods have been applied to investigations of bovine growth hormone (bGH), a medium-size protein (molecular weight rr 22 000, 191 amino acid residues) that is under development at Upjohn and other companies for its ability to increase milk production efficiency in dairy cattle. Recommendations are made for exploiting the structural information obtained from optical spectroscopy during development of a protein product. PRIMARY STRUCTURE Disulfide Bonds-Raman Spectroscopy. Disulfide bridges are an important element of primary structure that contribute significantly to the stability of the entire molecular structure (2). In the assessment of the structural integrity of a protein made by using recombinant DNA techniques, it is necessary to confirm both that the disulfide bonds are present and that these bridges are formed between the correct pair of cysteine residues. Laser Raman spectroscopy has been used to detect the presence or absence of cystine links ( 3 , 4 ) . The presence of an S-S stretch at about 500 cm-’ indicates an intact bridge, while the observation of an S-H stretch at about 2550 cm-I is indicative of the lack of a cystine bridge. The high symmetry of the S-S stretch makes it difficult to detect with IR, but the mode is easily observed in Raman spectra. We have used Raman spectroscopy to detect the presence of both disulfide bridges in bGH, and the presence or absence of one or both of the bridges in bGH analogues. BGH has two disulfide bridges that configure the molecule into two protein loops. A large loop is formed by a cystine link connecting residues 53 and 164. The second disulfide bridge produces a small loop at the carboxyl terminal end of bGH from residues 181 to 189. The Raman disulfide stretching region for untreated bGH (Figure 2A) exhibits a broad envelope centered at 535 cm-’. Vibrations observed in this region are usually assigned as disulfide stretches. However, fully reduced and carboxamidomethylated bGH (TSCAM), which has both disulfide bridges cleaved and prohibited from re-forming by carboxamidomethylation, exhibits a similar vibrational envelope in this region (Figure 2B). 0 1989 American Chemical Society
ANALYTICAL CHEMISTRY. VOL. 61. NO. 7. APRIL 1. 1989
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Table 1. Comparison of Spectroscopic Methods for Protein Structure Determination
technique absorption far-UV near-UV IR
protein structure level seconquaterprimary dary tertiary nary X
aromatic amino acid and amide transitions; sensitive to environment aromatic amino acid transitions; sensitive to environment backbone and side chain vibrations (amide and S-H): solid or solution state
X X
X
comments
X
circular dichroism far-lJV ~.~ .
amide transitions used for % a-helix. &pleated sheet, and disordered tryptophan, tyrosine, and phenylslanine bands shift with environment amide vibrations studied initially; advances needed in theory and experiment
X
near-UV
X
IR
X
fluorescence steady-state
. )
X
time-resolved
X
X
X X
X X
band shifts indicate tryptophan environment; quenching caused by proximal group excited-state lifetimes reflect tryptophan environment: anisotropy decays
light scattering
classical PCS mass Spectiometiy
X
NMR
X
X
X
X
X X
X X
X
X
X
Raman nonresonance reSOnsnee
X-rav diffraction
X
scattered light intensity related to molecular weight and radius of gyration analysis of autocorrelation function yields hydrodynamic radius amino acid sequencing; LC-MS tryptic digest maps; limited to M W 295 nm and an emission X > 350 nm. The effect of unfolding on,the tryptophan emission maxima of bGH is substantial as the environment changes from nonpolar in the native, folded state to polar in the unfolded state (26, 35). In addition, about a 4-fold increase in fluorescence intensity is seen for the unfolded state, probably due to quenching of emission by proximal groups in the native state. Fluorescence quenching by extrinsic agents provides information on the accessibility of a fluorophore to solution components. Quenching of the fluorescence of tryptophan in bGH has been employed to study differences in bGH conformations (35). The results were dependent upon the molecular size and chemical properties of the quencher. Figure 7 shows an example of the differences observed for acrylamide quenching of bGH in the native and unfolded states. The results show that the native-state tryptophan is protected from acrylamide penetration. Structural information about the noncovalent multimeric intermediate state of bGH has been obtained by use of a different fluorescence quencher, iodide (35). Iodide is a poor quencher of tryptophan fluorescence in this conformation indicating that the tryptophan is not accessible to iodide. These results provide evidence for negative charges near the tryptophan in the multimeric intermediate conformation. The interpretation of steady-state fluorescence results is often difficult and requires fluorescence lifetime information to provide more detailed knowledge about the ground and excited states of the fluorophore. Protein lifetime measure-
647
ments are complicated by the fact that even proteins with a single tryptophan residue are observed to have non-exponential decay kinetics (36),and due to low quantum yields and short lifetimes, picosecond laser systems are required. Because the fluorescence decays for tryptophan and tyrosine in solution are also nonexponential (due to different rotamers of the side chain in the ground state) it is likely that nearby side chains influence the nonexponentiality of protein fluorescence decays. Fluorescence lifetime measurements on bGH (35)have revealed that the native state is composed of at least three components with one component very fast (T N 100 ps). As the protein unfolds, the component lifetimes become longer and approach a two-component model. The effects described are due to reduced interactions of the tryptophan with fluorescence quenching groups in the unfolded state when compared to the native state. Near-UV Absorption Spectroscopy. Proteins absorb energy in the near-UV due to electronic transitions of aromatic amino acids (Arnm E 260-280 nm) and disulfide bonds (Arnm E 250 nm). Measurement of the absorbance near 280 nm is convenient for determination of protein concentration and for bGH tZm = 15400 (37). Near-UV absorption spectra of aromatic amino acids are sensitive to the polarity of their environment due to perturbations of electronic energy levels. For instance, the native bGH absorption spectrum (Figure 8A) exhibits a blue shift of about 5 nm upon unfolding (1). The unfolded bGH spectrum is indistinguishable from the spectrum of a mixture of aromatic amino acids contained in bGH. These results demonstrate how the absorption spectrum can be used to monitor tertiary structural changes. The different absorption bands of a protein in the near-UV are significantly overlapped, however. The resolution can be improved and peak positions located more accurately if the derivative of absorbance with respect to wavelength is calculated (38) as shown in Figure 8B for native bGH. By use of model compounds for tyrosine and tryptophan, it has been demonstrated (39)that increasing solvent polarity causes shifts in second-derivative band positions of tyrosine with little change in band intensities, while for tryptophan band intensities change more than band positions. These results have been used to interpret the second-derivative spectra observed for bGH in different conformational states (1): the native state has the most nonpolar tryptophan environment and the multimeric state has the most polar. Near-UV CD Spectroscopy. The overlap of electronic transitions of proteins in the near-UV, which leads to poor resolution of spectral features, is less pronounced in a CD spectrum as transitions can have different signs (compare part A with part C in Figure 8). A CD spectrum, therefore, has higher inherent resolution than an absorption spectrum. CD spectra were used to assign vibrational fine structure in absorption bands of aromatic amino acids (40-42). The bGH near-UV CD spectrum (Figure 8C) contains several vibronic bands which have been assigned by Puett and co-workers (21). The spectrum is indicative of the tertiary structure of the protein and has been used to follow the folding of bGH at equilibrium (43) and kinetically (44),especially by monitoring changes in the CD at 300 nm due to the population of the noncovalent multimeric state. Raman Spectroscopy. The resonance Raman effect has been used to assign electronic absorption bands since resonance-enhanced vibrations are coupled to electronic transitions. We have used resonance Raman spectroscopy to investigate the electronic absorption spectrum of bGH near 220 nm (45). To the extent that the electronic structures of amino acid residues are sensitive to their local environment, such spectra may (eventually) yield tertiary structure information.
648
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-
acids rather than protein secondary structure. The latter would be the case if the absorption were due to amide transitions. Both tryptophan and tyrosine display Raman bands that are sensitive to local environment (46,47). For instance, the sharp decrease in intensity of the 756-cm-' tryptophan resonance Raman vibration in bGH at pH 8 (Figure 5B),compared to bGH a t pH 2 (Figure 5C), indicates a change in the tryptophan environment during unfolding. Nonresonance Raman spectroscopy can be used to probe elements of protein tertiary structure as well. Information on tyrosine hydroxyl environment (amount of hydrogen bonding between tyrosine and another amino acid) can be gained from the intensity ratio of the tyrosine 850 to 830 cm-l band in a nonresonance spectrum (47). Irradiation of bGH with y-rays in the absence of oxygen yields an intensity ratio of 1.0, which is the same value observed in untreated bGH. Radiation in the presence of oxygen destroys the bands in this doublet, indicating that drastic changes have occurred affecting all six bGH tyrosines. The geometry about S-S bonds is another indicator of tertiary structure that can be obtained from nonresonance Raman spectroscopy. Studies involving conformational differences in cystine linkages (48-52) have indicated that disulfide stretches around 508 cm-' indicate a gauche-gauchegauche bridge geometry. Cystine stretches at about 525 cm-' suggest a trans-gauche-gauche geometry and vibrations around 540 cm-' are indicative of trans-gauche-trans conformations. The S-S stretching vibrations for both disulfide bridges in bGH occur at -508 cm-' and are therefore assigned to a gauche-gauche-gauche configuration. Photon Correlation Spectroscopy. The hydrodynamic size of macromolecules can be estimated through the use of a laser light scattering technique known alternatively as photon correlation spectroscopy (PCS), dynamic light scattering (DLS), or quasielastic light scattering (QELS) (53). PCS can be used to monitor protein denaturation (54) due to an ability to detect variations in hydrodynamic size. Protein samples are frequently polydisperse due to association processes, conformational inhomogeneity, and unwanted components. Therefore, different data analysis routines, such as exponential sampling (55) and regularization (56), in addition to the cumulants technique (57) may be required to accurately retrieve diffusion coefficient information from autocorrelation functions. We have achieved good results with Provencher's program CONTIN (58).
QUATERNARY STRUCTURE Photon Correlation Spectroscopy. PCS results can be used to monitor quaternary structure of proteins through measurement of hydrodynamic radii. In addition, models of diffusion coefficient behavior fit to PCS data can yield useful information about intermolecular interactions as a function of charge and ionic strength (59). Association equilibria under nondenaturing conditions have been examined as well. Example systems are antigen-antibody complexes (60) and dimerization processes (61). I t is important to keep in mind that in the latter case, the resolution of individual n-mers is extremely unlikely. Rather, a weight fraction average will be observed, thus any quantitative information extracted about the equilibrium process is contingent upon the assumption of an appropriate model. PCS has been used to monitor bGH unfolding (43) and has yielded convincing evidence for the formation of a noncovalent multimeric intermediate (Figure 9). During the unfolding process, the hydrodynamic radius increased with the size, achieving a maximum under partially denaturing conditions, signifying the presence of multimeric intermediate($. Further addition of denaturant produced a decrease in size that is
ANALYTICAL CHEMISTRY, VOL. 61, NO. 7, APRIL 1, 1989
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attributed to the disappearance of multimeric intermediate(s) and formation of fully unfolded protein. We have begun PCS studies of protein samples where amino acid replacements have been made within the wild-type bGH sequence (44)in order to determine the effects that these replacements have on the unfolding and multimeric intermediate-formation processes. Classical (intensity) light scttering also provides useful information about protein quaternary structure. The results complement those of PCS; radii of gyration are determined instead of hydrodynamic radii. Interaction effects (virial coefficients), estimations of polydispersity, and absolute molecular weights can also be obtained with this method. We have attempted to determine the number of monomers involved in the formation of bGH multimeric intermediateb) using classical light scattering but, due to experimental difficulties, can only estimate that between three and five monomer units are involved. Fluorescence Anisotropy. Measurements of fluorescence anisotropy (or polarization) decay allow the determination of the time scale of motions which depolarize fluorescence emission. Such motions include the overall rotation of the molecule (rotational correlation time) and local, segmental motion. Rotational correlation time measurements (a) are useful as an adjunct to other methods for sizing proteins, (b) confirm rotational correlation time measurements determined by NMR, and (c) can be used to study protein-protein interactions, such as the binding of a protein to its receptor.
SUMMARY The results obtained on bGH have demonstrated several specific analytical capabilities for optical spectroscopy of proteins. In general, we have found that optical methods are capable of detecting differences in protein structure more reliably than providing absolute structural information. Optical spectra can be used to investigate all levels of protein structure: primary, secondary, tertiary, and quaternary. It must be emphasized, however, that the degree to which structural differences can be quantitated may be molecule dependent. Indiscriminate application of results from one protein to another will likely lead to misinterpretation. Structural information from optical spectroscopy can expedite development of protein products. The areas of application are largely dependent on chemical and physical characteristics of the protein, the manufacturing process, and intended use of the product. These areas include the following: (a) Solution-State Structure. Detection of altered protein structure in solution, particularly the use of CD, IR, and
849
Raman spectroscopy for estimating secondary structure, Raman spectroscopy to examine any disulfide bonds, and absorption, fluorescence, near-UV CD, and resonance Raman spectroscopy to investigate aromatic amino acid environments. Our experience with bGH allows us to estimate that secondary structure changes on the order of 10-15% can be detected. Due to the selective nature of fluorescence and Raman spectroscopy, this value may drop to 5-10% for amino acid environments. (b) Solid- vs Solution-State Structure, Establishment of differences between solution- and solid-state structure by the use of IR and Raman spectroscopy. (c) Molecular Size Distribution and Solubility. Studies of the effects of solution constituents on molecular size distribution using a combination of light scattering and fluorescence anisotropy decay experiments; may provide information to interpret protein solubility in various solvent systems. (d) Protein Folding. Optical spectroscopic measurements are fast, nonintrusive, and provide several monitors of protein structure, making them valuable tools for protein folding studies. (e) Biological Activity. Determination of the relationship between protein structure and biological activity; spectroscopic methods can be used for activity assays in vitro, such as monitoring protein-receptor interactions. The challenges of protein structure determination are significant but they can be met if structural information is combined from several techniques. By contributing to an understanding of protein structure, optical spectroscopy can play an invaluable role in protein product developm-ent.
ACKNOWLEDGMENT We are grateful for expert technical assistance from Paul Elzinga, E. Wayne Kauffman, and Scott Plaisted. In addition, we wish to acknowledge numerous useful discussions with David Brems, John Dougherty, James Freeman, and Robert White. LITERATURE CITED Havel. H. A. The Impact of Chemistry on Biotechnology; Phlllips, M., Shoemaker, S.P., Miilekauff, R. D., Ottenbrite, R. M., Eds.; American Chemical Society: Washington, DC, 1988; Chapter 14. Flory, P. J. J. Am. Chem. SOC. 1058, 7 8 , 5222-5235. Sprio, T. G.; Gaber, B. P. Annu. Rev. Biochem. 1077, 4 6 , 553. Tu, A. T. Raman Spectroscopy in Biology: Principles and Applications; Wiley: New York, 1982. Christensen, A. E. I n Steriiization and Preservation of Biological Tissues by Ionizing Radiation; IAEA: Vienna, 1970; pp 1-13. StrbBk, V.; Macho, L.; Sedlik, J.; Hromadovi, M. Endocrinol. Exper. 1078, 70, 3-11. Parker, F. S. Application of Infrared Spectroscopy in Biochemistry, Biology and M i c i n e ; Plenum: New York, 1973. Byler, D. M.; Susi, H. Biopolymers 1086, 2 5 , 469-487. Surewicz, W.; Mantsch, H. H. Biochim. Biophys. Acta 1088, 952, 115-130. Chen, C.-j. H.; Sonenberg, M. Bbchemistry 1977, 76, 2110-2118. Sonenberg, M.; Beychok, S. Biochim. Bbphys. Acte 1971, 2 2 9 , 88-101. AbdeCMeguid, S.S.;Shieh, H.-S.; Smith, W. W.; Dayringer, H. E.; V i e land, B. N.; Bentle, L. A. R o c . Natl. Acad. Sci. U . S . A . 1087, 8 4 , 6434-6437. Parker, F. S. Applications of Infrared, Raman, and Resonance Ra man Spectroscopy in Biochemistry; Plenum: New York, 1983; Chapter 3. Asher, S. A.; Murtaugh, J. L. Appl. Spectrosc. l98& 4 2 , 83-90. Asher, S.A.; Ludwig, M. L.; Johnson, C. R. J. Am. Chem. SOC.1988, 708, 3186-3197. Caswell. D. S.; Spiro, T. 0. J. A m . Chem. SOC. 1087, 109, 2796-2800. Copeland, R. A.; Splro, T. G. Bbchemfstry 1987, 2 6 , 2134-2139. Greenfield, N.; Fasman, G. D. Biochemistry 1080, 8 , 4108-41 16. Provencher, S.W.; Glockner, J. Biochemistry 1081, 2 0 , 33-37. Compton, L. A.; Johnson, W. C., Jr. Anal. Biochem. 1086, 755, 155-167. Holladay, L. A.; Hammonds, R. G., Jr.; Puen, D. Biochemistry 1074, 73, 1653-1661. Kuwaiima. K.: Nina. K.: Yonevama.. M.:. Suoai. - . S.J. Mol. Bioi. 1078. 706,359-373. . Henkens, R. W.; Kitchell, 6. B.;Lotlch, S.C.; Stein, P. J.; Williams, T. J. Biochemistry 1982, 27, 5918-5923.
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Anal. Chem. 1989, 61, 650-656 BetIOn. J.-M.; Desmadrll, M.; Mitraki, A.; Yon, J. M. Biochemkby 1984, 23 6654-6681. Craig, S.;Hollecker, M.; Creighton, T. E.; Pain, R. H. J. Mol. Biol. 1985. 185. 681-687. Brems, D. N.; Plaisted, S.M.; Havel, H. A.; Kauffman, E. W.; Stodola, J. D.; Eaton, L. C.; White, R. D. Blochemisby 1985, 24, 7662-7668. Holzman. T. F.; Brems, D. N.; Douaherty. . J. J.. Jr. Biochemlst~y1986, 25, 6907-6917. Kuwaiima, K.; Hiraoka, Y.; Ikegushi, M.; Sugai, S.Biochemistry 1985, 24 074-88 1. Frontlcelli, C.; Bucci, E. Biophys. Chem. 1985, 23, 125-128. Brems, D. N.; Plaisted, S. M.; Dougherty. J. J., Jr.; Holzman. T. F. J. Bioi. Chem. 1987. 262, 2590-2596. Kuwajlma, K.; Yamaya, H.; Mlwa, S.;Sugal. S.;Nagamura, T. F€BS Lett. 1987, 221, 115-118. Rosenheck, K.; Doty, P. Proc. Natl. Acad. Sci. U . S . A . 1981, 4 7 , 1775- 1785. Lakowicz, J. R. Rlnclples of Fiuorescence Spectroscopy; Plenum: New York, 1983; Chapter 11. Teale, F. W. J.; Weber, G. Biochem. J. 1957, 65, 476-482. Havel, H. A.; Elzinga, P. A.; Kauffrnan, E. W. Biochim. Biophys. Acta 1988, 955, 154-163. Beechem, J. M.; Brand, L. Ann. Rev. Biochem. 1985, 54, 43-71. Burger, H. G.; Edelhoch, H.; Condliffe, P. G. J. Biol. Chem. 1988, 24 1 , 449-457. Butler, W. L. Methods Enzyrnol. 1979, 56, 510-515. Terada, H.; Inoue, Y.; Ichikawa, T. Chem. Pharm. Bull. 1984, 32, 585-590. Horwltz, J.; Strickland, E. H.; Billups, C. J. Am. Chem. SOC. 1969, 91, 184-190. Strickland, E. H.; Horwitz, H.; Billups, C. Siochemlsfry 1989, 8 , 3205-32 13. Horwltz, J.; Strkkland, E. H.; Billups, C. J. Am. Chem. SOC. 1970, 92, 2119-2129. Havei. H. A.; Kauffman, E. W.; Plaisted, S.M.; Brems, D. N. Biochemistry 1986, 25, 6533-6538. ~
(44) Brems, D. N.; Plaisted, S. M.; Havei. H. A,; Tomich, C.4. C. Proc. Natl. Acad. Sci. U . S . A . 1988, 85, 3367-3371. (45) Kauffman, E. W.; Thamann, T. J. I n Pittsburgh Conference & Exposition on Analytical Chemistry and Applied Spectroscopy; New Orleans, LA, 1968; Abstract 734. (46) Harada, I.; Muira, T.; Takeuchi, H. Spectrochim. Acta, Part A 1988, 42A, 307-312. (47) Siamwiza, M. N.; Lord, R. C.; Chen, M. C.; Takamatsu, T.; Harada, I.; Matsuura, H.; Shimanouchi, T. Biochemistry 1975, 74, 4870-4876. (48) Van Wart, H. E.; Scheraga, H. A. J . Phys. Chem. 1978, 8 0 , 1812-1822. (49) Van Wart, H. E.; Scheraga, H. A. J. Phys. Chem. 1978, 8 0 , 1823-1732. (50) Van Wart, H. E.; Scheraga, H. A. R o c . NaN. Acad. Sci. U . S . A . 1977, 7 4 , 13-17. (51) Van Wart, H. E.; Cardinaux, F.; Scheraga, H. A. J. fhys. Chem. 1978, 80,625-630. (52) Van Wart, H. E.; Scheraga, H. A.; Martin, R. 8. J. Phys. Chem. 1976, 80, 1832. (53) McConnell, M. L. Anal. Chem. 1981, 53, 1007A-1018A. (54) Nystrom, 8.; Roots, J. Chem. Phys. Lett. 1982, 97, 236-240. (55) Ostrowsky, N. D.; Sornett, P. P.; Pike, E. R. Opt. Acta 1981, 28, 1059-1070. (56) Provencher, S. W. Comput. M y s . Commun. 1982, 27, 213-227. (57) Koppei. D. E. J. Chem. Phys. 1972, 57, 4814-4820. (58) Provencher, S. W. Comput. Phys. Commun. 1982, 27, 229-242. (59) Dorshow, R.; Nicoli, D. F. J. Chem. Phys. 1981, 75, 5853-5856. (60) Yarmush, D. M.; Morel, G.; Yarmush, M. L. J. Biochem. Biophys. Methods 1987, 14, 279-289. (61) Wlllis, P. R.; Georgalis, Y. J. Phys. Chem. 1981, 85, 3978-3984.
RECEIVED November 9, 1988.
ARTICLES
Transient Infrared Emission Spectroscopy Roger W.Jones' and J o h n F. McClelland*JJ Center for New Industrial Materials and Ames Laboratory-USDOE, Iowa State University, Ames, Iowa 50011 Translent infrared emisslon spectroscopy (TIRES) is a new method that produces analytically useful emlsslon spectra from optlcally Ihlck, solid samples by greatly reducing selfabsorptlon of emitted radlatlon. The method reduces selfabsorption by creating a thln, short-llved, heated layer at the sample surface and coHectlng the transient emission from thls layer. The techdque requires no sample preparation and may be appiled to both moving and statlonary samples. The slngle-ended, noncontact TIRES measurement geometry Is Ideal for on-llne and other remote-senslng applications. TIRES spectra acquired via a Fourler transform infrared spectrometer on movlng samples of coal, plastic, and palnt are presented and compared to photoacoustlc absorptlon spectra of these materlals. The TIRES and photoacoustk results are In close agreement as predlcted by Klrchhoff's law. Center for N e w I n d u s t r i a l Materials. Ames Laboratory-USDOE.
INTRODUCTION Conventional infrared emission spectroscopy, with the sample held at an elevated uniform temperature, has not been a practical method for infrared analysis of most bulk materials due to the phenomenon of self-absorption. Self-absorption in optically thick samples causes severe truncation of strong bands and leads to emission spectra that closely resemble black-body emission spectra and contain little spectral structure characteristic of the material being analyzed (1,Z). The applications of emission spectroscopy could be greatly increased if self-absorption could be controlled. For example, remote, on-line infrared analysis of bulk materials requires a single-ended measurement geometry that could be uniquely satisfied by emission spectroscopy if self-absorption were sufficiently reduced. Conventionally, self-absorption is reduced by thinning the sample. High-quality spectra of free-standing films and thin layers on low-emittance substrates are routinely measured
0003-2700/89/0361-0650$01.50/00 1989 American Chemical Society
