CRYOBIOLOGY
12, 2633
Fluorimetric
(1975)
Evidence of Interactions Involving and Biomolecules M. J. RUWART,
J. I?. HOLLAND,
AND
Cryoprotectants
A. HAUG
of Biophysics, Department of Biochemistry, and MSU/AEC Plant Research Laboratory, Michigan State Unbersity, East Lansing, Michigan 48824
Department
A class of compounds known as cryo- than would be expected based upon the protectants minimizes freeze-thaw damage sum of the individual cryoprotectant effects in biological systems such as enzymes (2)) ( 4, 5). Other workers have found that cerplateIets (S), erythrocytes ( 14), bacteria tain cryoprotectants act in an antagonistic 1, S), spermatozoa ( 17), skin ( 20), and manner (6, ‘7). Such studies indicate that heart (13). cryoprotectants might interact with each other in a manner which alters their efFecMany of these protective compounds stabilize and/or activate enzymes (18) and tiveness. Since there are few instrumental techniques available with sufficient senprevent thermal denaturation of heat-sensitive proteins (16) at temperatures above sitivity to detect noncovalent interactions in aqueous solutions, and since these cryothe freezing point of the aqueous solutions. Cryoprotectants also prevent injury to sen- protectants exhibit complex behavior, elucisitive celIular components exposed to rela- dation of their mode of action has been extively high concentrations of salt, urea, or ceedingly difficult. Therefore, it is the purpose of the following investigation to report guanidine hydrochloride ( 12, 2)) minimizon experiments regarding cryoprotectant ing deleterious effects of injurious environments upon the structure and function of interaction. A computer centered spectrofluorimeter (10, 11) will be employed. This biological materials. The mechanism by which these com- instrument is capable of measuring the pounds exert their protective in%uence is partial quantum efficiency (PQ). The PQ unknown. The ability of cryoprotectants to value of a %uorophore in the presence of alter solvent structure and ordered water of two compounds can only be a linear extraplabile materials have been cited as a pos- olation of the PQ values determined when the fluorophore is exposed to each comsible stabilization mechanism ( 6). Organic ponent separately, unless interaction occurs molecules with cryoprotectant properties between the two compounds, This principle have excellent hydrogen-bonding capabilities, but their direct interaction or absorp- has been applied to direct noncovalent intion to sensitive biological molecules has teractions between cryoprotectants. not been observed, MATERIALS Addition of two cryoprotectants to a sysTyrosine, tryptophan, lysozyme, and tem undergoing freeze-thaw stress has polyvinylpyrrolidone ( average MW 40,000) sometimes resulted in better preservation were purchased from Sigma ChemicaI Co., Received April 19, 1974. St. Louis, Missouri. Bovine serum albumin 26 Copyright 1975 by Academic Press, Inc. All rights o8 reproduction in any form reserved.
INTERACTIONS
INVOLVING
CRYOPROTECTANTS
was obtained from Armour Chemical Co., Chicago, Illinois. Dextran (av, 86,000 MW) was purchased from K and K Laboratories, Plainview, New York. Ultrapure sucrose was a product of Schwartz-Mann, Orangeburg, New York. Glucose was obtained from Fisher Scientific, Fair Law, New Jersey. Xylose was purchased from Nutritional Biochemicals Corporation, Cleveland, Ohio. Glycerol, analytical grade, was a product of Mallinckrodt, St. Louis, Missouri, Spectroscopic grade dimethyl sulfoxide was purchased from AIdrich Chemical Co., Milwaukee, Wisconsin. Twenty-one-day-old red bIood cells were donated by the American Bed Cross Block Bank, Lansing, Michigan.
AND BIOMOLECULES
27
been normalized to PQ of tryptophan in water at 277 nm ( PQZT7), Excitation scans were taken as described (10). Data in Figs, 3 and 418were collected during operation of a time dependent fluorescence program. The fl uorophore was mixed with 2 ml of a cryoprotectant solution and additions of appropriate solutes were made at timed intervals. Throughout this procedure, sampling of absorbance and ffuorescence occurred at fixed excitation and emission wavelengths. The pH was observed to be independent of sohte concentration. Bed blood cells suspended in a mixture of physiological saline and appropriate cryoprotectants were frozen in &l-ml aliquots in Pyrex capiIIary tubes (0.3 cm i.d. x 10 cm). The samples were frozen by METHODS immersion in liquid nitrogen (SOO”C/min). Aqueous stock solutions of bovine serum Thawing was accomplished by allowing the albumin or tryptophan were prepared daily sample to remain at ambient temperature at 0°C. Buffered solutions (0.01 M phos- for 20 min (4O”Cjmin). phate) gave similar spectra, but results with aqueous solutions were preferred to RESULTS AND DISCUSSION minimize the number of species present. Fluorescence of Bovine Serum Albumin in Aliquots from these solutions were mixed Water with cryoprotectants. All spectra were determined at 7°C. Fluorescence emission Typical absorbance and partial quantum was monitored at 340 nm for bovine serum efficiency spectra of bovine serum albumin albumin and 350 nm for tryptophan. in water are shown in Fig. 1A. The excitaPartial quantum efficiency ( PQ) , cor- tion spectra of this prot.ein were monitored rected fluorescence ( F,,), and absorbance at its emission maximum of 340 nm. The (A) spectra were measured as described by protein has three absorbing and fluorescing Holland et al. (10, 11). These parameters species: phenylalanine, tyrosine, and tryptoare defined as follows. (a) F,, is fluores- phan. Since phenyIaIanine does not absorb cence corrected for the instrumental and light at a wavelength greater than 280 nm photophysical variables of the measuring (19), and does not fluoresce at an emission system including the absorption artifact: wavelength of 340 nm (22)) it will not con(b) PQ is a linear relation of the number tribute to the fluorescence excitation specof quanta fluoresced by the sample per trum of bovine serum albumin above 280 number of quanta absorbed by the sample nm. Below this wavelength, however, the and is presented as a function of the ex- absorbance of phenylalanine results in a citation wavelength. Partial quantum effi- decrease in relative PQ as compared to the ciency is directly proportional to total higher wavelengths where tryptophan and quantum yieId, and therefore independent tyrosine are the only absorbing species. of fluorophore concentration, Unless other- Tyrosine accounts for approximateIy 68% wise noted, all values of PQ and F,, have of bovine serum albumin absorbance at
RUWART,
28
260
230
300
260
WAVELENGTH
280
HOLLAND
300
(nm]
FIG. 1. A. Absorption and PQ spectra of bovine serum albumin in water, Emission was measured at 340 mn. PQ is normalized to PQm of tryptophan in HaO. B. Absorption and PQ spectra of lysozyme in water. Emission was measured at 340 nm. PQ is normalized to PQw of tryptophan in water. C. Absorption and PQ spectra of tryptophan in water, Emission was measured at 350 nm. PQ is normalized to P& of tryptophan in water. D. Absorption and PQ spectra of a solution of 0.08 M tryptophan and 0.24 M tryosine. Emission was measured at 350 nm.
280 nm with its maximum fluorescence emission at 305 nm. However, Teale (21) has shown that the small contribution of tyrosine to fluorescence at an emission wavelength of 340 nm is effectively quenched. Therefore, tyrosine and tryptophan are the onIy amino acids in bovine serum albumin that contribute to absorption in the region of interest (excitation wavelength of 270-320 nm ), while onIy tryptophan contributes to the fluorescence at an emission of 340 nm. If tryptophan were the only absorbing species present in bovine serum albumin, the partial quantum efficiency spectrum would be independent of excitation wavelength ( 10). Since tyrosine absorbance above 280 nm decreases more rapidly than that of tryptophan, an increase is observed in the PQ of bovine serum albumin between 280 and 300 nm (Fig. IA). A PQ spectrum similar to that of bovine serum
AND HAU’G
albumin in this region can be produced by mixing tyrosine and tryptophan in water in appropriate proportions (Fig. ID). The molar ratio of tyrosine to tryptophan needed to generat.e this PQ spectrum differs from the molar ratio of the two amino acids as found in bovine serum albumin, a situation to be expected since tryptophan may exist in differing environments in the macromolecule, In lysozyme, tryotophan accounts for over 90% of the absorbance at 277 nm ( 21), and then the minimal decrease in tyrosine absorbance does not result in a PQ peak at 293 nm, Indeed, the excitation spectrum of lysozyme (Fig. 13) is very similar to that of tryptophan in water (Fig. 1C). These data indicate that PQsB3 is probably the actual PQ of tryptophan in bovine serum albumin, whereas PQzT7 is a constant fraction thereof due to the absorbance of tryosine. Since PQsT7 is located at the center of the linear portion of the PQ spectrum, and is more accurately measured, the results presented in this communication have been reported as PQa77. Changes in PQ of Bovine Serum Albumin Due to Den&wants, Cryoprotectanh, and Combinations thereof High concentrations of urea (2-8 M) or guanidine hydrochloride (2-5 M) reduce bovine serum albumin fluorescence as much as 50% as shown in Fig, 2. These attenuations were found to produce no change in the general shape of the spectra. The ORD (9) and fluorescence data shown in Fig. 2 follow the same general trends although each method measures a different property of the protein. Since a lowered PQ or quantum yield suggests a less rigid orbital environment (23)) or a greater orbital interaction in the vicinity of the fluorophore, and since a lowered optical rotation suggests a relaxation of protein structure (3), the results obtained by both methods are consistent with the interpretation that
INTERACTIONS 1 -40
’
0
-
l
-20 -
, A
/
/ /
-lo-
’ _
/ . t’
:=15,$, 1;; _
1
f’
/.’
-SO -
a
1
l/ /
0’0
0
1
I’
-10 ” 0
‘,’
CRYOPROTECTANTS
//
/
-30 -
PQ2,,
INVOLVING
Y /*
’ 2466
UREA
’
..
l; i
’ 2
I 4
I 6
GLJANIOINE
I 6
HCI
MOLAR CONCENTRATION OF DENATURANT FIG. 2. Comparison of fluorimetric and optical rotary dispersion data. PQ is expressed as percent change in quantum yield relative to PQ of bovine serum albumin in water. [“I379 is expressed as change in optical rotation relative to the rotation of bovine serum albumin in 0.05 M Tris buffer, pH 7.6. Broken lines indicate general trends only.
denaturants cause an unfolding of the bovine serum albumin. Figure 3A illustrates the differences in PQ of bovine serum albumin in urea aIone ( l ), dimethy sulfoxide alone ( A ), and urea plus dimethyl sulfoxide in combination ( X ). The PQ of bovine serum albumin remains constant for dimethyl sulfoxide concentrations less than 4 M ( A ) . With no other solute present, the PQ of bovine serum albumin is enhanced as the urea concentration diminishes ( l ). It is obvious that the PQ obtained from the action of the two solutes in concert does not appear to be a linear extrapolation ( x ) of their individual effects. This is an example of an inclusive interdependent interaction with the fluorophore. Inclusive interdependent interactions are defined as those wherein the PQ of the fluorophore is affected by both solutes in a manner which is not the simple sum of the individual solute effects. As dimethyl sulfoxide is added to bovine serum albumin in the presence of high concentrations of urea (Fig. 3A), the protein’s PQ returns to the native species indicating that the interaction between urea and the fluorophore rnay have been ahered in a quantitative manner by the presence of
AND
BIOMOLECULES
29
increasing amounts of dimethyl suIfoxide. These results may be correlated to the observation of mutual antagonism found between dimethyl sulfoxide and urea in biological systems (6, 7). DimethyI sulfoxide, when used alone in the concentration region (O-4 M), where it is observed to have only a shght effect upon PQ, is an excellent cryoprotectant. A similar behavior is shown by urea alone in the range from (O-2 M ). The mechanism of the anatogistic action of these compounds is not clearly understood (6-8). Table I lists the relative changes in the PQ of bovine serum albumin as a result of cryoprotectant addition, In every case, the 1.25
I DEXTRAN ’ DMSO.. DEXTRA.N
DMSOOO 41 00
08 3.3 27
1.7 3.0
2.6 2.4
Q Q
5.2
6.3
Q
FIG. 3. A. X--P& of bovine serum albumin in urea and dimethyl suIfoxide mixtures in the molar ratios (DMSO/urea) marked on the abcissa. The molarity of dimethyl sulfoxide and urea in these mixtures are listed under A and l , respectively. A--PQ~TI of bovine serum albumin as a function of dimethyl sulfoxide molarity corresponding to the values on the abcissa. l --PQm of bovine serum albumin as a function of urea molarity corresponding to the values on the abcissa. B. PQm of bovine serum albumin in: X-mixtures of d&ran and dimethyl sulfoxide (dextran/DMSO X I@). A-dimethyl sulfoxide; l -dextran (X lo-‘) as in Fig. 3A.
RUWART,
30 TABLE
AND
HAVG
1
PQe,, OF BOVINE
SERUM ALBUMIN CRYOPROTECTANTS”
VARIOUS
HOLLAND
Cr~oprDtectant
IN
PChb ..-
_-
1.00
None
6.0% dextran 10.Oc/O dimethyl 5.0% glucose 5.oyo sucrose 5.of70 xylose 10.Oyo glycerol II~-.~-_-
1.37 0.88 0.95 0.86 0.86 0.00
sulfovide
.-
a Emission monitored at 340 nm. ShiftR in t.he emission maximum were not of sufficient magnitude to produce significant changes in PQzT,. in water. b Normalized to PQ2,, of tryptophan
general shape of the PQ spectrum remains unaltered. The low molecular weight cryoprotectants slightly depress the fluorescence of the albumin, whereas dextran (86,000 MW) increases PQ. During this study, it was observed that the low molecular weight cryoprotectants appear to stabilize a particular conformation of the protein over a wide range of cryoprotectant concentration. This effect is especiaIly pronounced with dimethyl sulfoxide (0.5-4.0 M) one of the most effective cryoprotectants known. When the concentration of dimethyl sulfoxide is very high (> 4.5 M), however, the PQ of bovine serum albumin is further diminTABLE I’$z,Y
2
OF TRYPTOPHAN IK VARIOUS CRYOPROTECTANTS~ ~.._. Cryoprotectsnt PQd .-
None 5.0yo ZO.Ooj, 5.0yo 6.0% 8.6% 10.0% 5.0%
glucose dimethgl glycerol dextran lactose sucrme xgose
stdfoxide
I .oo 0.96 1.15 1 .Ol 1.24 1.22 1.05 1.01
0 Emission wa.~ monitored at 350 nm. The solutiona tested did not shift the emission maximum enough to produce significant changes in PQ,,,. bE’&~ is normalized t’o PQpTi of tryptophan in water.
FIG. 4. A. X-PQB~ of tryptophan in glucose and dimethyl sulfoxide mixtures in the molar ratios (glucoseJDMS0) marked on the abcissa. The molarity of DMSO and glucose in these mixtures are listed under A and l , respectively. A-PQPI of tryptophan in 3.8 M dimethyl sulfoxide. aPQm of tryptophan in glucose as a function of molarity a indicated on the abcissa. B. PQm, of tryptophan in: X-mixtures of dimethyl sulfoxide and urea (urea/DMSO) A-&methyl sulfoxide; in: e-urea as in Fig. 4A. C. PQw of tryptophan X-mixtures of dextran and dimethyl sulfoxide ( DMSO/dextran X 10s) A-dimethyl sulfoxide; e-O.7 M X lo-” dextran as in Fig. 4A.
ished, suggesting that at high molarities, a different mechanism of interaction predominates. It is noteworthy that such high levels of dimethyl sulfoxide have been seldom, if ever, used in successful cryopreservation. The results of adding dextran ( a ) and dimethyl sulfoxide ( A ) individually and in combination ( x ) to bovine serum albumin are shown in Fig. 3B. The PQ of bovine serum albumin in the presence of both cryoprotectants is dependent solely on dextran concentration, except where the
INTERACTIONS
INVOLVING
CRYOPROTECTANTS
AND
BIOMOLECULES
31
dextran concentration is very low. These results indicate that when bovine serum albumin is exposed to dextran and dimethyl sulfoxide in combination the PQ of the ffuorophore does not reflect the presence of dimethyl sulfoxide except at extremely low dextran concentrations. This is an example of excZusivs interaction with the fluorophore. Exclusive interactions are dejined as MOLE FRACTION those whereby in a mixture of solutes, tIze DMSO PQ of the fluorophore responds as if on& FIG, 5. Fluorescence of tryptophan in urea and one solute toere present. The exclusive be- dimethyl sulfoxide as a function of the mole frachavior of the dextran with bovine serum tion of dimethyl sulfoxide. Dimethyl sulfoxide conalbumin in the presence of dimethyl sul- centration was increased as urea concentration was decreased, so that the total molarity of the mixture foxide is exhibited in all concentrations was always 6,. The solid lines indicate thePOrn greater than an approximate one-to-one expected in the presence of either compound dextran-bovine serum albumin ratio. alone; the dotted Iine indicates the PQSV expected in the mixture of the two compounds if no interThese results suggest that cryoprotection of large biological molecules by high mo- action occurs. The line formed by the circles indicates the experimentally determined PQm, lecular weight compounds might occur through the formation of a complex inert varied. In the absence of dimethyl sulfto other influences, as previously snggested oxide, the PQ of tryptophan is depressed (15). Upon removal of the cryoprotectant by increasing amounts of glucose ( l ) ; in through dilution, the complex dissociates, the presence of both solutes, however, the releasing the native protein. PQ is enhanced with further addition of glucose ( x ). This suggests that dimethy Efects of Various Cyoprotectants on sulfoxide may alter the association between Tryptophan the glucose and the tryptophan so that only Since the major fluorophore being obthe effects of solvent structuring are felt by served in bovine abumin studies is tryptothe fluorophore. This is an example of an phan, the effects of the cryoprotectants inclusive interdependent interaction with acting directly on this amino acid were in- the fluorophore. Similar interactions have vestigated to assist in interpreting its fluobeen observed when these two cryoprotecrescence behavior in the macromolecule. tants are combined to protect simple cells All compounds tested with the exception from freeze-thaw stress. Rat plateIets surof glucose raised the partial quantum effi- vive poorly ( l-.25% ) when frozen in either ciency of tryptophan (Table 2). These in- 5% dimethyl sulfoxide or 5% glucose, but creases in PQ are interpreted as increases TABLE 3 in the structure of the surrounding solvent. SIJRVIVAL OF RED Br,oon Cs~r.s AFTER FREEZING The effect of glucose on the PQ of tryptoAND THAWING (THE FREEZING RAW IS phan indicates the possibility of an intimate 500"C~'M~r~; THE THAWING RATE interaction between the two molecules reIS 409C:Mrs) sulting in m increase in radiationless processes. Evidence supporting this interpretation is presented in Fig. 4A. The con6.0% dextran 25 centration of dimethyl sulfoxide (A ) in 12.5% dimet,hyl sulfoxide 50 this experiment was kept constant while 6.0% dextran + 12.5y, dimethyl sulfoxide 74 the concentration of glucose ( l ) was
32
RUWART,
HOLLAND
AND HAUG
are better protected (‘IO-90%) by the two effects on proteins. Hence, the presence of effect on the cryoprotectants in concert (5). The pro- an inclusive Interdependent tozoa Entamoeba inoadens shows little sur- fluorophore does not necessarily predict a vival in 0.24 M glucose (0% ) or 2.1 M favorable or unfavorable cryoprotectant dimethyl sulfoxide (25% ) but retains its joint action, but it does indicate that an viability much better (457% ) when in the interaction is occurring which can alter the action of the combination. presence of both cryoprotectants (4). The cryoprotectant action of urea and dimethyl sulfoxide on The significant point is, in the limited bovine serum albumin (Fig. 3A) can also studies thus far, combinations of agents be termed in&&us and interdependent. that have shown inclusive, interdependent Urea and dimethyl sulfoxide also show effects on the fluorophore have invariably exhibited anomalous cryoprotectant action inclusive interdependent effects on tryptophan alone (Fig. 4B). Increasing urea con- when used in concert. On the other hand, the action of dextran centrations ( l ) cause increases in PQ of and dimethyl s&oxide on the fluorophore tryptophan. Decreases in dimethyl sulfoxide concentrations cause decreases in tryptophan appears to be the simple sum of the individual solutes ( Fig. 4C). The dexPQ ( A ). The PQ of tryptophan in mixtures of these compounds is invariably higher tran concentration was kept constant ( m ), than the sum of the individual solute effects while the dimethyl sulfoxide concentration ( x ). This interaction, as indicated by PQ, was varied ( A ). These cryoprotectants in between urea and dimethyl sulfoxide has combination resulted in a PQ of tryptophan been observed to be greatest when these which was the sum of the individual effects ( x ). Inclusive and independent inleracsolutes are in a one-to-one ratio (Fig. 5). While the inclusive interdependent in- tions are defined as those wherein the PQ of the jluorophore is affected by both teractions of glucose and dimethyl sulfoxide solutes in a manner which is the simple produced a complimentary cryoprotectant solute effects. The effect, interactions observed between di- sum of the individual methyl sulfoxide and urea reflect the an- survival of red blood cells in the presence of dimethyl sulfoxide and dextran also aptagonistic behavior of these two compounds pears to approximate the sum of the indiwhen used in combination to influence vidual effects (Table 3) for the concentrafreeze-thaw survival, Erythrocyte protections studied. These results indicate that tion by dimethyl sulfoxide was adversely affected when urea was added in equal or cryoprotectants which exert in.&siue indegreater concentrations (7). Thus, the critipendent effects on tryptophan will to a first act in an additive manner cal ratio of urea to hydrogen-binding sites approximation, appears to be one-to-one. This is also the when used in concert to protect simple cells ratio at which the greatest fluorimetric in- against freeze-thaw damage. Although free teraction is observed between the two com- tryptophan was used in these studies, any fluorophore which responds primarily to pounds (Fig. 5). changes in solvent structure should give Thus, compounds which exhibit ilaclusiue interdependent interactions appear to affect analogous results. cryoprotection of simple cells in a manner In summary, the proper cryoprotectants which is not the simple sum of their indifor preservation of a biological system will vidual effects. Whether the combination depend upon the effects they have on molecules crucial to the cell’s survival. Comwill be more or less conducive to freezethaw survival cannot be determined from binations of cryoprotectants could interact their effect on tryptophan fluorescence, but with each other or with the crucial molemight be inferred from their individual cules simultaneously to produce effects
INTERACTIONS
INVOLVING
CRYOPROTEXTANTS
other than those which would occur with individual use (inclusive and inttwdependent}. One cryoprotectant of the combination might dominate the other (exclusiue) or both might act to produce an additive effect (incZusive and independent). Apparent correlations can be drawn between these interactions and the effects of cryoprotectants on simple bioIogical systems subjected to freeze-thaw stress, Continuing physicochemical investigations of this type may eventually aid in elucidation of CVOprotectant mechanism. ACKNOWLEDGMENT This work was supported by the U.S. Atomic Energy Commission Contract No. AT-( ll-I)-1338. M. J. Ruwart was supported by the U.S. Public Health Service Training Grant No. GM-01422-06 from the National Institutes of Health. REFERENCES I. Ashwood-Smith, M. J., and Warby, C. A species of Pseudomonlu; a most useful bacteria far cryobiological studies. Cryobiology 8,208-210 (1971). 2. Chilson, 0. P., Costello, L. A., and Kaplan, N. 0. Effect of freezing on enzymes. Fed. Proc. 24, s-55-65 ( 196.5). 3. Cohen, C. Optical rotation and polypeptide configuration in proteins. Nature (London) 175, 129-130 ( 1955). 4. Diamond, L. W. Freeze-preservation of protozoa. Cr&ioEogy 1,95-102 ( 1964). 5. Djerassi, I., and Roy, A. A method for preservation of viable platelets: Combined effects of sugars and dimethylsulfoxide. Blood 22, 703-717 ( 1963). 6. Doebbler, G. F’., and Rinfret, A. P. Influence of protective compounds and cooling and warming conditions on hemolysis of erythrocytes by freezing and thawing. &o&m. Bbphys. Actu 58, 449-458 ( 19621, ‘7. Doebbler, G. F., and Rinfret, A. P. Rapid freezing of human blood. Cryobiology 1, 205-211 (1965). 8. Doebbler, G. I?., Rowe, A. W., and Rinfret, A. P. Freezing of mammalian bIood and its
9.
10.
II, 12.
13.
14.
15. 16. 17.
18.
19.
20.
21.
22.
23.
AND
BIOMOLECULES
33
constituents. In “Cryobiology” ( H. Meryman, Ed.) pp. 407-440. Academic Press, New York, 1906. Gordon, J. A., and Jencks, W. P. The relationship of structure to the effectiveness of denaturing reagents for proteins. Biochmisty g47-57 ( 1963) * Holland, J. F., Teets, R. E., and Timnick, A. A unique computer-centered instrument for simultaneous absorbance and fluorescence measurements. AnraE. &em. 45, 145-153 (1973). Holland, J. F., Teets, R. E., and Timnick, A. Annal. Chem. submitted (1974). Lovelock, J. E. The haemolysis of red blood cells by freezing and thawing. Bbchim. Biophys. Acta 10, 414-426 (1953). Luyet, B. A review of research in the preservation of hearts in the frozen state. Cryobiology 8, 190-207 ( 1971), Luyet, B., and Rapatz, G. A review of basic researches on the cryopreservation of red blood cells. Cryobiology 6, 425-482 ( 1970). Meryman, H. T. “Cryobiology,” pp+ 83-64, Academic Press, New York, 1966. M&tone, H. Purification of thrombin. 1. Clin. F’hysiol. 25, 679-687 ( 1942). Pologe, C., and Soltys, M. A. In “Research in Freezing and Drying” (A. S. Farkes and A. U. Smith, Eds.) pp. 87-100. Blackwell, London, 1960. Ruwart, M. J., and Suelter, C. H.: Activation of yeast pyruvate kinase by natural and artificial cryoprotectants. 1. Biol. Chem. 246,5990-5993 (1971). Sober, II. A. In “Handbook of Biochemistry,” p. B76, ChemicaI Rubber Co., CIeveland, 1970. Taylor, A. C., and Gerstner, R. Tissue survival after exposure to low temperatures and the effectiveness of protective pretreatments. J. Cell. Camp. Physiol. 46, 477-502 (1955). Teale, F. W. J. The ultraviolet fluorescence of proteins in neutral soIution. Biochem J. 76, 381388 (1960). Teale, F. W. J., and Weber, G. The ultraviolet fluorescence of aromatic amino acids. Biochem. J. 65,476482 ( 1957). Wehry, E. L., and Rogers, L. B. In “Fluoressence and Phosphorescence” (D. M. Hercules, Ed.) pp. 113-142, Interscience, New York, 1970.
12, 2633
Fluorimetric
(1975)
Evidence of Interactions Involving and Biomolecules M. J. RUWART,
J. I?. HOLLAND,
AND
Cryoprotectants
A. HAUG
of Biophysics, Department of Biochemistry, and MSU/AEC Plant Research Laboratory, Michigan State Unbersity, East Lansing, Michigan 48824
Department
A class of compounds known as cryo- than would be expected based upon the protectants minimizes freeze-thaw damage sum of the individual cryoprotectant effects in biological systems such as enzymes (2)) ( 4, 5). Other workers have found that cerplateIets (S), erythrocytes ( 14), bacteria tain cryoprotectants act in an antagonistic 1, S), spermatozoa ( 17), skin ( 20), and manner (6, ‘7). Such studies indicate that heart (13). cryoprotectants might interact with each other in a manner which alters their efFecMany of these protective compounds stabilize and/or activate enzymes (18) and tiveness. Since there are few instrumental techniques available with sufficient senprevent thermal denaturation of heat-sensitive proteins (16) at temperatures above sitivity to detect noncovalent interactions in aqueous solutions, and since these cryothe freezing point of the aqueous solutions. Cryoprotectants also prevent injury to sen- protectants exhibit complex behavior, elucisitive celIular components exposed to rela- dation of their mode of action has been extively high concentrations of salt, urea, or ceedingly difficult. Therefore, it is the purpose of the following investigation to report guanidine hydrochloride ( 12, 2)) minimizon experiments regarding cryoprotectant ing deleterious effects of injurious environments upon the structure and function of interaction. A computer centered spectrofluorimeter (10, 11) will be employed. This biological materials. The mechanism by which these com- instrument is capable of measuring the pounds exert their protective in%uence is partial quantum efficiency (PQ). The PQ unknown. The ability of cryoprotectants to value of a %uorophore in the presence of alter solvent structure and ordered water of two compounds can only be a linear extraplabile materials have been cited as a pos- olation of the PQ values determined when the fluorophore is exposed to each comsible stabilization mechanism ( 6). Organic ponent separately, unless interaction occurs molecules with cryoprotectant properties between the two compounds, This principle have excellent hydrogen-bonding capabilities, but their direct interaction or absorp- has been applied to direct noncovalent intion to sensitive biological molecules has teractions between cryoprotectants. not been observed, MATERIALS Addition of two cryoprotectants to a sysTyrosine, tryptophan, lysozyme, and tem undergoing freeze-thaw stress has polyvinylpyrrolidone ( average MW 40,000) sometimes resulted in better preservation were purchased from Sigma ChemicaI Co., Received April 19, 1974. St. Louis, Missouri. Bovine serum albumin 26 Copyright 1975 by Academic Press, Inc. All rights o8 reproduction in any form reserved.
INTERACTIONS
INVOLVING
CRYOPROTECTANTS
was obtained from Armour Chemical Co., Chicago, Illinois. Dextran (av, 86,000 MW) was purchased from K and K Laboratories, Plainview, New York. Ultrapure sucrose was a product of Schwartz-Mann, Orangeburg, New York. Glucose was obtained from Fisher Scientific, Fair Law, New Jersey. Xylose was purchased from Nutritional Biochemicals Corporation, Cleveland, Ohio. Glycerol, analytical grade, was a product of Mallinckrodt, St. Louis, Missouri, Spectroscopic grade dimethyl sulfoxide was purchased from AIdrich Chemical Co., Milwaukee, Wisconsin. Twenty-one-day-old red bIood cells were donated by the American Bed Cross Block Bank, Lansing, Michigan.
AND BIOMOLECULES
27
been normalized to PQ of tryptophan in water at 277 nm ( PQZT7), Excitation scans were taken as described (10). Data in Figs, 3 and 418were collected during operation of a time dependent fluorescence program. The fl uorophore was mixed with 2 ml of a cryoprotectant solution and additions of appropriate solutes were made at timed intervals. Throughout this procedure, sampling of absorbance and ffuorescence occurred at fixed excitation and emission wavelengths. The pH was observed to be independent of sohte concentration. Bed blood cells suspended in a mixture of physiological saline and appropriate cryoprotectants were frozen in &l-ml aliquots in Pyrex capiIIary tubes (0.3 cm i.d. x 10 cm). The samples were frozen by METHODS immersion in liquid nitrogen (SOO”C/min). Aqueous stock solutions of bovine serum Thawing was accomplished by allowing the albumin or tryptophan were prepared daily sample to remain at ambient temperature at 0°C. Buffered solutions (0.01 M phos- for 20 min (4O”Cjmin). phate) gave similar spectra, but results with aqueous solutions were preferred to RESULTS AND DISCUSSION minimize the number of species present. Fluorescence of Bovine Serum Albumin in Aliquots from these solutions were mixed Water with cryoprotectants. All spectra were determined at 7°C. Fluorescence emission Typical absorbance and partial quantum was monitored at 340 nm for bovine serum efficiency spectra of bovine serum albumin albumin and 350 nm for tryptophan. in water are shown in Fig. 1A. The excitaPartial quantum efficiency ( PQ) , cor- tion spectra of this prot.ein were monitored rected fluorescence ( F,,), and absorbance at its emission maximum of 340 nm. The (A) spectra were measured as described by protein has three absorbing and fluorescing Holland et al. (10, 11). These parameters species: phenylalanine, tyrosine, and tryptoare defined as follows. (a) F,, is fluores- phan. Since phenyIaIanine does not absorb cence corrected for the instrumental and light at a wavelength greater than 280 nm photophysical variables of the measuring (19), and does not fluoresce at an emission system including the absorption artifact: wavelength of 340 nm (22)) it will not con(b) PQ is a linear relation of the number tribute to the fluorescence excitation specof quanta fluoresced by the sample per trum of bovine serum albumin above 280 number of quanta absorbed by the sample nm. Below this wavelength, however, the and is presented as a function of the ex- absorbance of phenylalanine results in a citation wavelength. Partial quantum effi- decrease in relative PQ as compared to the ciency is directly proportional to total higher wavelengths where tryptophan and quantum yieId, and therefore independent tyrosine are the only absorbing species. of fluorophore concentration, Unless other- Tyrosine accounts for approximateIy 68% wise noted, all values of PQ and F,, have of bovine serum albumin absorbance at
RUWART,
28
260
230
300
260
WAVELENGTH
280
HOLLAND
300
(nm]
FIG. 1. A. Absorption and PQ spectra of bovine serum albumin in water, Emission was measured at 340 mn. PQ is normalized to PQm of tryptophan in HaO. B. Absorption and PQ spectra of lysozyme in water. Emission was measured at 340 nm. PQ is normalized to PQw of tryptophan in water. C. Absorption and PQ spectra of tryptophan in water, Emission was measured at 350 nm. PQ is normalized to P& of tryptophan in water. D. Absorption and PQ spectra of a solution of 0.08 M tryptophan and 0.24 M tryosine. Emission was measured at 350 nm.
280 nm with its maximum fluorescence emission at 305 nm. However, Teale (21) has shown that the small contribution of tyrosine to fluorescence at an emission wavelength of 340 nm is effectively quenched. Therefore, tyrosine and tryptophan are the onIy amino acids in bovine serum albumin that contribute to absorption in the region of interest (excitation wavelength of 270-320 nm ), while onIy tryptophan contributes to the fluorescence at an emission of 340 nm. If tryptophan were the only absorbing species present in bovine serum albumin, the partial quantum efficiency spectrum would be independent of excitation wavelength ( 10). Since tyrosine absorbance above 280 nm decreases more rapidly than that of tryptophan, an increase is observed in the PQ of bovine serum albumin between 280 and 300 nm (Fig. IA). A PQ spectrum similar to that of bovine serum
AND HAU’G
albumin in this region can be produced by mixing tyrosine and tryptophan in water in appropriate proportions (Fig. ID). The molar ratio of tyrosine to tryptophan needed to generat.e this PQ spectrum differs from the molar ratio of the two amino acids as found in bovine serum albumin, a situation to be expected since tryptophan may exist in differing environments in the macromolecule, In lysozyme, tryotophan accounts for over 90% of the absorbance at 277 nm ( 21), and then the minimal decrease in tyrosine absorbance does not result in a PQ peak at 293 nm, Indeed, the excitation spectrum of lysozyme (Fig. 13) is very similar to that of tryptophan in water (Fig. 1C). These data indicate that PQsB3 is probably the actual PQ of tryptophan in bovine serum albumin, whereas PQzT7 is a constant fraction thereof due to the absorbance of tryosine. Since PQsT7 is located at the center of the linear portion of the PQ spectrum, and is more accurately measured, the results presented in this communication have been reported as PQa77. Changes in PQ of Bovine Serum Albumin Due to Den&wants, Cryoprotectanh, and Combinations thereof High concentrations of urea (2-8 M) or guanidine hydrochloride (2-5 M) reduce bovine serum albumin fluorescence as much as 50% as shown in Fig, 2. These attenuations were found to produce no change in the general shape of the spectra. The ORD (9) and fluorescence data shown in Fig. 2 follow the same general trends although each method measures a different property of the protein. Since a lowered PQ or quantum yield suggests a less rigid orbital environment (23)) or a greater orbital interaction in the vicinity of the fluorophore, and since a lowered optical rotation suggests a relaxation of protein structure (3), the results obtained by both methods are consistent with the interpretation that
INTERACTIONS 1 -40
’
0
-
l
-20 -
, A
/
/ /
-lo-
’ _
/ . t’
:=15,$, 1;; _
1
f’
/.’
-SO -
a
1
l/ /
0’0
0
1
I’
-10 ” 0
‘,’
CRYOPROTECTANTS
//
/
-30 -
PQ2,,
INVOLVING
Y /*
’ 2466
UREA
’
..
l; i
’ 2
I 4
I 6
GLJANIOINE
I 6
HCI
MOLAR CONCENTRATION OF DENATURANT FIG. 2. Comparison of fluorimetric and optical rotary dispersion data. PQ is expressed as percent change in quantum yield relative to PQ of bovine serum albumin in water. [“I379 is expressed as change in optical rotation relative to the rotation of bovine serum albumin in 0.05 M Tris buffer, pH 7.6. Broken lines indicate general trends only.
denaturants cause an unfolding of the bovine serum albumin. Figure 3A illustrates the differences in PQ of bovine serum albumin in urea aIone ( l ), dimethy sulfoxide alone ( A ), and urea plus dimethyl sulfoxide in combination ( X ). The PQ of bovine serum albumin remains constant for dimethyl sulfoxide concentrations less than 4 M ( A ) . With no other solute present, the PQ of bovine serum albumin is enhanced as the urea concentration diminishes ( l ). It is obvious that the PQ obtained from the action of the two solutes in concert does not appear to be a linear extrapolation ( x ) of their individual effects. This is an example of an inclusive interdependent interaction with the fluorophore. Inclusive interdependent interactions are defined as those wherein the PQ of the fluorophore is affected by both solutes in a manner which is not the simple sum of the individual solute effects. As dimethyl sulfoxide is added to bovine serum albumin in the presence of high concentrations of urea (Fig. 3A), the protein’s PQ returns to the native species indicating that the interaction between urea and the fluorophore rnay have been ahered in a quantitative manner by the presence of
AND
BIOMOLECULES
29
increasing amounts of dimethyl suIfoxide. These results may be correlated to the observation of mutual antagonism found between dimethyl sulfoxide and urea in biological systems (6, 7). DimethyI sulfoxide, when used alone in the concentration region (O-4 M), where it is observed to have only a shght effect upon PQ, is an excellent cryoprotectant. A similar behavior is shown by urea alone in the range from (O-2 M ). The mechanism of the anatogistic action of these compounds is not clearly understood (6-8). Table I lists the relative changes in the PQ of bovine serum albumin as a result of cryoprotectant addition, In every case, the 1.25
I DEXTRAN ’ DMSO.. DEXTRA.N
DMSOOO 41 00
08 3.3 27
1.7 3.0
2.6 2.4
Q Q
5.2
6.3
Q
FIG. 3. A. X--P& of bovine serum albumin in urea and dimethyl suIfoxide mixtures in the molar ratios (DMSO/urea) marked on the abcissa. The molarity of dimethyl sulfoxide and urea in these mixtures are listed under A and l , respectively. A--PQ~TI of bovine serum albumin as a function of dimethyl sulfoxide molarity corresponding to the values on the abcissa. l --PQm of bovine serum albumin as a function of urea molarity corresponding to the values on the abcissa. B. PQm of bovine serum albumin in: X-mixtures of d&ran and dimethyl sulfoxide (dextran/DMSO X I@). A-dimethyl sulfoxide; l -dextran (X lo-‘) as in Fig. 3A.
RUWART,
30 TABLE
AND
HAVG
1
PQe,, OF BOVINE
SERUM ALBUMIN CRYOPROTECTANTS”
VARIOUS
HOLLAND
Cr~oprDtectant
IN
PChb ..-
_-
1.00
None
6.0% dextran 10.Oc/O dimethyl 5.0% glucose 5.oyo sucrose 5.of70 xylose 10.Oyo glycerol II~-.~-_-
1.37 0.88 0.95 0.86 0.86 0.00
sulfovide
.-
a Emission monitored at 340 nm. ShiftR in t.he emission maximum were not of sufficient magnitude to produce significant changes in PQzT,. in water. b Normalized to PQ2,, of tryptophan
general shape of the PQ spectrum remains unaltered. The low molecular weight cryoprotectants slightly depress the fluorescence of the albumin, whereas dextran (86,000 MW) increases PQ. During this study, it was observed that the low molecular weight cryoprotectants appear to stabilize a particular conformation of the protein over a wide range of cryoprotectant concentration. This effect is especiaIly pronounced with dimethyl sulfoxide (0.5-4.0 M) one of the most effective cryoprotectants known. When the concentration of dimethyl sulfoxide is very high (> 4.5 M), however, the PQ of bovine serum albumin is further diminTABLE I’$z,Y
2
OF TRYPTOPHAN IK VARIOUS CRYOPROTECTANTS~ ~.._. Cryoprotectsnt PQd .-
None 5.0yo ZO.Ooj, 5.0yo 6.0% 8.6% 10.0% 5.0%
glucose dimethgl glycerol dextran lactose sucrme xgose
stdfoxide
I .oo 0.96 1.15 1 .Ol 1.24 1.22 1.05 1.01
0 Emission wa.~ monitored at 350 nm. The solutiona tested did not shift the emission maximum enough to produce significant changes in PQ,,,. bE’&~ is normalized t’o PQpTi of tryptophan in water.
FIG. 4. A. X-PQB~ of tryptophan in glucose and dimethyl sulfoxide mixtures in the molar ratios (glucoseJDMS0) marked on the abcissa. The molarity of DMSO and glucose in these mixtures are listed under A and l , respectively. A-PQPI of tryptophan in 3.8 M dimethyl sulfoxide. aPQm of tryptophan in glucose as a function of molarity a indicated on the abcissa. B. PQm, of tryptophan in: X-mixtures of dimethyl sulfoxide and urea (urea/DMSO) A-&methyl sulfoxide; in: e-urea as in Fig. 4A. C. PQw of tryptophan X-mixtures of dextran and dimethyl sulfoxide ( DMSO/dextran X 10s) A-dimethyl sulfoxide; e-O.7 M X lo-” dextran as in Fig. 4A.
ished, suggesting that at high molarities, a different mechanism of interaction predominates. It is noteworthy that such high levels of dimethyl sulfoxide have been seldom, if ever, used in successful cryopreservation. The results of adding dextran ( a ) and dimethyl sulfoxide ( A ) individually and in combination ( x ) to bovine serum albumin are shown in Fig. 3B. The PQ of bovine serum albumin in the presence of both cryoprotectants is dependent solely on dextran concentration, except where the
INTERACTIONS
INVOLVING
CRYOPROTECTANTS
AND
BIOMOLECULES
31
dextran concentration is very low. These results indicate that when bovine serum albumin is exposed to dextran and dimethyl sulfoxide in combination the PQ of the ffuorophore does not reflect the presence of dimethyl sulfoxide except at extremely low dextran concentrations. This is an example of excZusivs interaction with the fluorophore. Exclusive interactions are dejined as MOLE FRACTION those whereby in a mixture of solutes, tIze DMSO PQ of the fluorophore responds as if on& FIG, 5. Fluorescence of tryptophan in urea and one solute toere present. The exclusive be- dimethyl sulfoxide as a function of the mole frachavior of the dextran with bovine serum tion of dimethyl sulfoxide. Dimethyl sulfoxide conalbumin in the presence of dimethyl sul- centration was increased as urea concentration was decreased, so that the total molarity of the mixture foxide is exhibited in all concentrations was always 6,. The solid lines indicate thePOrn greater than an approximate one-to-one expected in the presence of either compound dextran-bovine serum albumin ratio. alone; the dotted Iine indicates the PQSV expected in the mixture of the two compounds if no interThese results suggest that cryoprotection of large biological molecules by high mo- action occurs. The line formed by the circles indicates the experimentally determined PQm, lecular weight compounds might occur through the formation of a complex inert varied. In the absence of dimethyl sulfto other influences, as previously snggested oxide, the PQ of tryptophan is depressed (15). Upon removal of the cryoprotectant by increasing amounts of glucose ( l ) ; in through dilution, the complex dissociates, the presence of both solutes, however, the releasing the native protein. PQ is enhanced with further addition of glucose ( x ). This suggests that dimethy Efects of Various Cyoprotectants on sulfoxide may alter the association between Tryptophan the glucose and the tryptophan so that only Since the major fluorophore being obthe effects of solvent structuring are felt by served in bovine abumin studies is tryptothe fluorophore. This is an example of an phan, the effects of the cryoprotectants inclusive interdependent interaction with acting directly on this amino acid were in- the fluorophore. Similar interactions have vestigated to assist in interpreting its fluobeen observed when these two cryoprotecrescence behavior in the macromolecule. tants are combined to protect simple cells All compounds tested with the exception from freeze-thaw stress. Rat plateIets surof glucose raised the partial quantum effi- vive poorly ( l-.25% ) when frozen in either ciency of tryptophan (Table 2). These in- 5% dimethyl sulfoxide or 5% glucose, but creases in PQ are interpreted as increases TABLE 3 in the structure of the surrounding solvent. SIJRVIVAL OF RED Br,oon Cs~r.s AFTER FREEZING The effect of glucose on the PQ of tryptoAND THAWING (THE FREEZING RAW IS phan indicates the possibility of an intimate 500"C~'M~r~; THE THAWING RATE interaction between the two molecules reIS 409C:Mrs) sulting in m increase in radiationless processes. Evidence supporting this interpretation is presented in Fig. 4A. The con6.0% dextran 25 centration of dimethyl sulfoxide (A ) in 12.5% dimet,hyl sulfoxide 50 this experiment was kept constant while 6.0% dextran + 12.5y, dimethyl sulfoxide 74 the concentration of glucose ( l ) was
32
RUWART,
HOLLAND
AND HAUG
are better protected (‘IO-90%) by the two effects on proteins. Hence, the presence of effect on the cryoprotectants in concert (5). The pro- an inclusive Interdependent tozoa Entamoeba inoadens shows little sur- fluorophore does not necessarily predict a vival in 0.24 M glucose (0% ) or 2.1 M favorable or unfavorable cryoprotectant dimethyl sulfoxide (25% ) but retains its joint action, but it does indicate that an viability much better (457% ) when in the interaction is occurring which can alter the action of the combination. presence of both cryoprotectants (4). The cryoprotectant action of urea and dimethyl sulfoxide on The significant point is, in the limited bovine serum albumin (Fig. 3A) can also studies thus far, combinations of agents be termed in&&us and interdependent. that have shown inclusive, interdependent Urea and dimethyl sulfoxide also show effects on the fluorophore have invariably exhibited anomalous cryoprotectant action inclusive interdependent effects on tryptophan alone (Fig. 4B). Increasing urea con- when used in concert. On the other hand, the action of dextran centrations ( l ) cause increases in PQ of and dimethyl s&oxide on the fluorophore tryptophan. Decreases in dimethyl sulfoxide concentrations cause decreases in tryptophan appears to be the simple sum of the individual solutes ( Fig. 4C). The dexPQ ( A ). The PQ of tryptophan in mixtures of these compounds is invariably higher tran concentration was kept constant ( m ), than the sum of the individual solute effects while the dimethyl sulfoxide concentration ( x ). This interaction, as indicated by PQ, was varied ( A ). These cryoprotectants in between urea and dimethyl sulfoxide has combination resulted in a PQ of tryptophan been observed to be greatest when these which was the sum of the individual effects ( x ). Inclusive and independent inleracsolutes are in a one-to-one ratio (Fig. 5). While the inclusive interdependent in- tions are defined as those wherein the PQ of the jluorophore is affected by both teractions of glucose and dimethyl sulfoxide solutes in a manner which is the simple produced a complimentary cryoprotectant solute effects. The effect, interactions observed between di- sum of the individual methyl sulfoxide and urea reflect the an- survival of red blood cells in the presence of dimethyl sulfoxide and dextran also aptagonistic behavior of these two compounds pears to approximate the sum of the indiwhen used in combination to influence vidual effects (Table 3) for the concentrafreeze-thaw survival, Erythrocyte protections studied. These results indicate that tion by dimethyl sulfoxide was adversely affected when urea was added in equal or cryoprotectants which exert in.&siue indegreater concentrations (7). Thus, the critipendent effects on tryptophan will to a first act in an additive manner cal ratio of urea to hydrogen-binding sites approximation, appears to be one-to-one. This is also the when used in concert to protect simple cells ratio at which the greatest fluorimetric in- against freeze-thaw damage. Although free teraction is observed between the two com- tryptophan was used in these studies, any fluorophore which responds primarily to pounds (Fig. 5). changes in solvent structure should give Thus, compounds which exhibit ilaclusiue interdependent interactions appear to affect analogous results. cryoprotection of simple cells in a manner In summary, the proper cryoprotectants which is not the simple sum of their indifor preservation of a biological system will vidual effects. Whether the combination depend upon the effects they have on molecules crucial to the cell’s survival. Comwill be more or less conducive to freezethaw survival cannot be determined from binations of cryoprotectants could interact their effect on tryptophan fluorescence, but with each other or with the crucial molemight be inferred from their individual cules simultaneously to produce effects
INTERACTIONS
INVOLVING
CRYOPROTEXTANTS
other than those which would occur with individual use (inclusive and inttwdependent}. One cryoprotectant of the combination might dominate the other (exclusiue) or both might act to produce an additive effect (incZusive and independent). Apparent correlations can be drawn between these interactions and the effects of cryoprotectants on simple bioIogical systems subjected to freeze-thaw stress, Continuing physicochemical investigations of this type may eventually aid in elucidation of CVOprotectant mechanism. ACKNOWLEDGMENT This work was supported by the U.S. Atomic Energy Commission Contract No. AT-( ll-I)-1338. M. J. Ruwart was supported by the U.S. Public Health Service Training Grant No. GM-01422-06 from the National Institutes of Health. REFERENCES I. Ashwood-Smith, M. J., and Warby, C. A species of Pseudomonlu; a most useful bacteria far cryobiological studies. Cryobiology 8,208-210 (1971). 2. Chilson, 0. P., Costello, L. A., and Kaplan, N. 0. Effect of freezing on enzymes. Fed. Proc. 24, s-55-65 ( 196.5). 3. Cohen, C. Optical rotation and polypeptide configuration in proteins. Nature (London) 175, 129-130 ( 1955). 4. Diamond, L. W. Freeze-preservation of protozoa. Cr&ioEogy 1,95-102 ( 1964). 5. Djerassi, I., and Roy, A. A method for preservation of viable platelets: Combined effects of sugars and dimethylsulfoxide. Blood 22, 703-717 ( 1963). 6. Doebbler, G. F’., and Rinfret, A. P. Influence of protective compounds and cooling and warming conditions on hemolysis of erythrocytes by freezing and thawing. &o&m. Bbphys. Actu 58, 449-458 ( 19621, ‘7. Doebbler, G. F., and Rinfret, A. P. Rapid freezing of human blood. Cryobiology 1, 205-211 (1965). 8. Doebbler, G. I?., Rowe, A. W., and Rinfret, A. P. Freezing of mammalian bIood and its
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10.
II, 12.
13.
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15. 16. 17.
18.
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21.
22.
23.
AND
BIOMOLECULES
33
constituents. In “Cryobiology” ( H. Meryman, Ed.) pp. 407-440. Academic Press, New York, 1906. Gordon, J. A., and Jencks, W. P. The relationship of structure to the effectiveness of denaturing reagents for proteins. Biochmisty g47-57 ( 1963) * Holland, J. F., Teets, R. E., and Timnick, A. A unique computer-centered instrument for simultaneous absorbance and fluorescence measurements. AnraE. &em. 45, 145-153 (1973). Holland, J. F., Teets, R. E., and Timnick, A. Annal. Chem. submitted (1974). Lovelock, J. E. The haemolysis of red blood cells by freezing and thawing. Bbchim. Biophys. Acta 10, 414-426 (1953). Luyet, B. A review of research in the preservation of hearts in the frozen state. Cryobiology 8, 190-207 ( 1971), Luyet, B., and Rapatz, G. A review of basic researches on the cryopreservation of red blood cells. Cryobiology 6, 425-482 ( 1970). Meryman, H. T. “Cryobiology,” pp+ 83-64, Academic Press, New York, 1966. M&tone, H. Purification of thrombin. 1. Clin. F’hysiol. 25, 679-687 ( 1942). Pologe, C., and Soltys, M. A. In “Research in Freezing and Drying” (A. S. Farkes and A. U. Smith, Eds.) pp. 87-100. Blackwell, London, 1960. Ruwart, M. J., and Suelter, C. H.: Activation of yeast pyruvate kinase by natural and artificial cryoprotectants. 1. Biol. Chem. 246,5990-5993 (1971). Sober, II. A. In “Handbook of Biochemistry,” p. B76, ChemicaI Rubber Co., CIeveland, 1970. Taylor, A. C., and Gerstner, R. Tissue survival after exposure to low temperatures and the effectiveness of protective pretreatments. J. Cell. Camp. Physiol. 46, 477-502 (1955). Teale, F. W. J. The ultraviolet fluorescence of proteins in neutral soIution. Biochem J. 76, 381388 (1960). Teale, F. W. J., and Weber, G. The ultraviolet fluorescence of aromatic amino acids. Biochem. J. 65,476482 ( 1957). Wehry, E. L., and Rogers, L. B. In “Fluoressence and Phosphorescence” (D. M. Hercules, Ed.) pp. 113-142, Interscience, New York, 1970.
