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An Efficient and Cost-Effective Procedure forPreparing Samples for Differential Scanning

tions of the protein solutions or suspensions providedby companies to the buffers of choice. For comparing

Calorimetry Experiments

Ashutosh Tiwari,1 D. Prasanna Kumar,and Rajiv Bhat2

Centre for Biotechnology, Jawaharlal Nehru University,New Delhi 110067, India

Received October 28, 1999

It has been a regular practice for years to carry outdialysis of protein samples against appropriate bufferconditions for equilibration and for their use in differ-ential scanning calorimetry (DSC)3 experiments (1–4).This has been both time-consuming and problematic attimes, especially when one is dealing with highly con-centrated solutions of denaturants, salts, or other co-solvents present in the buffer. Due to the excess ofsolvent volume required for equilibration, in additionto the large costs of the solvent additives involved,evaporational losses have often been observed, leadingto erratic results for protein denaturation (5). We re-port here the use of Centricon membrane concentratorsfor equilibration of protein samples with appropriatebuffer conditions and compare the results obtainedfrom DSC experiments using this procedure with thoseusing conventional dialysis. The procedure requires arefrigerated centrifuge with appropriate rotors forholding the Centricon concentrators available in mostbiochemistry laboratories. We have used ribonucleaseA (RNase A) as a model protein and Centricon concen-trators of 10-kDa cutoff. This procedure is also helpfulin getting rid of the various low molecular weight ad-ditives used in the formulation of proteins and en-zymes by various companies for stabilization purposes.For DSC experiments, it is most essential to get rid ofthese additives since their high concentrations wouldoften affect the DSC transitions observed. Further-more, often it is desirable to change the buffer condi-

1 Present address: Department of Neurology, University of Mas-sachusetts Medical Center, Worcester, MA 01655.

2 To whom correspondence should be addressed. Fax: 191-11-6165886. E-mail: rajiv@jnuniv.ernet.in.

3 Abbreviations used: DSC, differential scanning calorimetry;RNase A, ribonuclease A.

406

DSC results, we have carried out experiments withRNase A at pH 2.5, 4.0, and pH 7.0 as well as in thepresence of 0.5 M urea at pH 2.5 using both the Cen-tricon equilibration procedure and conventional dialy-sis. We show here that both procedures yield identicalresults for thermodynamic parameters such as thetransition temperature, Tm, and the enthalpy of dena-turation, DH d.

Materials and Methods

Materials. RNase A was from Sigma Chemical Co.Dialysis tubing (8- to 10-kDa cutoff) was Cellusep T2from Membrane Filtration Products Inc. Disodiumphosphate and monosodium hydrogen phosphate werefrom Merck. Glycine and sodium acetate were fromSisco Research Laboratories Pvt. Ltd. and acetic acidwas from Spectrochem Pvt. Ltd. The Centricon concen-trators (10-kDa cutoff) used were from Amicon Inc. Allchemicals used were of the highest purity grade avail-able and were used without any further purification.Glass double distilled water was used to make solu-tions and buffers. The pH of the buffer solutions wasadjusted on a Control Dynamics Model APX 175 pHmeter. The pH standards used for calibrating the pHmeter were from Sigma Chemical Co. The followingsolutions were prepared for the experiments carriedout: 20 mM phosphate buffer, pH 7.0; 20 mM acetatebuffer, pH 4.0; 20 mM glycine–HCl buffer, pH 2.5; and20 mM glycine–HCl buffer containing 0.5 M urea at pH2.5. The preparations of the solutions and the settingsof the pH values were carried out at room temperature(25°C). For the determination of the concentration ofprotein solutions, matched pairs of stoppered quartzcuvettes from Sigma Chemical Co. were used. After thepreparation of the buffers and the solutions, they werefiltered using a 0.45-mm filter (Sartorius) and stored instoppered reagent bottles. Further processing of RNaseA solutions was carried out as described below:

Dialysis. RNase A was dialyzed using a dialysistubing of molecular weight cutoff of 8–10 kDa againstthe buffer of choice overnight at 4°C in a cold room.Two changes of buffer, 500 ml each, were given forequilibration of the protein with the buffer or buffercontaining 0.5 M urea. The protein sample was care-fully collected from the dialysis tubing and was centri-

Analytical Biochemistry 284, 406–408 (2000)

doi:10.1006/abio.2000.4703

0003-2697/00 $35.00Copyright © 2000 by Academic Press

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fuged at 15,000g for 10 min at 4°C in a Sorvall RC 5Crefrigerated centrifuge. This was done to get rid of any

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407NOTES & TIPS

suspended impurities, which might contribute to noisein the DSC scans. The protein sample and the corre-sponding buffer were then degassed before loading intothe DSC cells. The protein concentrations used for thestudy were between 1.0 and 1.2 mg/ml. Experimentswere carried out in triplicate for each of the conditionsused.

Centricon concentrator procedure. The protein solu-tion was dissolved in 2 ml of buffer or solvent of choiceand was loaded in Centricon concentrators (Amicon,Inc.) followed by giving a wash with the buffer of choiceby centrifugation at 5000g at 4°C in the Sorvall centri-uge. The protein solution was equilibrated using ap-roximately 20 ml of the buffer of choice by passing theolution through the Centricon concentrator two tohree times. A Centricon concentrator of 10-kDa cutoffas used for the processing of RNase A. After process-

ng of the sample, the protein was diluted to appropri-te concentrations (1–1.2 mg/ml) and was subjected toentrifugation at 15,000g for 10 min at 4°C. The solu-

tion was then degassed for 10 min before loading intothe DSC cell.

Differential scanning calorimetry. An MC-2 differ-ential scanning calorimeter interfaced to a 486-DXcomputer was used for carrying out the thermal dena-turation of RNase A. A scan rate of 60°C/h was used.The principle and operation of the instrument havebeen described elsewhere (6, 7).

Results and Discussion

Figures 1A and 1B present the DSC curves forRNase A at different pH conditions in the presence andabsence of 0.5 M urea using the Centricon procedureand conventional dialysis, respectively. In addition,DSC curves for the protein after exchanging the bufferfrom pH 4.0 to 2.5 either by Centricon or by dialysisequilibration are also presented. Table 1 shows thevalues of Tm and DH d for RNase A unfolding evaluatedfrom the DSC curves using the two procedures. The Tm

and DH d values for RNase A processed by both theethods at several pH conditions do not show any

ignificant difference and the values are within thexperimental uncertainties expected. The values form and DH d evaluated under certain conditions alsoatch well with those reported in the literature by

ther workers using DSC (1, 2) and by us using spec-roscopy (8). The shape of the DSC curves obtained islso identical in both methods of preparation of theample used. The direct addition of lyophilized RNasepowder to the buffer without Centricon processing or

ialysis resulted in slightly different Tm and DH d val-ues relative to the one with the two procedures. Sincethe MC-2 differential scanning calorimeter is unable to

determine directly the absolute values of the heat ca-pacity as a function of temperature, unlike the VP-DSC(9) and nano DSC (10) available currently, the heatcapacity values are not presented in the table. How-ever, the original curves showing the temperature de-pendence of the heat capacity of the protein relative tothe buffer taken as zero at the starting temperatureare presented in Figs. 1A and 1B.

The results obtained suggest that processing of sam-ples using Centricon concentrators is as effective asprocessing by using conventional dialysis. However,processing with Centricon concentrators can be consid-ered superior to dialysis in many respects for DSCexperiments under certain conditions. First, it requiresvery small volumes of solution compared to dialysisand is thus very economical in the case of sampleswhich may be very costly to prepare in large volumes.Second, in the case of concentrated solutions, there are

FIG. 1. Calorimetric transitions of RNase A under the followingconditions of buffer and pH: (1) 20 mM glycine–HCl buffer, pH 2.5 10.5 M urea; (2) 20 mM glycine–HCl buffer, pH 2.5; (3) pH exchangefrom 20 mM sodium acetate buffer, pH 4.0, to 20 mM glycine–HClbuffer, pH 2.5; (4) 20 mM sodium acetate buffer, pH 4.0; (5) 20 mMphosphate buffer, pH 7.0. (A) Transitions for the protein processedusing Centricon concentrators; (B) transitions for the protein pro-cessed using conventional dialysis membranes.

4. Santoro, M. M., Liu, Y., Khan, S. M. A., Hou, L.-X., and Bolen, D. W.(1992) Increased thermal stability of proteins in the presence ofnaturally occurring osmolytes. Biochemistry 31, 5278–5283.

1

TABLE 1

Various Thermodynamic Parameters Evaluated from the

p

2

2

2

408 NOTES & TIPS

bound to be large errors in the evaluated thermody-namic parameters due to evaporational losses as re-ported for dialysis (5). This is essentially due to thelong duration of experiments. Also, for proteins whichare thermolabile and need quick processing, the Cen-tricon procedure should be preferred. Even though thecurrently available high-sensitivity VP-DSC and nanoDSC may be able to pick up slight differences in theheat capacity of denaturation in different buffer sys-tems by using the two procedures, for most practicalpurposes, Centricon processing of samples would beadvantageous since it requires smaller solvent vol-umes, is economical, and is less time-consuming.

Conclusion. Considering the above results, it hasbeen demonstrated that the use of a Centricon concen-trator setup is as effective as dialysis in terms of theDSC results obtained. The Centricon method can beconsidered much superior to and more advantageousthan the dialysis procedure since it requires very littlevolume of buffer or cosolvent system for equilibrationof samples and is also much less time-consuming.

Acknowledgment. This work was supported by a grant from theDepartment of Science and Technology, Government of India, underwhich the DSC equipment was procured.

REFERENCES

1. Privalov, P. L. (1979) Stability of proteins. Small globular pro-teins. Adv. Protein Chem. 33, 167–241.

2. Makhatadze, G. I., and Privalov, P. L. (1992) Protein interactionswith urea and guanidinium chloride. A calorimetric study. J.Mol. Biol. 226, 491–505.

3. Plaza del Pino, I. M., Pace, C. N., and Freire, E. (1992) Temper-ature and guanidine hydrochloride dependence of the structuralstability of ribonuclease T1. Biochemistry 31, 11196–11202.

DSC Transitions for RNase A after Processing the Protein bythe Centricon Concentrator and by Dialysis

Solvent pH Tm (°C)DH d

(kcal mol21)

20 mM glycine–HCl buffer 2.5 39.2 6 0.1a 79 6 339.3 6 0.1b 78 6 2

H exchange from 20 mMsodium acetate, pH 4.0, to20 mM glycine–HCl, pH2.5 2.5 39.1 6 0.1a 76 6 2

39.3 6 0.1b 75 6 20 mM glycine–HCl bufferwith 0.5 M urea 2.5 35.5 6 0.1a 68 6 2

35.4 6 0.1b 70 6 20 mM sodium acetate buffer 4.0 57.9 6 0.1a 94 6 3

57.8 6 0.1b 92 6 20 mM phosphate buffer 7.0 64.6 6 0.05a 123 6 5

64.6 6 0.05b 118 6 3

a Centricon-processed samples.b Dialyzed samples.

5. Johnson, C. M., and Fersht, A. R. (1995) Protein stability as afunction of denaturant concentration: The thermal stability ofbarnase in the presence of urea. Biochemistry 34, 6795–6804.

6. Krishnan, K. S., and Brandts, J. F. (1978) Scanning calorimetry.Methods Enzymol. 49, 3–14.

7. Sturtevant, J. M. (1987) Biochemical applications of differentialscanning calorimetry. Annu. Rev. Phys. Chem. 38, 463–488.

8. Kaushik, J. K., and Bhat, R. (1998) Thermal stability of proteins inaqueous polyol solutions: Role of the surface tension of water in thestabilizing effect of polyols. J. Phys. Chem. B 102, 7058–7066.

9. Plotnikov, V. V., Brandts, J. M., Lin, L. N., and Brandts, J. F.(1997) A new ultrasensitive scanning calorimeter. Anal. Bio-chem. 250, 237–244.

0. Privalov, G., Kavina, V., Freire, E., and Privalov, P. L. (1995)Precise scanning calorimeter for studying thermal properties ofbiological macromolecules in dilute solution. Anal. Biochem. 232,79–85.

Internal Alu-Polymerase Chain Reaction:A Sensitive Contamination Monitoring Protocolfor DNA Extracted from PrehistoricAnimal Bones

Carsten M. Pusch,* Lutz Bachmann,†,1

Martina Broghammer,* and Michael Scholz‡*Molecular Genetics Laboratory, University Eye Hospital,University of Tubingen, Auf der Morgenstelle 15, D-72076Tubingen, Germany; †Pritzker Laboratory for MolecularSystematics and Evolution, Field Museum, 1400 SouthLake Shore Drive, Chicago, Illinois 60605-2496; and‡Institute of Proto- and Prehistory, Department ofArcheobiology, University of Tubingen, Eugenstrasse 40,D-72072 Tubingen, Germany

Received January 24, 2000

DNA extracted from prehistoric material is oftencontaminated by extraneous biomolecules. Because theseparation of ancient and contemporary/vintage DNAis currently not possible, techniques for detection ofcontaminated samples are important. We present aquick PCR-mediated method for the identification ofDNA contamination derived from primate nucleic acidsin nonprimate specimens. Internal Alu-PCR (IAP)2

amplifies Alu repetitive elements that are present at acopy number in excess of 500,000 per haploid humangenome equivalent, producing PCR products that are

1 To whom correspondence should be addressed. Fax: (312) 6657754. E-mail: bachmann@fmppr.fmnh.org.

2 Abbreviations used: IAP, internal Alu-PCR; mt, mitochondrial.

Analytical Biochemistry 284, 408–411 (2000)doi:10.1006/abio.2000.4666

0003-2697/00 $35.00Copyright © 2000 by Academic Press

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