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An improved technique for the near infrared study of water binding by proteins
Biochimica et Biophysica Acta, 295 (1973) 30-36 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m -- P r i n t e d in T h e N e t h e r l a n d s
BBA 36305 AN IMPROVED T E C H N I Q U E FOR T H E N E A R I N b ' R A R E D STUI)Y ()V W A T E R B I N D I N G BY P R O T E I N S I L L U S T R A T I O N W I T H GELATIN
N. I),ESSLER AND Z I A U D D I N
Departments of Pathology and Biochemistry, University oJ illinois Medical Ce~ter, Chicago, Ill. 6o6~2 (U.S.A.) (Received J u l y 2oth, 1972)
SUMMARY
A description is presented of cells designed to minimize certain limitations of sampling methods generally used for near infrared studies of water binding by proteins or other polymers. The g per cm 2 of both protein and of bound water in the optical path can be varied over a considerable range, and the amount of each can be determined independently by appropriate weighings. When the method was applied to a film consisting of I8. 4 mg of gelatin per cm 2, bands due to the presence of bound water were exhibited near 1.45, 1.6, 1.8, 1.95, 2.1, 2.25, and 2.5 # m (6897, 6250, 5556, 5128, 4762, 4444, and 4000 cm -1, respectively). Comparisons to previously reported results, the dependence of the resolution of these bands upon the percent of water present, and the possible forms of water which some of the bands may represent, are discussed.
INTRODUCTION
The near infrared region of the spectrum includes combination and overtone bands, various low energy charge transfer bands, etc. 1. Numerous studies of hydrogen bonds 2, and of the structure of water a have been advantageously conducted in this range. The near infrared has also been used, to a limited extent, for the investigation of water binding to proteins or nucleic acids, but the large majority of infrared water binding studies have been in the fundamental or mid infrared region (the latter is commonly taken as 2.5 5o #m or 4o0o-2oo cm 1). The infrared investigation of water binding to proteins has usually involved the casting of an aqueous solution of the protein into a thin film. The latter can then be scanned while exposed to various atmospheres, which differ in particular relative humidities 4. In order to apply this procedure to the near, rather than to the mid infrared region, films of considerably greater thickness are necessary for optimal spectral
31
NEAR INFRARED STUDY OF WATER BINDING
measurements, since the absorbances are correspondingly less in the near infrared. As the thickness of the film is increased, however, the relation between the amount of water bound to the polymer and the relative humidity becomes more indirect 5, and the actual water content becomes more uncertain (when a nearly dry film containing 16 mg of gelatin per cm * was kept in a vacuum of about o.I mTorr Hg for 16 h at room temperature, for example, infrared water bands were still not completely eliminated). Investigators have suggested that the near infrared offers important advantages for the study of water binding to polymersS, 6. It is possible that such limitations in sampling techniques may partially account for the relatively meager use of the near infrared for this purpose. Consequently, an attempt was made to develop a sampling technique in which these limitations would be minimized. A special cell was constructed which could be opened in order to cast a particular number of ml/cm 2 of a given polymer solution on its inside optical surface, or to remove all or a portion of its water by use of a vacuum oven at 55 °C. It could be tightly closed for near infrared scanning or storage. By weighing the cell when empty, when it contained a protein film with a particular amount of bound water, and after the film was dried, the g of protein and of water per cm 2 of optical surface could be calculated for any particular scan. MATERIALS AND METHODS
Preparation of cells Polycarbonate centrifuge tubes with screw caps (Sargent-Welch no. S-I78848o-R, 2.59 cm inside diameter) and Coming microscope slides were used for construction of the cells. The centrifuge tubes were machine-cut into two portions, a few mm beneath the bottom of the screwed caps. The bottom portions were discarded, and the cut surface of each upper portion (containing the cap) was cemented on to a microscope slide with an epoxy adhesive. The top, flat portion of each screw cap was then cut out, leaving an open circle about 2.1 cm in diameter. A microscope slide, cut to about 3 cm square, was then cemented over each circular opening. The cell
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SIDE VII~W
Fig. I. Cell for p r o t e i n - w a t e r b i n d i n g spectra. I t is s h o w n on t h e right, as it positions a n enclosed p r o t e i n film p e r p e n d i c u l a r to s p e c t r o p h o t o m e t e r light b e a m . W h e n t h e p r o t e i n solution w a s initially p i p e t t e d into t h e cell, t h e w i n d o w e d cap h a d been r e m o v e d , a n d t h e microscope slide (on t h e left) w a s in a h o r i z o n t a l position.
g2
N. RESSLER, ZIAUDDIX
(Fig. i) thus consisted of a portion of a centrifuge tube which could be screwed opened, or shut so that it is airtight (verifiable by the constancy of the weight with water inside). The inside air space should not be too great, in order to aw~id significant deviations due to an equilibrimn between the film and the inside air. The two optical ends consist of microscope slide glass, which is low alkali, striation-free, and ttat. The glass did not exhibit any gross peaks between I and 2.8 #m, and the baseline, with e m p t y cells in both reference and sample positions, was indistinghishable from t h a t obtained with air vs air. The cells were positioned with each microscope slide surface perpendicular to the sample or reference beam ill a fixed manner, so t h a t cells could be removed and put back in the same position (the cells can be "fixed" against (:ell c o m p a r t m e n t structures with slits and attached clamps, or magnets, and with appropriate stops). Interference fringe effects were not observed when the cells were t(roperly aligned. Procedure The use of these cells can be illustrated with studies of water binding by gelatin, performed with a Beckman D K - 2 A spectrophotometer. A n u m b e r of cells were weighed and then placed upon a level surface with tile tops removed (in order to minimize any increase in weight due to accumulation of material on the outside, they were protected with dust covers during standing, and were never handled with bare hands). I ml of a IO(~) aqueous solution of gelatin (Fisher Scientific Co., no. G-7), dissolved by heating, was pipetted while still warm into each cell with a Cornwall syringe type pipet and stainless steel plunger, hmnediately after the addition, each cell was p r o m p t l y closed and left undisturbed at room temperature for a lbw hours or more. This permitted the solutions to form gels firm enough for handling (a nonabsorbing, gelling matrix might be incorporated into tile solutions for polymers which do not gel; see below). The accuracy of the pipetting was then monitored by a subsequent weighing and any cells which contained protein solutions which differed b y more t h a n o.2 mg were discarded. The cells were then dehydrated to various degrees, ranging from tile m i n i m u m necessary for absorbance readings to completely dry. This was done by initially removing water gradually with a slight vacuum at room temperature, and finally keeping them in a v a c u u m oven at 55 ~C for appropriate periods of time. In each case, the temperature was reduced to room teinpcrature before tile v a c u u m was removed. The cells were then proinptly closed, weighed and scanned at temperatures close to e 3 °C The same cell could, of course be scanned, then dehydrated and weighed further in successive steps. Each cell was eventually weighed and scanned after being dried completely, in order to verify t h a t the amount of gelatin in all of t h e m was equivalent on the bases of both near infrared scans and of weight (i.e:. the weight of the ('ell with completely dried gelatin mi,zus tile weight of the e m p t y celll. RESULTS AND I)ISCUSSION
The near infrared spectrum of proteins have been discussed by Ellis and B a t h ~ and subsequently by Hecht and W o o d ~. The type of scans which can be obtained by the procedure described here are illustrated in Fig. z. A cell containing gelatin with bound water can be read against an e m p t y cell, against a cell with gelatin containing
33
NEAR INFRARED STUDY OF WATER BINDING
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Fig. 2. I l l u s t r a t i o n of the t y p e of scans obtained b y the procedure described. The top 3 curves are absolute spectra (read against an e m p t y cell in the reference beam). T h e y all h a v e 18.4 mg/cm2 of gelatin, b u t differ in % of w a t e r (i.e., g of w a t e r per g of gelatin plus w a t e r × ioo), as indicated. The b o t t o m 2 curves are w a t e r difference spectra, between gelatin films with different a m o u n t s of water, or between gelatin with w a t e r vs d r y gelatin. The curves h a v e been separated in the vertical direction for p u r p o s e s of presentation, b u t each one approaches lOO% T (transmission) at low wavelengths. The ordinate scale expansion is O-lOO% T for the t o p 3 curves, and 75I 2 5 % T for the b o t t o m 2.
a different amount of water or against one with dry gelatin. Since the cells differ in the amount of water but not in amount of gelatin, the latter two difference curves represent water difference spectra. Spectra of cells containing gelatin with various amounts of bound water, read against a cell with the same amount of gelatin but completely dry, are presented in Fig. 3. In each case, after the gelatin with bound water had been scanned, it was subsequently dried and re-scanned against the same dried, reference film. A straight line, indistinguishable from an air v s air baseline, was consistently obtained, which confirms that each of the bands is due to the presence of water. The curves in Fig. 3 are samples taken from about 50 scans at various water levels. In all instances the consistencies between the water-difference peaks and the water content would mitigate against artifacts as a significant factor in the results. The most intense bands consist of the familiar water peaks near 1.45 # m (6897 cm -1) and 1.95 ffm (5128 cm-1), and one in the 2.5 ffm region (4000 cm-1). As the water level decreases, the presence of additional peaks on the red side of the 1.45 and 1.95 ffm peaks become resolved. These include rather broad bands near 1.6, 1.8, 2.1 and 2.25 # m (6250, 5556, 4762, and 4444 cm-1, respectively). When the water level increases sufficiently to obscure the resolution of adjacent bands, the apparent inclusion of the 1.6 or 2.1 ffm peaks into, respectively, the 1.45 or 1.95 ffm envelopes could relate to the a s y m m e t r y of the latter (in reference to the 1.45 and 1.6 ,urn bands, for example, compare the scans at 28.2 and 2.9% water). Apparent shifts in the frequency of certain bands, with variation in water content, might also be caused by the asymmetric overlapping of adjacent bands at
34
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27
Fig. 3. Water difference spectra (gelatin with bound water v s dry gelatin) at various water levels. In each case, the amount of gelatin was I8. 4 mg/cm ~. The }[ of water and the ordinate scale expansion are given on the left. The curves have been separated vertically, for purposes of presentation. The bottom, dashed curve is an absolute spectrum of dry gelatin, given for comparison.
higher water levels. For e x a m p l e , the apparent red shift, w i t h increasing water content, of the b a n d near 2.5 # m is p r o b a b l y due to an a s y m m e t r i c overlapping w i t h the shoulder on the blue side of the water peak around 2.86 # m . A n y o f the water-difference bands m a y , o f course, be due to an alteration in the absorption o f protein groups caused b y interactions w i t h the water, as well as b y the absorption of water itself. The possibility could be considered, for e x a m p l e , t h a t the b a n d s near 1.6 or 2.5 # m are due to alterations, respectively, in the absorption o f the N - H b a n d near 1.5 # m or the C - H b a n d around 2.52 ffm 4'8. The peaks w h i c h develop from the red side of the 1.45 # m b a n d w i t h decreasing
NEAR INFRARED STUDY OF WATER BINDING
35
water content (1.6 and 1.8 #m) m a y represent more firmly bound forms of water than that at 1.45 pm, since their intensities, relative to that of the 1.45/~m band, increase with less water (compare, for example, the curves at water contents of 11.6 and 2.9%). A band near 1.67/~m in water has been attributed to a hydrogen bonded form of OH 9. A protein-bound water band near the 1.79 # m has been discussed by Ellis and Bath 7. The band in the 2.5/zm region would also appear to be associated with a more firmly bound form of water, since it is still detected in gelatin with as little water as o.3~o. 2.5 # m has generally been an upper limit for near infrared protein-water binding studies, and a lower wavelength limit for mid infrared studies, and few water 'difference curves which extend across this wavelength region have been published. A band at 2.5 # m has been described in ice 1° and as an "association" band in water ~. As far as we are aware, bands near 1.6, 2.1, 2.25, and 2.5/zm in protein-water difference curves have not previously been reported. One of the limitations of the method as described is its restriction to proteins or other polymers which form a clear gel or film. The possibility is being examined of adding a non-absorbing, gelling matrix to the protein solution, so that other nongelling proteins can be studied in this manner. The results will be reported in another communication. The use of the procedure does, of course, require certain precautions appropriate for quantitative spectrophotometric and gravimetric operations; i.e. precise and reproducible positioning of the cells perpendicular to the spectrophotometer's light beams, protection of the cells from dust or other contacts which could change their weights, etc. The tubes must be sufficiently air tight so that any changes in weight between the conclusions of the removal of all or part of the water and the spectral scans are insufficient to alter the results. In order to obtain films which are undistorted and of uniform thickness, it is important to pipet the completely dissolved polymer solution into accurately leveled cells, and subsequently to protect these solutions from any physical disturbance until a firm gel has been formed. Careful handling m a y still be necessary until a certain amount of the water has been removed. By observing these precautions, significant errors due to non-uniformity of the films can be avoided. Criteria for the absence of such errors m a y include plots of the absorbance of water peaks vs water content, or the linearity of the scan when cells with the same protein and water content are in both the sample and reference compartments. (The uniformity of the films can also be studied by reducing the area covered by the light beam of the spectrophotometer with masking tape, and scanning different portions of the optical surface of the cell and of the film, one at a time. The beam can be temporarily visualized at 546 nm, but the cell must be perpendicular to the beam in all cases). A 55 °C temperature, combined with a vacuum, for 24 h is sufficient to remove all near infrared water bands and studies at various temperatures indicated that the spectra of gelatin were not altered at 55 °C. This temperature is only necessary for complete drying and, in the case of more labile proteins, a vacuum at room temperature or less could be used for the spectra of partially dehydrated samples. The method does not, of course, involve any solvent exclusion error, or organic solvents or other substances which could alter the protein structure. I t is hoped that the versatility in the amount of protein and water which can be used, and the ability to independently determine the g/cm 2 of each with an accuracy limited primarily by weighing, m a y be useful for water binding or other near infrared investigations.
36
y. RI,2SSLER, ZIAUDDIN
T h e m e t h o d w o u l d a p p e a r t o be a d a p t a b l e t o s t u d i e s o f n e a r i n f r a r e d d i e h r o i s m , H - 2 H e x c h a n g e , v a r i a t i o n s in t e m p e r a t u r e , o r o t h e r a u x i l i a r y p r o c e d u r e s . REFERENCES I 2 3 4 5 6 7 8 9 IO ii
Hanlon, S. and Klotz, 1. M. (1968) Dev. Appl. Specif. O, 2i 9 235 Burneau, A. and Corset, J. (197 I) .]. Chem. Phys. 56, 662-663 McCabe, W. C., and Fisher, H. F. (~97o) J. Phys. Chem. 74, 299o 2998 Susi, H., Ard, J. S. and Carroll, R. J. (197I) Biopolymers lO, 1597-16o4 Chirgadze, Yu. N., Venyaminov, S. Yu and Zimont, S. L. (1969) in Water in Biological Systems, (Kayushin, L. P., ed.) pp. 51-53, Plenum Press, New York Hecht, K. T. and Wood, D. L. (1956) Proc. R. Soc. Ser. A, 235, 174 183 Ellis, J. w . and Bath, J. (1938 ) J. Chem. Phys. 6, 723-729 Bradbury, E. M., Burgc, R. E.. Randall, J. T. and Wilkinson, G. R. (1958) Far, Soc. Disc. 25, I73-~85 Worley, J. 1). and Klotz, A. M. ([906) J. Chem. Phys. 45, 2868-z871 Haas, C. and Hornig, 1). F. (196o) J. Chem. Phys. 32, 1763 I769 Williams, 1). (1966) Nature 21o, 194 195
BBA 36305 AN IMPROVED T E C H N I Q U E FOR T H E N E A R I N b ' R A R E D STUI)Y ()V W A T E R B I N D I N G BY P R O T E I N S I L L U S T R A T I O N W I T H GELATIN
N. I),ESSLER AND Z I A U D D I N
Departments of Pathology and Biochemistry, University oJ illinois Medical Ce~ter, Chicago, Ill. 6o6~2 (U.S.A.) (Received J u l y 2oth, 1972)
SUMMARY
A description is presented of cells designed to minimize certain limitations of sampling methods generally used for near infrared studies of water binding by proteins or other polymers. The g per cm 2 of both protein and of bound water in the optical path can be varied over a considerable range, and the amount of each can be determined independently by appropriate weighings. When the method was applied to a film consisting of I8. 4 mg of gelatin per cm 2, bands due to the presence of bound water were exhibited near 1.45, 1.6, 1.8, 1.95, 2.1, 2.25, and 2.5 # m (6897, 6250, 5556, 5128, 4762, 4444, and 4000 cm -1, respectively). Comparisons to previously reported results, the dependence of the resolution of these bands upon the percent of water present, and the possible forms of water which some of the bands may represent, are discussed.
INTRODUCTION
The near infrared region of the spectrum includes combination and overtone bands, various low energy charge transfer bands, etc. 1. Numerous studies of hydrogen bonds 2, and of the structure of water a have been advantageously conducted in this range. The near infrared has also been used, to a limited extent, for the investigation of water binding to proteins or nucleic acids, but the large majority of infrared water binding studies have been in the fundamental or mid infrared region (the latter is commonly taken as 2.5 5o #m or 4o0o-2oo cm 1). The infrared investigation of water binding to proteins has usually involved the casting of an aqueous solution of the protein into a thin film. The latter can then be scanned while exposed to various atmospheres, which differ in particular relative humidities 4. In order to apply this procedure to the near, rather than to the mid infrared region, films of considerably greater thickness are necessary for optimal spectral
31
NEAR INFRARED STUDY OF WATER BINDING
measurements, since the absorbances are correspondingly less in the near infrared. As the thickness of the film is increased, however, the relation between the amount of water bound to the polymer and the relative humidity becomes more indirect 5, and the actual water content becomes more uncertain (when a nearly dry film containing 16 mg of gelatin per cm * was kept in a vacuum of about o.I mTorr Hg for 16 h at room temperature, for example, infrared water bands were still not completely eliminated). Investigators have suggested that the near infrared offers important advantages for the study of water binding to polymersS, 6. It is possible that such limitations in sampling techniques may partially account for the relatively meager use of the near infrared for this purpose. Consequently, an attempt was made to develop a sampling technique in which these limitations would be minimized. A special cell was constructed which could be opened in order to cast a particular number of ml/cm 2 of a given polymer solution on its inside optical surface, or to remove all or a portion of its water by use of a vacuum oven at 55 °C. It could be tightly closed for near infrared scanning or storage. By weighing the cell when empty, when it contained a protein film with a particular amount of bound water, and after the film was dried, the g of protein and of water per cm 2 of optical surface could be calculated for any particular scan. MATERIALS AND METHODS
Preparation of cells Polycarbonate centrifuge tubes with screw caps (Sargent-Welch no. S-I78848o-R, 2.59 cm inside diameter) and Coming microscope slides were used for construction of the cells. The centrifuge tubes were machine-cut into two portions, a few mm beneath the bottom of the screwed caps. The bottom portions were discarded, and the cut surface of each upper portion (containing the cap) was cemented on to a microscope slide with an epoxy adhesive. The top, flat portion of each screw cap was then cut out, leaving an open circle about 2.1 cm in diameter. A microscope slide, cut to about 3 cm square, was then cemented over each circular opening. The cell
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SIDE VII~W
Fig. I. Cell for p r o t e i n - w a t e r b i n d i n g spectra. I t is s h o w n on t h e right, as it positions a n enclosed p r o t e i n film p e r p e n d i c u l a r to s p e c t r o p h o t o m e t e r light b e a m . W h e n t h e p r o t e i n solution w a s initially p i p e t t e d into t h e cell, t h e w i n d o w e d cap h a d been r e m o v e d , a n d t h e microscope slide (on t h e left) w a s in a h o r i z o n t a l position.
g2
N. RESSLER, ZIAUDDIX
(Fig. i) thus consisted of a portion of a centrifuge tube which could be screwed opened, or shut so that it is airtight (verifiable by the constancy of the weight with water inside). The inside air space should not be too great, in order to aw~id significant deviations due to an equilibrimn between the film and the inside air. The two optical ends consist of microscope slide glass, which is low alkali, striation-free, and ttat. The glass did not exhibit any gross peaks between I and 2.8 #m, and the baseline, with e m p t y cells in both reference and sample positions, was indistinghishable from t h a t obtained with air vs air. The cells were positioned with each microscope slide surface perpendicular to the sample or reference beam ill a fixed manner, so t h a t cells could be removed and put back in the same position (the cells can be "fixed" against (:ell c o m p a r t m e n t structures with slits and attached clamps, or magnets, and with appropriate stops). Interference fringe effects were not observed when the cells were t(roperly aligned. Procedure The use of these cells can be illustrated with studies of water binding by gelatin, performed with a Beckman D K - 2 A spectrophotometer. A n u m b e r of cells were weighed and then placed upon a level surface with tile tops removed (in order to minimize any increase in weight due to accumulation of material on the outside, they were protected with dust covers during standing, and were never handled with bare hands). I ml of a IO(~) aqueous solution of gelatin (Fisher Scientific Co., no. G-7), dissolved by heating, was pipetted while still warm into each cell with a Cornwall syringe type pipet and stainless steel plunger, hmnediately after the addition, each cell was p r o m p t l y closed and left undisturbed at room temperature for a lbw hours or more. This permitted the solutions to form gels firm enough for handling (a nonabsorbing, gelling matrix might be incorporated into tile solutions for polymers which do not gel; see below). The accuracy of the pipetting was then monitored by a subsequent weighing and any cells which contained protein solutions which differed b y more t h a n o.2 mg were discarded. The cells were then dehydrated to various degrees, ranging from tile m i n i m u m necessary for absorbance readings to completely dry. This was done by initially removing water gradually with a slight vacuum at room temperature, and finally keeping them in a v a c u u m oven at 55 ~C for appropriate periods of time. In each case, the temperature was reduced to room teinpcrature before tile v a c u u m was removed. The cells were then proinptly closed, weighed and scanned at temperatures close to e 3 °C The same cell could, of course be scanned, then dehydrated and weighed further in successive steps. Each cell was eventually weighed and scanned after being dried completely, in order to verify t h a t the amount of gelatin in all of t h e m was equivalent on the bases of both near infrared scans and of weight (i.e:. the weight of the ('ell with completely dried gelatin mi,zus tile weight of the e m p t y celll. RESULTS AND I)ISCUSSION
The near infrared spectrum of proteins have been discussed by Ellis and B a t h ~ and subsequently by Hecht and W o o d ~. The type of scans which can be obtained by the procedure described here are illustrated in Fig. z. A cell containing gelatin with bound water can be read against an e m p t y cell, against a cell with gelatin containing
33
NEAR INFRARED STUDY OF WATER BINDING
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t
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_o
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i
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~ i
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i
~
i
- -
i
i
Wave Lengih, microns
>
Fig. 2. I l l u s t r a t i o n of the t y p e of scans obtained b y the procedure described. The top 3 curves are absolute spectra (read against an e m p t y cell in the reference beam). T h e y all h a v e 18.4 mg/cm2 of gelatin, b u t differ in % of w a t e r (i.e., g of w a t e r per g of gelatin plus w a t e r × ioo), as indicated. The b o t t o m 2 curves are w a t e r difference spectra, between gelatin films with different a m o u n t s of water, or between gelatin with w a t e r vs d r y gelatin. The curves h a v e been separated in the vertical direction for p u r p o s e s of presentation, b u t each one approaches lOO% T (transmission) at low wavelengths. The ordinate scale expansion is O-lOO% T for the t o p 3 curves, and 75I 2 5 % T for the b o t t o m 2.
a different amount of water or against one with dry gelatin. Since the cells differ in the amount of water but not in amount of gelatin, the latter two difference curves represent water difference spectra. Spectra of cells containing gelatin with various amounts of bound water, read against a cell with the same amount of gelatin but completely dry, are presented in Fig. 3. In each case, after the gelatin with bound water had been scanned, it was subsequently dried and re-scanned against the same dried, reference film. A straight line, indistinguishable from an air v s air baseline, was consistently obtained, which confirms that each of the bands is due to the presence of water. The curves in Fig. 3 are samples taken from about 50 scans at various water levels. In all instances the consistencies between the water-difference peaks and the water content would mitigate against artifacts as a significant factor in the results. The most intense bands consist of the familiar water peaks near 1.45 # m (6897 cm -1) and 1.95 ffm (5128 cm-1), and one in the 2.5 ffm region (4000 cm-1). As the water level decreases, the presence of additional peaks on the red side of the 1.45 and 1.95 ffm peaks become resolved. These include rather broad bands near 1.6, 1.8, 2.1 and 2.25 # m (6250, 5556, 4762, and 4444 cm-1, respectively). When the water level increases sufficiently to obscure the resolution of adjacent bands, the apparent inclusion of the 1.6 or 2.1 ffm peaks into, respectively, the 1.45 or 1.95 ffm envelopes could relate to the a s y m m e t r y of the latter (in reference to the 1.45 and 1.6 ,urn bands, for example, compare the scans at 28.2 and 2.9% water). Apparent shifts in the frequency of certain bands, with variation in water content, might also be caused by the asymmetric overlapping of adjacent bands at
34
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27
Fig. 3. Water difference spectra (gelatin with bound water v s dry gelatin) at various water levels. In each case, the amount of gelatin was I8. 4 mg/cm ~. The }[ of water and the ordinate scale expansion are given on the left. The curves have been separated vertically, for purposes of presentation. The bottom, dashed curve is an absolute spectrum of dry gelatin, given for comparison.
higher water levels. For e x a m p l e , the apparent red shift, w i t h increasing water content, of the b a n d near 2.5 # m is p r o b a b l y due to an a s y m m e t r i c overlapping w i t h the shoulder on the blue side of the water peak around 2.86 # m . A n y o f the water-difference bands m a y , o f course, be due to an alteration in the absorption o f protein groups caused b y interactions w i t h the water, as well as b y the absorption of water itself. The possibility could be considered, for e x a m p l e , t h a t the b a n d s near 1.6 or 2.5 # m are due to alterations, respectively, in the absorption o f the N - H b a n d near 1.5 # m or the C - H b a n d around 2.52 ffm 4'8. The peaks w h i c h develop from the red side of the 1.45 # m b a n d w i t h decreasing
NEAR INFRARED STUDY OF WATER BINDING
35
water content (1.6 and 1.8 #m) m a y represent more firmly bound forms of water than that at 1.45 pm, since their intensities, relative to that of the 1.45/~m band, increase with less water (compare, for example, the curves at water contents of 11.6 and 2.9%). A band near 1.67/~m in water has been attributed to a hydrogen bonded form of OH 9. A protein-bound water band near the 1.79 # m has been discussed by Ellis and Bath 7. The band in the 2.5/zm region would also appear to be associated with a more firmly bound form of water, since it is still detected in gelatin with as little water as o.3~o. 2.5 # m has generally been an upper limit for near infrared protein-water binding studies, and a lower wavelength limit for mid infrared studies, and few water 'difference curves which extend across this wavelength region have been published. A band at 2.5 # m has been described in ice 1° and as an "association" band in water ~. As far as we are aware, bands near 1.6, 2.1, 2.25, and 2.5/zm in protein-water difference curves have not previously been reported. One of the limitations of the method as described is its restriction to proteins or other polymers which form a clear gel or film. The possibility is being examined of adding a non-absorbing, gelling matrix to the protein solution, so that other nongelling proteins can be studied in this manner. The results will be reported in another communication. The use of the procedure does, of course, require certain precautions appropriate for quantitative spectrophotometric and gravimetric operations; i.e. precise and reproducible positioning of the cells perpendicular to the spectrophotometer's light beams, protection of the cells from dust or other contacts which could change their weights, etc. The tubes must be sufficiently air tight so that any changes in weight between the conclusions of the removal of all or part of the water and the spectral scans are insufficient to alter the results. In order to obtain films which are undistorted and of uniform thickness, it is important to pipet the completely dissolved polymer solution into accurately leveled cells, and subsequently to protect these solutions from any physical disturbance until a firm gel has been formed. Careful handling m a y still be necessary until a certain amount of the water has been removed. By observing these precautions, significant errors due to non-uniformity of the films can be avoided. Criteria for the absence of such errors m a y include plots of the absorbance of water peaks vs water content, or the linearity of the scan when cells with the same protein and water content are in both the sample and reference compartments. (The uniformity of the films can also be studied by reducing the area covered by the light beam of the spectrophotometer with masking tape, and scanning different portions of the optical surface of the cell and of the film, one at a time. The beam can be temporarily visualized at 546 nm, but the cell must be perpendicular to the beam in all cases). A 55 °C temperature, combined with a vacuum, for 24 h is sufficient to remove all near infrared water bands and studies at various temperatures indicated that the spectra of gelatin were not altered at 55 °C. This temperature is only necessary for complete drying and, in the case of more labile proteins, a vacuum at room temperature or less could be used for the spectra of partially dehydrated samples. The method does not, of course, involve any solvent exclusion error, or organic solvents or other substances which could alter the protein structure. I t is hoped that the versatility in the amount of protein and water which can be used, and the ability to independently determine the g/cm 2 of each with an accuracy limited primarily by weighing, m a y be useful for water binding or other near infrared investigations.
36
y. RI,2SSLER, ZIAUDDIN
T h e m e t h o d w o u l d a p p e a r t o be a d a p t a b l e t o s t u d i e s o f n e a r i n f r a r e d d i e h r o i s m , H - 2 H e x c h a n g e , v a r i a t i o n s in t e m p e r a t u r e , o r o t h e r a u x i l i a r y p r o c e d u r e s . REFERENCES I 2 3 4 5 6 7 8 9 IO ii
Hanlon, S. and Klotz, 1. M. (1968) Dev. Appl. Specif. O, 2i 9 235 Burneau, A. and Corset, J. (197 I) .]. Chem. Phys. 56, 662-663 McCabe, W. C., and Fisher, H. F. (~97o) J. Phys. Chem. 74, 299o 2998 Susi, H., Ard, J. S. and Carroll, R. J. (197I) Biopolymers lO, 1597-16o4 Chirgadze, Yu. N., Venyaminov, S. Yu and Zimont, S. L. (1969) in Water in Biological Systems, (Kayushin, L. P., ed.) pp. 51-53, Plenum Press, New York Hecht, K. T. and Wood, D. L. (1956) Proc. R. Soc. Ser. A, 235, 174 183 Ellis, J. w . and Bath, J. (1938 ) J. Chem. Phys. 6, 723-729 Bradbury, E. M., Burgc, R. E.. Randall, J. T. and Wilkinson, G. R. (1958) Far, Soc. Disc. 25, I73-~85 Worley, J. 1). and Klotz, A. M. ([906) J. Chem. Phys. 45, 2868-z871 Haas, C. and Hornig, 1). F. (196o) J. Chem. Phys. 32, 1763 I769 Williams, 1). (1966) Nature 21o, 194 195