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Effect of glycerol on protein aggregation: Quantitation of thermal aggregation of proteins from CHO cells and analysis of aggregated proteins
J. therm. BioL Vol. 18, No. 1, pp. 41-48, 1993
0306-4565/93 $6.00+ 0.00 Copyright © 1993Pergamon Press Ltd
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EFFECT OF GLYCEROL ON PROTEIN AGGREGATION: QUANTITATION OF THERMAL AGGREGATION OF PROTEINS FROM CHO CELLS A N D ANALYSIS OF A G G R E G A T E D PROTEINS DOOHA KIM and YONG J. LEE Department of Radiation Oncology, Research Laboratory, William Beaumont Hospital, 3601 West Thirteen Mile Road, Royal Oak, MI 48073, U.S.A (Received 15 August 1992; accepted in revised form 7 October 1992) Abstract--1. In an effort to elucidate the mechanism by which glycerol protects cells against heat killing,
the effects of glycerol on heat-induced protein aggregation were studied by employing a cell free system containing cytosolic proteins from CHO cells. 2. Heat-induced unfolding and subsequent aggregation of cytosolic proteins were quantified by a novel turbidimetric assay system utilizing rhodanese (RDN) in 6 M guanidinium chloride. The turbidity caused by protein-RDN aggregation showed a correlation to the amount of aggregated proteins. 3. Aggregation of the proteins was suppressed by glycerol present during the heating in concentration up to 15% (w/v). Glycerol suppressed protein aggregation at various heat doses (0-30 rain at 45.5°C) and temperatures (42-45°C), implying that glycerol stabilizes proteins against thermal denaturation. 4. The aggregated proteins were analyzed by an SDS-PAGE and individual proteins were quantified. 5. An unknown 28 kDa protein and a 70 kDa protein showed the most pronounced change in aggregation after 30 min heating at 45.5°C. The 28 kDa protein showed a 5.7-fold increase in control, but only 1.9-fold increase in 10% glycerol. The 70 kDa protein showed a 6.8-fold increase in control, while a 3.5-fold increase in 10% glycerol. 6. Other proteins such as 24, 33, 43, 46, 50, and 55 kDa protein showed 28, 38, 55, 39, 30, and I% respective reduction in aggregation by 10% glycerol. 7. These results demonstrate that glycerol affords a differential heat protection for each protein. Key Word Index: Protein aggregation; turbidimetric assay; glycerol; heat protector
other sugars and sugar analogs have been found to protect in cell heat killing (Henle et al., 1983, 1985), while alcohols with mono- and dihydroxyl groups sensitized cells against heat killing (Henle, 1981). Henle and Warters (1982) observed that characteristics of glycerol protection were similar to that of heat-induced thermotolerance in many respects, and they suggested that there is a common mechanistic basis for the two phenomena. Subsequently, Henle and Warters (1982) and Henle et al. (1982) suggested that thermal protection by glycerol was presumably exerted by stabilization of either protein or membranes. Lin et al. (1984) also suggested that glycerol protects some heat sensitive proteins. By employing a differential scanning calorimeter and Chinese hamster lung V79-WNRE cells, Lepock et al. (1990) clearly showed that glycerol protects cellular proteins from denaturation. However, the greatest drawback of cell experiments with polyol compounds is that intracellular concentration of these compounds cannot be controlled because of the limitation in
INTRODUCTION
Glycerol is known to protect cells from hyperthermic cell killing. Various cell lines such as Chinese hamster ovary ceils (Henle, 1981; Henle and Warters, 1982; Henle et al., 1983; Mivechi and Dewey, 1984), Chinese hamster V79 cells (Lin et al., 1984), HeLa cells (Henle and Warters, 1982), HeLa $3 cells (Kampinga et al., 1989), BP-8 murine sarcoma cells (Mivechi and Hofer, 1983), and Reuber H35 rat hepatoma cells (Rijn et al., 1984) have been tested for glycerol-induced thermal protection. Henle and Warters (1982) reported that CHO and HeLa cells were protected from thermal killing by glycerol and the protection was increased in a concentration dependent manner up to approx. 10% glycerol. The protection was exerted only when glycerol was present during heating. They demonstrated that metabolites of glycerol or cell cycle redistribution was not involved in the protection of cell killing. Other polyhydroxyl compounds such as adonitol, erythritol, and 41
42
DOOHAKIM and YONGJ. LEE
membrane permeability and the osmolarity of the medium. Thus, the observed protection in heat killing or protein denaturation might be dependent on intracellular concentration as well as characteristics of the polyols. In this study, an attempt was made to investigate the effect of glycerol on proteins in a cell free system with controlled glycerol concentration. More specifically, glycerol effects on heat-induced aggregation of proteins were tested by employing a turbidimetric assay system (Kim et al., 1992a). In order to determine the extent of thermal damage of proteins, we used a novel assay technique employing rhodanese. By utilizing the characteristics of rhodanese, which tends to aggregate when diluted into a buffer solution from a denatured form in 6 M guanidinium chloride (Horowitz and Criscimagna, 1986, 1990; Tandon and Horowitz, 1989), the extent of denaturation or partial unfolding of proteins and subsequent aggregation could be determined by measuring the turbidity caused by RDN-protein aggregation. Furthermore, aggregated proteins were analyzed with an SDSpolyacrylamide gel electrophoresis in an effort to investigate the possible target protein(s) of hyperthermic cell killing. MATERIALS AND METHODS
Cell culture and preparation of proteins Chinese hamster ovary cells (CHO) were grown in McCoy's 5A media with 10% bovine calf serum (iron supplemented) and 26 mM sodium bicarbonate. Cells grown to 90% confluence in flasks were trypsinized and rinsed twice with the Spinner salt solution (SSS) to remove residual trypsin as previously reported (Lee et al., 1991a). Harvested cells were incubated for 10 min in an ice bath in a hypotonic solution (buffer C; 10raM Tris-HC1, pH 7.5, 0.1 mM PMSF, I mlper 4E7 cells), homogenized with a Dounce homogenizer, and centrifuged for 10 min at 1000g as reported previously (Kim et al., 1991). The supernatant was pooled and adjusted to buffer R (30 mM Tris-HCl, 50 mM KCI, pH 7.2) with 10 x buffer R. The protein content was determined by the Bradford (1976) method and diluted to 0.5 mg/ml with buffer R.
Turbidimetric assay of protein-RDN aggregation Protein samples (0.5 mg/ml in buffer R) with and without glycerol were heated (at 45.5°C if not specified) for 0-30 rain. Microcentrifuge tubes conraining I ml of protein samples were totally submerged in a water bath which was controlled within _0.05°C. To 1 ml of the heated protein solution, 0.01 ml of rhodanese (2.5 mg/ml in 6 M guanidinium chloride) was added, mixed rapidly, and the ab-
sorbance at 320 nm was monitored either continuously for 5min or after 5min by a u.v./vis spectrophotometer (Model DU65, Beckman Co.). A blank with buffer alone and a control with protein but without heating were run in each experiment as a control. The absorbance values at 5 min incubation were corrected by subtracting the control value.
Quantitation of aggregated proteins Samples containing protein-RDN aggregates were spun at 14,000g for 5 min with a microcentrifuge in a cold room and the protein amounts in the pellet were assayed by the Bradford method (Bradford, 1976).
SDS-polyacrylamide gel electrophoresis and densitometer scanning The proteins in the pellet obtained by centrifugation after rhodanese aggregation as described above were analyzed with an SDS-PAGE as previously reported (Lee et ai., 1991b; Kim et aL, 1991). A gradient acrylamide gel from 10 to 18% for separating gel and a 5% stacking gel were employed. The stacking gel and separating gel contained an additional 2 mM EDTA and 5% glycerol, respectively, in the Laemmli gel system (Laemmli, 1970). Gels after electrophoresis were stained with Coomassie blue R250 and scanned with a laser densitometer (Model 300A, Molecular Dynamics Co.). The relative intensity (%) of protein bands were calculated from the scanned peak areas of unheated and heated samples. RESULTS
Determination of effect of glycerol on aggregation of proteins by employing turbidimetric assay system Proteins with or without glycerol (10%) were heated for 30 rain at 45.5°C and protein-RDN aggregation was determined by measuring the absorbance at 320 rim. Figure 1 shows the profile of absorbance change of the control and glycerol containing samples after R D N addition. Proteins containing 5 or 10% glycerol showed significantly less absorbance after R D N addition, compared to the control sample without glycerol, demonstrating that glycerol protected proteins from thermal damage. Under the assay condition the turbidity (as denoted by absorhance at 320 nm) of most of the samples gradually increased and reached a plateau approx, 5 rain after RDN dilution, The absorbance change for 5 w.in was utilized as a parameter of thermal damage of proteins. In Fig. 2(A), it is clearly shown that the absorbance is proportionally increased with increased time of heating at 45.5°C. Proteins containing 10%
Glycerol effect on protein aggregation glycerol showed less absorbance change with increasing heat dose. As shown in Fig. 2(B), the amount of aggregated protein recovered by centrifugation proportionally increased with increasing heat dose, implying that the absorbance increase was caused by p r o t e i n - R D N aggregation. Furthermore, the amount of aggregated protein was less in samples containing 10% glycerol than in samples without glycerol.
43
Analysis of aggregated proteins with SDS-PAGE Aggregated proteins were pelleted by centrifugation and analyzed with an SDS-polyacrylamide gel electrophoresis following Coomassie blue staining. Stained gels were scanned with a laser densitometer and individual proteins were quantitated. Figure 5 shows profiles of the aggregated proteins after heat shock at various heat doses with and without glycerol (10%). The total amount of aggregated proteins was
Effect of various concentrations of glycerol on aggregation of proteins Proteins with various concentrations of glycerol (0-15%) were heated for 30min at 45.5°C and the protcin-RDN aggregation was determined. Figure 3 clearly shows that the aggregation decreased with increasing concentration of glycerol, indicating that glycerol protected proteins from thermal denaturation.
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Time after RDN Dilution (sec) Fig. 1. Determination of effect of glycerol on aggregation of proteins by employing turbidimetric assay system. Samples containing proteins (0.5 mg/mi) with and without glycerol (5-10%) were heated for 30rain at 45.5°C. Rhodanese (25/zg in 10 #l of 6 M guanidinium chloride) was added into I mi of the solution, mixed rapidly, and the absorbance at 320 nm was recorded for 5 rain.
Fig. 2. Quantitation of aggregation of proteins after various heat doses. (A) Proteins (0.5 mg/ml) from CHO cells with or without glycerol (10%) were heated at 45.5°C for various times (0-30min), mixed with rhodanes¢ (25#g in 10#1 of 6 M guanidinium chloride) and incubated for 5 rain, and the absorbance (320 nm) was measured. The absorbance at 5 min was corrected by subtracting that of the unheated control. 0B) Samples in (A) were spun at 14,000g for 5 rain and the protein amount in the pellet was determined. The protein amount was corrected by subtracting that of the unheated control.
44
DOOHAKIM and YOr~GJ. LEE
significantly lower in samples containing glycerol (10%) than in control samples throughout different heat doses. The amounts of individual proteins in different gel lanes were quantitated in arbitrary units by a laser densitometer and the relative amounts compared to that of the unheated sample were calculated. Figure 6 shows aggregation of individual proteins after heat shock with and without glycerol. Several proteins with molecular weights of 28, 43, and 70 kDa showed remarkably reduced aggregation in the presence of glycerol (10%) during heat shock, while several proteins with molecular weights of 24, 33, 46, and 50 kDa showed only moderate protection by glycerol. However, a protein with molecular weight of 55 kDa showed no significant difference between the control and the glycerol treated sample. DISCUSSION
Our observations that glycerol inhibits heatinduced aggregation of proteins in a cell free system seems to be consistent to other studies which show that glycerol protected heat-induced denaturation of proteins (Gerlsma and Stuur, 1972; Back et al., 1979; Gekko and Timasheff, 1981a, b; Beaudette et al., 1982; Na, 1986; Levine et al., 1980). Since thermal denaturation or unfolding of proteins generally induce protein aggregation, the quantity of protein aggregation is correlated to the quantity and extent of protein denaturation (Kim et al., 1992a). Quanti0.3
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Glycerol (%) Fig. 3. Effect of various concentrations of glycerol on aggregation of proteins. Proteins from CHO cells (0.5 mg/mi) containing various concentrations of glycerol (0-15%, w/v) were heated for 30 rain at 45.5°C. Rhodancse (25 #g in 10 ?tl of 6 M guanidinium chloride) was added to I ml of sample and the absorbanc¢ (320 nm) was ~ u r e d following 5 rain incubation at room temperature. Experiments were done with duplicate samples. The line was
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Temperature (%) Fig. 4. Effect of glycerol on aggregation of proteins at various temperatures. Proteins from CHO cells (0.5 mg/ml) containing various concentrations of glycerol (0-10%) were heated for 30 min at various temperatures (42-45°C), mixed with 25/tg of rhodanese in 10#1 of 6M guanidinium chloride, and the absorbance (320 nm) was measured after 5 min incubation. The dotted lines were drawn by a computer aided curve fitting with a linear regression method.
tation of protein aggregation and subsequent analysis of the aggregated proteins might greatly contribute to studies of target proteins of hyperthermic cell killing. Glycerol, which is known as a stabilizer of protein denaturation and a protector for thermal cell killing, was employed to modulate protein denaturation and subsequent aggregation of proteins. As expected, glycerol showed preferential suppression of aggregation of several heat sensitive proteins. Glycerol is known as a protein stabilizing agent and has been utilized as an additive to protein or enzyme solutions (Jarabak, 1972; Bradbury and Jakoby, 1972; Myers and Jakoby, 1973; Shifirin and Parott, 1975). Gekko and Timasheff (1981a, b) reported that glycerol strengthens the hydrophobic interactions in proteins. The interaction between hydrophobic domains in a protein and the glycerol is thermodynamically unfavorable, and consequently glycerol stabilizes proteins against heat denaturation. Glycerol and other polyols stabilize proteins or enzymes against chemical or thermal denaturation and protect cells from thermal killing, implying that protection of heat sensitive proteins confer protection of cells from heat killing. Many investigators (Rosenberg et al., 1971; Dewey et al., 1980; Warters et al., 1980; l.,¢poek et al., 1990) suggested that the target of thermal cell killing is protein(s) and thus hyperthermic cell killing is dependent on the intracellular amount or activity of the target proteins. Assuming the target protein hypothesis, two different modes of hyperthermic cell killing
Glycerol effect on protein aggregation
45
sensitive and thus, identification of heat sensitive proteins, although the amount may be very limited but not impossible to detect, might reveal valuable information in regards to target proteins of heat killing. In this respect, the experimental approach of this study, i.e. analysis of the aggregated proteins following heat and R D N dilution, might be useful in studies of hyperthermic target proteins. As shown in Fig. 1, the turbidity caused by RDN-protein aggregation increased rapidly and reached a plateau approx. 5 rain after R D N dilution.
might be considered. First, an individual target protein independently causes cell death. Any individual proteins may cause cell death whenever the effective amount or activity drops below a critical level. Second, instead of a single target protein, simultaneous damage of multiple target proteins cooperatively cause cell death. Our observation that glycerol affords a differential protection for each protein was, however, not sufficient to suggest a possible mechanism of hyperthermic cell killing. Target proteins of thermal cell killing are supposed to be relatively heat
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Fig. 5. Analysis of aggregated proteins with SDS-PAGE. Proteins from CHO cells (0.5 mg/ml) with and without glycerol (10%, w/v) were heated for various times (0-30 rain) at 45.5°C. Rhodanese (25 #g in 10 gl of 6 M guanidinium chloride) was added to 1 ml of heated samples and incubated for 5 rain at room temperature. Samples were spun at 14,000g for 5 min and the protein in the pellet was analyzed with an SDS-PAGE. Individual proteins were quantitated with a laser densitometer following Coomassie blue staining. Lanes 1--4(0, 10, 20, and 30 min, respectively) and lanes 5-8 (0, I0, 20, and 30 rain, respectively) correspond to samples from the control and glycerol treated, respectively. CONT and GLY indicate the control samples without glycerol and samples with 10% glycerol, respectively. Numbers at left and fight of lanes 1 and 8 indicate molecular weights (kDa) of standard marker proteins and protein bands analyzed with the laser densitometer. RDN indicates rhodanese.
46
DOOHA KIM and YONG J. LEE
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Fig. 6. Effectof glycerolon aggregationof individual proteins. Proteins were heated at 45.5°C for 0-30 min with and without glycerol (10%) and then aggregated with RDN a s described in Materials and Methods. The protein-RDN aggregates were analyzed with SDS-PAGE and quantified with a laser densitometer. Relative values were calculated by dividing the peak area of protein bands with that of the unheated control. Thus the absorbance at 5 min after RDN dilution was taken as an end point of aggregation, and it was found to be proportional to the extent of protein denaturation (Kim et al., 1992a). The fact that the turbidity represents the RDN-protein aggregation was proved by measuring the amount of aggregated proteins [see Fig. 2(A, B)]. Furthermore, Fig. 2 proved that the effect of glycerol on aggregation was actually exerted by stabilization of proteins. Stabilization of proteins by glycerol seemed to be a general phenomenon in various proteins, temperatures, and glycerol concentrations (see Figs 4 and 5). Our data (Fig. 3) shows that the heat-induced (45.5°C, 30 min) aggregation of proteins, denoted by turbidity, decreases with increasing glycerol concentrations. The killing cell fraction (KF = 1 - SF) after similar heat dose (45°C, 30 min) showed a similar trend, i.e. the slope of KF against glycerol concentration was very close to that of turbidity vs glycerol concentration up to approx. 5% glycerol (data not shown). Our observation was consistent to Henle and Warters (1982), who showed a similar correlation of survival vs glycerol concentrations (data not shown). The slope of KF vs glycerol concentration was steeper than that of protein aggregation in glycerol concentration of 5-10% [when data were redrawn from Henle et al. (1982) and Rijn et aL (1984)]. The larger discrepancy in higher glycerol concentration
(5-10%) might be due to the limitation in intracellular glycerol concentration. Surprisingly, small sized proteins showed a more pronounced dose-response of aggregation to heat dose than larger proteins. Most of these smaller proteins most likely originated from subunits of much larger oligomeri¢ proteins which are supposed to be more heat sensitive. Among several proteins so far analyzed, a 28 kDa protein and a 70 kDa protein showed the most pronounced changes of aggregation by heat shock. We identified the 70 kDa protein as the constitutive heat shock protein by employing an antibody (data not shown). Several proteins with molecular weight 24, 33, 43, 46, and 50 kDa showed relatively great heat sensitivity and great heat protection by glycerol among proteins so far analyzed. Some of these proteins might be possible candidates for hyperthermic target proteins. Further characterizations, such as identification of these proteins by molecular biological techniques, might bring us more valuable information about hyperthermic target proteins. The 43 kDa protein, probably actin, was greatly stabilized by glycerol. Actin is a major component of cytoskeleton and may play many important roles in cell survival. However, the role of actin to heat cell killing is not known. We interpreted that the constitutive 70 kDa heat-shock protein recognized and bound partially unfolded protein and co-aggregated with protein-RDN complex (Kim et al., 1992b). This
Glycerol effect on protein aggregation assumption was partly supported by the fact that the 70 k D a protein was preferentially aggregated by heat shock, while the constitutive HSP70 is very heat stable in physiological condition (Palleros et al., 1991). Further studies employing two-dimensional gel analysis and through the identification of these heat sensitive proteins might help reveal heat sensitive target proteins in hyperthermic cell killing. Acknowledgements--This research was supported by NCI Grants CA 48000 and William Beaumont Hospital Research Institute Grants 91-10, 92-27, and 92-28.
REFERENCES
Back J. F., Okenfull D. and Smith M. B. (1979) Increased thermal stability of proteins in the presence of sugars and polyols. Biochemistry 18, 5191-5196. Beaudette N. V., Okabayashi H. and Fasman G. D. (1982) Conformational effects of organic solvents on histone complexes. Biochemistry 21, 1765-1772. Bradbury S. L. and Jakohy W. B. (1972) Glycerol as an enzyme-stabilizingagent: effects on aldehyde dehydrogenase. Proc. hath. Acad. Sci. U.S.A. 69, 2373-2376. Bradford M. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. Dewey W. C., Freeman M. L., Raaphorst G. P., Clark E. P., Wong R. S. L., Highfield D. P., Spiro I, J., Tomasovic S. P., Denman D. L. and Coss R. A. (1980) Cell biology of hyperthermia and radiation. In Radiation Biology in Cancer Research (Edited by Meyn R. E. and Withers H. R.), pp. 589-621. Raven Press, New York. Gekko K. and Timasheff S. N. (1981a) Mechanism of protein stabilization by glycerol: preferential hydration in glycerol-water mixtures. Biochemistry 20, 4667-4676. Gekko K. and Timasheff S. N. ( 198 lb) Thermodynamic and kinetic examination of protein stabilization by glycerol. Biochemistry 20, 4677-4686. Gerlsma S. Y. and Stuur E. R. (1972) The effect of polyhydric and monohydric alcohols on the heat-induced reversible denaturation of lysozyme and ribonuclease. Int. J. Pept. Protein Res. 4, 377-383. Henle K. J. (1981) Interaction of mono- and polyhydroxy alcohols with hyperthermia in CHO cells. Radiat. Res. 88, 392-402. Henle K. J. and Waters R. (1982) Heat protection by glycerol in vitro. Cancer Res. 42, 2171-2176. Henle K. J., Peck J. W. and Higashikubo R. (1983) Protection against heat-induced cell killing by polyols/n vitro. Cancer Res. 43, 1624-1627. Henle K. J., Nagle W. A., Moss A. J. Jr and Herman T. S. (1982) Polyhydroxy compounds and thermotolerance: a proposed concatenation. Radiat. Res. 92, 445-451. Henle K. J., Monson T. P., Nagle W. A. and Moss A. J. (1985) Heat protection by sugars and sugar analogues. Int. J. Hyperther. I, 371-382. Horowitz P. M. and Criscimagna N. L. (1986) Low concentrations of guanidinium chloride expose apolar surfaces and cause differential perturbation in catalytic intermediates of rhodanese. J. biol. Chem. 261, 15,652-15,658. TB
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Horowitz P. M. and Criscimagna N. L. (1990) Stable intermediates can be trapped during the reversible refolding of urea-denatured rhodanese. J. biol. Chem. 265, 2576-2583. Jarabak J. (1972) Human placental 15-hydroxyprostaglandin dehydrogenase. Proc. natn. Acad. Sci. U.S.A. 69, 533-534. Kampinga H. H., Turkel-Uygur N., Roti Roti J. L. and Konings A. W. (1989) The relationship of increased nuclear protein content induced by hyperthermia to killing of HeLa $3 cells. Radiat. Res. 117, 511-522. Kim D., Lee Y. J. and Corry P. M. (1992a) Employment of a turbidimetric assay system to measure heat-induced protein aggregation. J. therm. Biol. 17, 297-303. Kim D., Lee Y. J. and Corry P. M. (1992b) Employment of a turbidimetric assay system to study the biochemical role of HSP70 in heat-induced protein aggregation. In Hyperthermic Oncology 1992. Vol. 1, Summary Papers (Edited by Gerner E.). Proceedings of the 6th International Congress of Hyperthermic Oncology, Tucson, Arizona. 27 April-I May, 1992. Kim D., Lee Y. J., Rausch C. M. and Borrelli M. J. (1991) Electroporation of extraneous proteins into CHO ceils: increased efficiency by utilizing centrifugal force and microsecond electrical pulses. Expl. Cell Res. 197, 207-212. Laemmii U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lee Y. J., Kim D. H., Hou Z. and Corry P. M. (1991a) Differences in thermotolerance induced by heat or sodium arsenite: correlation between redistribution of a 26 kDa protein and development of protein synthesis-independent thermotolerance in CHO cells. Radiat. Res. 127, 325-334. Lee Y. J., Kim D. H., Hou Z. and Corry P. M. (1991b) Effect of tunicamycin on glycosylation of a 50 kDa protein and thermotolerance development. J. cell. Physiol. 149, 202-207. Lepock J. R., Frey H. E., Heynen M. P., Nishio J., Waters B., Ritchie K. P. and Kruuv J. (1990) Increased thermostability of thermotolerant CHL V79 cells as determined by differential scanning calorimetry. J. cell. Physiol. 142, 628-634. Levine B. J., Orphanos P. D., Fisclunann B. S. and Beychok S. (1980) Physicochemical properties and interactions of Eseherichia coil ribonucleic acid polymerase holoenzyme, core enzyme, subunits, and subassembly ~2fl.Biochemistry 19, 4808-4814. Lin P. S., Hefter K. and Ho K. C. (1984) Modification of membrane function, protein synthesis, and heat killing effect in cultured chinese hamster ceils by glycerol and D20. Cancer ICes. 44, 5776-5784. Mivechi N. F. and Hofer K.G. (1983) Evidence for separate mode of action in thermal radiosensitization and direct thermal cell death. Cancer 51, 38--43. Mivechi N. F. and Dewey W. C. (1984) Effects of glycerol and low pH on heat-induced cell killing and loss of cellular DNA polymerase activities in Chinese hamster ovary cells. Radiat. Res. 99, 352-362. Myers J. S. and Jakoby W. B. (1973) Effect of polyhydric alcohols on kinetic parameters of enzymes. Biochem. biophys. Res. Commun. 51, 631-636. Na G. C. (1986) Interaction of calf skin collagen with glycerol: linked function analysis. Biochemistry 25, 967-973.
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DOOI-L~KIM and Yor~o J. LEE
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0306-4565/93 $6.00+ 0.00 Copyright © 1993Pergamon Press Ltd
Printed in Great Britain.All rights reserved
EFFECT OF GLYCEROL ON PROTEIN AGGREGATION: QUANTITATION OF THERMAL AGGREGATION OF PROTEINS FROM CHO CELLS A N D ANALYSIS OF A G G R E G A T E D PROTEINS DOOHA KIM and YONG J. LEE Department of Radiation Oncology, Research Laboratory, William Beaumont Hospital, 3601 West Thirteen Mile Road, Royal Oak, MI 48073, U.S.A (Received 15 August 1992; accepted in revised form 7 October 1992) Abstract--1. In an effort to elucidate the mechanism by which glycerol protects cells against heat killing,
the effects of glycerol on heat-induced protein aggregation were studied by employing a cell free system containing cytosolic proteins from CHO cells. 2. Heat-induced unfolding and subsequent aggregation of cytosolic proteins were quantified by a novel turbidimetric assay system utilizing rhodanese (RDN) in 6 M guanidinium chloride. The turbidity caused by protein-RDN aggregation showed a correlation to the amount of aggregated proteins. 3. Aggregation of the proteins was suppressed by glycerol present during the heating in concentration up to 15% (w/v). Glycerol suppressed protein aggregation at various heat doses (0-30 rain at 45.5°C) and temperatures (42-45°C), implying that glycerol stabilizes proteins against thermal denaturation. 4. The aggregated proteins were analyzed by an SDS-PAGE and individual proteins were quantified. 5. An unknown 28 kDa protein and a 70 kDa protein showed the most pronounced change in aggregation after 30 min heating at 45.5°C. The 28 kDa protein showed a 5.7-fold increase in control, but only 1.9-fold increase in 10% glycerol. The 70 kDa protein showed a 6.8-fold increase in control, while a 3.5-fold increase in 10% glycerol. 6. Other proteins such as 24, 33, 43, 46, 50, and 55 kDa protein showed 28, 38, 55, 39, 30, and I% respective reduction in aggregation by 10% glycerol. 7. These results demonstrate that glycerol affords a differential heat protection for each protein. Key Word Index: Protein aggregation; turbidimetric assay; glycerol; heat protector
other sugars and sugar analogs have been found to protect in cell heat killing (Henle et al., 1983, 1985), while alcohols with mono- and dihydroxyl groups sensitized cells against heat killing (Henle, 1981). Henle and Warters (1982) observed that characteristics of glycerol protection were similar to that of heat-induced thermotolerance in many respects, and they suggested that there is a common mechanistic basis for the two phenomena. Subsequently, Henle and Warters (1982) and Henle et al. (1982) suggested that thermal protection by glycerol was presumably exerted by stabilization of either protein or membranes. Lin et al. (1984) also suggested that glycerol protects some heat sensitive proteins. By employing a differential scanning calorimeter and Chinese hamster lung V79-WNRE cells, Lepock et al. (1990) clearly showed that glycerol protects cellular proteins from denaturation. However, the greatest drawback of cell experiments with polyol compounds is that intracellular concentration of these compounds cannot be controlled because of the limitation in
INTRODUCTION
Glycerol is known to protect cells from hyperthermic cell killing. Various cell lines such as Chinese hamster ovary ceils (Henle, 1981; Henle and Warters, 1982; Henle et al., 1983; Mivechi and Dewey, 1984), Chinese hamster V79 cells (Lin et al., 1984), HeLa cells (Henle and Warters, 1982), HeLa $3 cells (Kampinga et al., 1989), BP-8 murine sarcoma cells (Mivechi and Hofer, 1983), and Reuber H35 rat hepatoma cells (Rijn et al., 1984) have been tested for glycerol-induced thermal protection. Henle and Warters (1982) reported that CHO and HeLa cells were protected from thermal killing by glycerol and the protection was increased in a concentration dependent manner up to approx. 10% glycerol. The protection was exerted only when glycerol was present during heating. They demonstrated that metabolites of glycerol or cell cycle redistribution was not involved in the protection of cell killing. Other polyhydroxyl compounds such as adonitol, erythritol, and 41
42
DOOHAKIM and YONGJ. LEE
membrane permeability and the osmolarity of the medium. Thus, the observed protection in heat killing or protein denaturation might be dependent on intracellular concentration as well as characteristics of the polyols. In this study, an attempt was made to investigate the effect of glycerol on proteins in a cell free system with controlled glycerol concentration. More specifically, glycerol effects on heat-induced aggregation of proteins were tested by employing a turbidimetric assay system (Kim et al., 1992a). In order to determine the extent of thermal damage of proteins, we used a novel assay technique employing rhodanese. By utilizing the characteristics of rhodanese, which tends to aggregate when diluted into a buffer solution from a denatured form in 6 M guanidinium chloride (Horowitz and Criscimagna, 1986, 1990; Tandon and Horowitz, 1989), the extent of denaturation or partial unfolding of proteins and subsequent aggregation could be determined by measuring the turbidity caused by RDN-protein aggregation. Furthermore, aggregated proteins were analyzed with an SDSpolyacrylamide gel electrophoresis in an effort to investigate the possible target protein(s) of hyperthermic cell killing. MATERIALS AND METHODS
Cell culture and preparation of proteins Chinese hamster ovary cells (CHO) were grown in McCoy's 5A media with 10% bovine calf serum (iron supplemented) and 26 mM sodium bicarbonate. Cells grown to 90% confluence in flasks were trypsinized and rinsed twice with the Spinner salt solution (SSS) to remove residual trypsin as previously reported (Lee et al., 1991a). Harvested cells were incubated for 10 min in an ice bath in a hypotonic solution (buffer C; 10raM Tris-HC1, pH 7.5, 0.1 mM PMSF, I mlper 4E7 cells), homogenized with a Dounce homogenizer, and centrifuged for 10 min at 1000g as reported previously (Kim et al., 1991). The supernatant was pooled and adjusted to buffer R (30 mM Tris-HCl, 50 mM KCI, pH 7.2) with 10 x buffer R. The protein content was determined by the Bradford (1976) method and diluted to 0.5 mg/ml with buffer R.
Turbidimetric assay of protein-RDN aggregation Protein samples (0.5 mg/ml in buffer R) with and without glycerol were heated (at 45.5°C if not specified) for 0-30 rain. Microcentrifuge tubes conraining I ml of protein samples were totally submerged in a water bath which was controlled within _0.05°C. To 1 ml of the heated protein solution, 0.01 ml of rhodanese (2.5 mg/ml in 6 M guanidinium chloride) was added, mixed rapidly, and the ab-
sorbance at 320 nm was monitored either continuously for 5min or after 5min by a u.v./vis spectrophotometer (Model DU65, Beckman Co.). A blank with buffer alone and a control with protein but without heating were run in each experiment as a control. The absorbance values at 5 min incubation were corrected by subtracting the control value.
Quantitation of aggregated proteins Samples containing protein-RDN aggregates were spun at 14,000g for 5 min with a microcentrifuge in a cold room and the protein amounts in the pellet were assayed by the Bradford method (Bradford, 1976).
SDS-polyacrylamide gel electrophoresis and densitometer scanning The proteins in the pellet obtained by centrifugation after rhodanese aggregation as described above were analyzed with an SDS-PAGE as previously reported (Lee et ai., 1991b; Kim et aL, 1991). A gradient acrylamide gel from 10 to 18% for separating gel and a 5% stacking gel were employed. The stacking gel and separating gel contained an additional 2 mM EDTA and 5% glycerol, respectively, in the Laemmli gel system (Laemmli, 1970). Gels after electrophoresis were stained with Coomassie blue R250 and scanned with a laser densitometer (Model 300A, Molecular Dynamics Co.). The relative intensity (%) of protein bands were calculated from the scanned peak areas of unheated and heated samples. RESULTS
Determination of effect of glycerol on aggregation of proteins by employing turbidimetric assay system Proteins with or without glycerol (10%) were heated for 30 rain at 45.5°C and protein-RDN aggregation was determined by measuring the absorbance at 320 rim. Figure 1 shows the profile of absorbance change of the control and glycerol containing samples after R D N addition. Proteins containing 5 or 10% glycerol showed significantly less absorbance after R D N addition, compared to the control sample without glycerol, demonstrating that glycerol protected proteins from thermal damage. Under the assay condition the turbidity (as denoted by absorhance at 320 nm) of most of the samples gradually increased and reached a plateau approx, 5 rain after RDN dilution, The absorbance change for 5 w.in was utilized as a parameter of thermal damage of proteins. In Fig. 2(A), it is clearly shown that the absorbance is proportionally increased with increased time of heating at 45.5°C. Proteins containing 10%
Glycerol effect on protein aggregation glycerol showed less absorbance change with increasing heat dose. As shown in Fig. 2(B), the amount of aggregated protein recovered by centrifugation proportionally increased with increasing heat dose, implying that the absorbance increase was caused by p r o t e i n - R D N aggregation. Furthermore, the amount of aggregated protein was less in samples containing 10% glycerol than in samples without glycerol.
43
Analysis of aggregated proteins with SDS-PAGE Aggregated proteins were pelleted by centrifugation and analyzed with an SDS-polyacrylamide gel electrophoresis following Coomassie blue staining. Stained gels were scanned with a laser densitometer and individual proteins were quantitated. Figure 5 shows profiles of the aggregated proteins after heat shock at various heat doses with and without glycerol (10%). The total amount of aggregated proteins was
Effect of various concentrations of glycerol on aggregation of proteins Proteins with various concentrations of glycerol (0-15%) were heated for 30min at 45.5°C and the protcin-RDN aggregation was determined. Figure 3 clearly shows that the aggregation decreased with increasing concentration of glycerol, indicating that glycerol protected proteins from thermal denaturation.
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Fig. 2. Quantitation of aggregation of proteins after various heat doses. (A) Proteins (0.5 mg/ml) from CHO cells with or without glycerol (10%) were heated at 45.5°C for various times (0-30min), mixed with rhodanes¢ (25#g in 10#1 of 6 M guanidinium chloride) and incubated for 5 rain, and the absorbance (320 nm) was measured. The absorbance at 5 min was corrected by subtracting that of the unheated control. 0B) Samples in (A) were spun at 14,000g for 5 rain and the protein amount in the pellet was determined. The protein amount was corrected by subtracting that of the unheated control.
44
DOOHAKIM and YOr~GJ. LEE
significantly lower in samples containing glycerol (10%) than in control samples throughout different heat doses. The amounts of individual proteins in different gel lanes were quantitated in arbitrary units by a laser densitometer and the relative amounts compared to that of the unheated sample were calculated. Figure 6 shows aggregation of individual proteins after heat shock with and without glycerol. Several proteins with molecular weights of 28, 43, and 70 kDa showed remarkably reduced aggregation in the presence of glycerol (10%) during heat shock, while several proteins with molecular weights of 24, 33, 46, and 50 kDa showed only moderate protection by glycerol. However, a protein with molecular weight of 55 kDa showed no significant difference between the control and the glycerol treated sample. DISCUSSION
Our observations that glycerol inhibits heatinduced aggregation of proteins in a cell free system seems to be consistent to other studies which show that glycerol protected heat-induced denaturation of proteins (Gerlsma and Stuur, 1972; Back et al., 1979; Gekko and Timasheff, 1981a, b; Beaudette et al., 1982; Na, 1986; Levine et al., 1980). Since thermal denaturation or unfolding of proteins generally induce protein aggregation, the quantity of protein aggregation is correlated to the quantity and extent of protein denaturation (Kim et al., 1992a). Quanti0.3
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Glycerol (%) Fig. 3. Effect of various concentrations of glycerol on aggregation of proteins. Proteins from CHO cells (0.5 mg/mi) containing various concentrations of glycerol (0-15%, w/v) were heated for 30 rain at 45.5°C. Rhodancse (25 #g in 10 ?tl of 6 M guanidinium chloride) was added to I ml of sample and the absorbanc¢ (320 nm) was ~ u r e d following 5 rain incubation at room temperature. Experiments were done with duplicate samples. The line was
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tation of protein aggregation and subsequent analysis of the aggregated proteins might greatly contribute to studies of target proteins of hyperthermic cell killing. Glycerol, which is known as a stabilizer of protein denaturation and a protector for thermal cell killing, was employed to modulate protein denaturation and subsequent aggregation of proteins. As expected, glycerol showed preferential suppression of aggregation of several heat sensitive proteins. Glycerol is known as a protein stabilizing agent and has been utilized as an additive to protein or enzyme solutions (Jarabak, 1972; Bradbury and Jakoby, 1972; Myers and Jakoby, 1973; Shifirin and Parott, 1975). Gekko and Timasheff (1981a, b) reported that glycerol strengthens the hydrophobic interactions in proteins. The interaction between hydrophobic domains in a protein and the glycerol is thermodynamically unfavorable, and consequently glycerol stabilizes proteins against heat denaturation. Glycerol and other polyols stabilize proteins or enzymes against chemical or thermal denaturation and protect cells from thermal killing, implying that protection of heat sensitive proteins confer protection of cells from heat killing. Many investigators (Rosenberg et al., 1971; Dewey et al., 1980; Warters et al., 1980; l.,¢poek et al., 1990) suggested that the target of thermal cell killing is protein(s) and thus hyperthermic cell killing is dependent on the intracellular amount or activity of the target proteins. Assuming the target protein hypothesis, two different modes of hyperthermic cell killing
Glycerol effect on protein aggregation
45
sensitive and thus, identification of heat sensitive proteins, although the amount may be very limited but not impossible to detect, might reveal valuable information in regards to target proteins of heat killing. In this respect, the experimental approach of this study, i.e. analysis of the aggregated proteins following heat and R D N dilution, might be useful in studies of hyperthermic target proteins. As shown in Fig. 1, the turbidity caused by RDN-protein aggregation increased rapidly and reached a plateau approx. 5 rain after R D N dilution.
might be considered. First, an individual target protein independently causes cell death. Any individual proteins may cause cell death whenever the effective amount or activity drops below a critical level. Second, instead of a single target protein, simultaneous damage of multiple target proteins cooperatively cause cell death. Our observation that glycerol affords a differential protection for each protein was, however, not sufficient to suggest a possible mechanism of hyperthermic cell killing. Target proteins of thermal cell killing are supposed to be relatively heat
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Fig. 5. Analysis of aggregated proteins with SDS-PAGE. Proteins from CHO cells (0.5 mg/ml) with and without glycerol (10%, w/v) were heated for various times (0-30 rain) at 45.5°C. Rhodanese (25 #g in 10 gl of 6 M guanidinium chloride) was added to 1 ml of heated samples and incubated for 5 rain at room temperature. Samples were spun at 14,000g for 5 min and the protein in the pellet was analyzed with an SDS-PAGE. Individual proteins were quantitated with a laser densitometer following Coomassie blue staining. Lanes 1--4(0, 10, 20, and 30 min, respectively) and lanes 5-8 (0, I0, 20, and 30 rain, respectively) correspond to samples from the control and glycerol treated, respectively. CONT and GLY indicate the control samples without glycerol and samples with 10% glycerol, respectively. Numbers at left and fight of lanes 1 and 8 indicate molecular weights (kDa) of standard marker proteins and protein bands analyzed with the laser densitometer. RDN indicates rhodanese.
46
DOOHA KIM and YONG J. LEE
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Fig. 6. Effectof glycerolon aggregationof individual proteins. Proteins were heated at 45.5°C for 0-30 min with and without glycerol (10%) and then aggregated with RDN a s described in Materials and Methods. The protein-RDN aggregates were analyzed with SDS-PAGE and quantified with a laser densitometer. Relative values were calculated by dividing the peak area of protein bands with that of the unheated control. Thus the absorbance at 5 min after RDN dilution was taken as an end point of aggregation, and it was found to be proportional to the extent of protein denaturation (Kim et al., 1992a). The fact that the turbidity represents the RDN-protein aggregation was proved by measuring the amount of aggregated proteins [see Fig. 2(A, B)]. Furthermore, Fig. 2 proved that the effect of glycerol on aggregation was actually exerted by stabilization of proteins. Stabilization of proteins by glycerol seemed to be a general phenomenon in various proteins, temperatures, and glycerol concentrations (see Figs 4 and 5). Our data (Fig. 3) shows that the heat-induced (45.5°C, 30 min) aggregation of proteins, denoted by turbidity, decreases with increasing glycerol concentrations. The killing cell fraction (KF = 1 - SF) after similar heat dose (45°C, 30 min) showed a similar trend, i.e. the slope of KF against glycerol concentration was very close to that of turbidity vs glycerol concentration up to approx. 5% glycerol (data not shown). Our observation was consistent to Henle and Warters (1982), who showed a similar correlation of survival vs glycerol concentrations (data not shown). The slope of KF vs glycerol concentration was steeper than that of protein aggregation in glycerol concentration of 5-10% [when data were redrawn from Henle et al. (1982) and Rijn et aL (1984)]. The larger discrepancy in higher glycerol concentration
(5-10%) might be due to the limitation in intracellular glycerol concentration. Surprisingly, small sized proteins showed a more pronounced dose-response of aggregation to heat dose than larger proteins. Most of these smaller proteins most likely originated from subunits of much larger oligomeri¢ proteins which are supposed to be more heat sensitive. Among several proteins so far analyzed, a 28 kDa protein and a 70 kDa protein showed the most pronounced changes of aggregation by heat shock. We identified the 70 kDa protein as the constitutive heat shock protein by employing an antibody (data not shown). Several proteins with molecular weight 24, 33, 43, 46, and 50 kDa showed relatively great heat sensitivity and great heat protection by glycerol among proteins so far analyzed. Some of these proteins might be possible candidates for hyperthermic target proteins. Further characterizations, such as identification of these proteins by molecular biological techniques, might bring us more valuable information about hyperthermic target proteins. The 43 kDa protein, probably actin, was greatly stabilized by glycerol. Actin is a major component of cytoskeleton and may play many important roles in cell survival. However, the role of actin to heat cell killing is not known. We interpreted that the constitutive 70 kDa heat-shock protein recognized and bound partially unfolded protein and co-aggregated with protein-RDN complex (Kim et al., 1992b). This
Glycerol effect on protein aggregation assumption was partly supported by the fact that the 70 k D a protein was preferentially aggregated by heat shock, while the constitutive HSP70 is very heat stable in physiological condition (Palleros et al., 1991). Further studies employing two-dimensional gel analysis and through the identification of these heat sensitive proteins might help reveal heat sensitive target proteins in hyperthermic cell killing. Acknowledgements--This research was supported by NCI Grants CA 48000 and William Beaumont Hospital Research Institute Grants 91-10, 92-27, and 92-28.
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DOOI-L~KIM and Yor~o J. LEE
Palleros D. R., Welch W. J. and Fink A. L. (1991) Interaction of HSP70 with unfolded proteins: effects of temperature and nucleotides on the kinetics of binding. Prec. hath. Acad. Sci. U.S.A. 88, 5719-5723. Rijn J. V., Berg J. V. D., Schamhart D. H. J. and Wijk R. V. (1984) Morphological response and survival of hepatoma cells during fractionated hyperthermia: effects of glycerol. Radiat. Res. 98, 471-478. Rosenberg B., Kemery G., Switzer R. C. and Hamilton T. C. (1971) Quantitative evidence for protein denatura-
tion as the cause of thermal death. Nature (Lend.) 232, 471-473. Shifirin S. and Parrott C. L. (1975) Influence of giycerol and other polyhydric alcohol on the quaternary structure of an oligomeric protein. Archs Biochem. Biophys. 166, 426-432. Tandon S. and Horowitz P. M. (1989) Reversible folding of rhodanese. J. biol. Chem. 264, 9859-9866. Warters R. L., Roti Roti J. L. and Winward R. T. (1980) Nucleosome structure in chromatin from heated cells. Radiat. Res. 84, 504-513.