NotesEffect of Glass on the Polymerization of G-Actinto F-Actin

Priya S. Niranjan, † Jeffrey G. Forbes, † andSandra C. Greer* ,†,‡

Department of Chemistry and Biochemistry and Department ofChemical Engineering, The University of Maryland at CollegePark, College Park, Maryland 20742-2111

Received March 6, 2000Revised Manuscript Received April 17, 2000

1. Introduction

Under appropriate conditions of temperature, concentrationof actin, and concentration of salts, the globular proteinG-actin aggregates reversibly to form the filamentary poly-mer F-actin.1 Since actin is an important protein, this processhas been studied extensively, but the details of this reactionmechanism are still not well understood.2 The polymerizationof actin, in vivo and in vitro, is induced by the presence ofsalts. We report here new experiments which show that inthe absence of such added salts, the polymerization of rabbitmuscle actin is induced by borosilicate glass containers butis not induced by vitreous silica containers. This result is ofconsiderable importance in studies of actin polymerization,since the container is normally assumed to be inert.

We have studied the fluorescence of pyrene-labeled rabbitmuscle G-actin as a function of temperature.3 At lowtemperatures, the actin is below its polymerization temper-ature and does not polymerize.4,5 As the temperature increasesand exceeds the polymerization or “floor” temperature, anincrease in fluorescence indicates that polymerization istaking place.6 When KCl is added to freshly prepared actinin buffer solution in a vitreous silica vessel, we see clearevidence of polymerization of the G-actin; this has beenobserved by neutron scattering4 as well as by fluorescencelabeling.3 For freshly prepared G-actin in a vitreous silicacell and in the absence of any added salts, we observe noevidence of significant polymerization at temperatures abovethe polymerization temperature. However, when the actin iscontained in a glass cell in the absence of added salt, wesee clear evidence of polymerization at the higher temper-atures.

2. Materials and Methods

2.1. General.The fluorescence of pyrene-labeled actin inbuffer was measured as the temperature was increased. Thesamples were contained in cells made from vitreous silica,from glass, and from silanized glass.

2.2. Actin Preparation. As in our previous work,4 rabbitmuscle acetone powder was prepared as described by Pardeeand Spudich.7 The actin was extracted from the acetonepowder into buffer A (4 mM Tris, 0.2 mM Na2ATP, 0.5mM 2-mercaptoethanol, 0.2 mM CaCl2, 0.005% azide, inH2O). The resulting G-actin solution was polymerized, byadding KCl to a final concentration of 100 mM and byincreasing the ATP concentration to 1 mM and the MgCl2

concentration to 2 mM, and then stored at 4°C as F-actinstock solution at about 3 mg/mL actin.

The stock solution was diluted to about 0.5 mg/mL; moreKCl, ATP, and MgCl2 were added to ensure full polymer-ization, and the solution was ultracentrifuged at 150 000gto make a pellet of F-actin. The pellet was resuspended inbuffer A and then depolymerized by dialysis in a collodionbag (10 000 Da cut off) against buffer at 4°C with rapidstirring, for approximately 12 h. The resulting G-actinsolution was then centrifuged at 120 000g for 1.5 h at 4°Cto pellet any remaining F-actin. The supernatant solution ofG-actin was further purified by size exclusion chromatog-raphy with Sephadex G-150, using the same buffer A. Thepurified G-actin was studied within 48 h. All the vessels usedin the actin purification were of plastic, except for the glassSephadex column.

G-Actin concentrations were obtained from the UVabsorbance at 290 nm, using an extinction coefficient ofε290

) 0.63 cm2/mg.8,9 Actin purity was assessed by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The actin purity was analyzed before and after theexperiments described below; both analyses showed verypure (>95%) actin.

The fluorescence of pyrene-labeled actin is the mostsensitive and accurate assay for actin polymerization.6 TheF-actin was labeled for the fluorescence studies using themethod used by Kouyama and Mihashi.10 F-Actin stocksolution was diluted to 0.5 mg/mL, completely polymerizedby again adding KCl, ATP, and MgCl2, and then dialyzedagainst buffer F (which is the same as buffer A but containsno 2-mercaptoethanol).N-(1-Pyrenyl)iodoacetamide (Mo-lecular Probes, Eugene, OR) was added to the dialyzedG-actin solution in a 4:1 molar ratio of dye to actin andallowed to react overnight on ice. Dithiothreitol was addedto a final concentration of 1 mM to remove unreacted dye.The sample was then ultracentrifuged at 120 000g for 1.5 hat 4 °C. A yellow pellet was obtained which was homo-genized and depolymerized by dialysis against buffer A asdescribed above. The dialyzed, labeled G-actin was purifiedon a Sephadex column as above. The labeled G-actinconcentration was calculated by measuring the UV absor-bance at 344 nm and using an extinction coefficient of 2.2× 104 M-1 cm-1.10 The labeled and purified G-actin was

* Corresponding author. Telephone: (301) 405-1895. Fax: (301) 405-0523. Email: sg28@umail.umd.edu.

† Department of Chemistry and Biochemistry.‡ Department of Chemical Engineering.

506 Biomacromolecules 2000,1, 506-508

10.1021/bm005553g CCC: $19.00 © 2000 American Chemical SocietyPublished on Web 06/10/2000

mixed with unlabeled purified G-actin to produce a mixtureof 3% labeled actin and 97% unlabeled actin. The totalG-actin concentration in the samples studied here was 3.1mg/mL, and 0.25 mL of sample was injected into each cell.We chose this rather high concentration of actin in order tobe able to compare these experiments to our earlier neutronscattering experiments, for which we needed high concentra-tions to have large scattering signals.4

2.3. Cells. The spectrometer cells (Starna Cells, Inc,Atascadero, CA) were made from (1) “special optical glass”,(2) “Spectrosil vitreous silica”, and (3) silanized “specialoptical glass” (see below). “Special optical glass” is aborosilicate glass (Schott Glass, Mainz). The optical windowsof the Spectrosil cells are of Spectrosil and the other wallsare of Vitreosil. “Spectrosil vitreous silica” is a syntheticquartz made from silicon dioxide formed by vapor phasehydrolysis of silicon. All cells had interior dimensions of 4mm × 4 mm × 45 mm and nominal volumes of 0.56 mL.The cells were constructed by fusing the walls; thus noadhesives were present. The glass and vitreous silica cellswere rinsed several times with 10% HCl and then cleanedby sonication in deionized water. All cells were oven-driedat 120 °C before use. Both glass and vitreous silica cellsfilled with Nanopure water showed no fluorescence at 407nm.

The glass cell to be silanized was first cleaned thoroughlyand oven-dried. It was then placed for 20 min in a 2%solution of octadecyltrichlorosilane (Fluka) in hexane, afterwhich it was rinsed in hexane and dried at 120°C for 30min.

2.4. Measurement of the Extent of Polymerization.Thefluorescence was measured by an Aminco Bowman Series2 luminescence spectrometer with the excitation wavelength,λex, set at 365 nm, resulting in emission wavelengths atλem

) 387 and 407 nm. The temperature at the start of eachexperiment was 0.5°C; the temperature was increased insteps of 2°C to a maximum of 30°C, with a 25 minequilibration time after each temperature change. Theincrease in polymer concentration was determined bymeasuring the increase in the fluorescence signal at 407 nm.

After the temperature reached about 30°C, the samplewas completely polymerized by bringing the concentrationof MgCl2 to 15 mM. We then calculated the extent ofpolymerization of actin from the ratio of the fluorescence ata given temperature to the fluorescence of the fully polym-erized sample.

3. Results

Figure 1 shows the extent of polymerization of G-actincontainingno initiating saltas a function of temperature forsamples in cells made of (1) Spectrosil vitreous silica, (2)borosilicate glass, and (3) silanized borosilicate glass. Themeasurements shown were all made on aliquots of G-actintaken from the same preparative batch. Also shown in Figure1 for comparison are (4) our measurements of the extent ofpolymerization of G-actin from another preparative batch of2.9 mg/mL actin with 9 mM KCl as initiating salt.3

We first note that negligible polymerization took place inthe G-actin sample (1) that was studied in the vitreous silica

cell. The nonzero extent of polymerization for that sample(and the other samples) reflects the fluorescence of G-actin,which has not been subtracted. Second, it is clear thatconsiderable polymerization of the actin occurred in theborosilicate glass cell (2) as the temperature was increased,even though no initiating salts had been added to the actin.Compare the polymerization measured in sample (4) of 2.9mg/mL G-actin in buffer A with 9 mM (0.04%) KCl addedas an initiator for the polymerization. Third, the sample inthe silanized borosilicate glass (3) also polymerized, but thepolymerization began at a higher temperature than for theunsilanized borosilicate glass. Samples 1-3 were lateranalyzed by atomic absorption spectroscopy (GalbraithLaboratories) for Ca2+, Na+, and K+; analysis showed nopresence of these species in any of the samples, at detectionlimits of 0.01%, except that Na+ was detected at 0.01% forthe unsilanized borosilicate glass sample. The behavior inFigure 1 was reproduced with a second preparative batch ofG-actin.

Figure 2 shows the time development of samples 1, 2,and 4 in Figure 1, after an increase in temperature of 2°Cfrom 20 °C. Sample 1 in a vitreous silica cell without saltremained unpolymerized.Sample 4 containing KCl in avitreous cell showed an increase in extent of polymerizationand reached a new equilibrium after about 30 min. Bycontrast, sample 2 in the glass cell with no salt showed anincrease in extent of polymerization which had not reachedequilibrium even after 70 min. Thus the data in Figure 1 forthe glass cell (2) do not represent an equilibrated system.

Figure 1. Extent of polymerization as a function of temperature forsamples of 3.1 mg/mL rabbit muscle G-actin in buffer (see text), asmeasured by labeled fluorescence spectroscopy. The actin sampleswere held in cells made of (1) Spectrosil vitreous silica, (2) borosilicateglass, and (3) silanized borosilicate glass. Also shown are (4)measurements of the extent of polymerization of 2.9 mg/mL G-actinin buffer containing 9 mM KCl as initiating salt.3 The nonzero extentof polymerization at low temperatures reflects the fluorescence ofG-actin, which has not been subtracted.

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4. Discussion and Conclusions

Borosilicate glass vessels can cause the polymerization ofrabbit muscle actin. We do not know the exact mechanismof this process. Silanization of the glass produces a hydro-phobic coating which prevents the actin from contacting theglass surface. The actin in the silanized cell still polymerized,but that polymerization began at a temperature about 12°Chigher than in the case of the unsilanized cell. This resultsuggests that the polymerization in the borosilicate glass cellis due to the leaching of ions from the glass into the solution,rather than the polymerization of the actin by a process onthe surface of the cell. This view is supported by the detectionof a small amount of Na+ in the sample from the unsilanizedcell and by the failure of that sample to reach an equilibratedlevel of polymerization even after 70 min.

The known properties of borosilicate glass and of vitreoussilica are consistent with our observations. Borosilicate glassgenerally contains Na2O, K2O, B2O3, and Al2O3, in additionto SiO2.11,12 It is known that water leaches ions from glass,that the alkali metals are preferentially removed, and that

cleaning with acid serves to remove alkali metals.12 Spectrosilvitreous silica (or fused silica) is nearly pure SiO2, with lessthan 0.1 ppm Na, 0.3 ppm Ca, 0.1 ppm K, and 0.5 ppmMg.13,14 Vitreosil is similar to Spectrosil, but does not haveas good optical properties; Vitreosil has less than 2 ppm Na,1.5 ppm Ca, 1.8 ppm K, and no significant Mg.14 Vitreoussilica “is relatively unaffected by any acidic medium” exceptHF; HCl can remove traces of alkaline materials fromvitreous silica.13

We suggest that studies of the polymerization of actin arebetter done in vitreous silica or plastic containers and thatthe inertness of any cell should be verified.

Acknowledgment. This work was supported by theNational Institute of Arthritis and Musculoskeletal and SkinDiseases, a part of the National Institutes of Health. We thankN. Blough for the use of his luminescence spectrometer andA. K. Hulme of Optiglass Ltd. (Essex, U.K.) for informationon the cell materials.

References and Notes(1) Oosawa, F.; Asakura, S.Thermodynamics of the Polymerization of

Protein; Academic Press: New York, 1975.(2) Sheterline, P.; Clayton, J.; Sparrow, J. C.Actins; 3rd ed.; Academic

Press: San Diego, CA, 1996.(3) Niranjan, P. S.; Forbes, J. G.; Greer, S. C. To be published.(4) Ivkov, R.; Forbes, J. G.; Greer, S. C.J. Chem. Phys.1998, 108,

5599-5607.(5) Greer, S. C.J. Phys. Chem.1998, 102, 5413-5422.(6) Cooper, J. A.; Pollard, T. D. InMethods in Enzymology: Structural

and Contractile Proteins, Part B, The Contractile Apparatus andthe Cytoskeleton; Frederiksen, D. W., Cunningham, L. W., Eds.;Academic Press: New York, 1982; Vol. 85, pp 182-210.

(7) Pardee, J. D.; Spudich, J. A.; InMethods in Enzymology: Structuraland Contractile Proteins, Part B, The Contractile Apparatus andthe Cytoskeleton; Frederiksen, D. W., Cunningham, L. W., Eds.;Academic Press: New York, 1982; Vol. 85, pp 164-181.

(8) Maclean-Fletcher, S.; Pollard, T. D.Biochem. Biophys. Res. Commun.1980, 96, 18-27.

(9) Houk, T. W.; Ue, K.Anal. Biochem.1974, 62, 66-74.(10) Kouyama, T.; Mihashi, K.Eur. J. Biochem.1981, 114, 33-38.(11) Moore, J. H.; Davis, C. C.; Coplan, M. A.Building Scientific

Apparatus: A Practical Guide to Design and Construction; 2nd ed.;Addison-Wesley: New York, 1989.

(12) Adams, P. B.Ultrapurity Methods and Techniques; Zief, M., Speights,R., Eds.; Marcel Dekker: New York, 1972; pp 293-351.

(13) Hetherington, G.; Bell, L. W. InUltrapurity Methods and Techniques;Zief, M., Speights, R., Eds.; Marcel Dekker: New York, 1972; pp353-400.

(14) Hulme, A. K. Personal communication.

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Figure 2. Extent of polymerization as a function of time for samplesof 3 mg/mL rabbit muscle G-actin in buffer, as in Figure 1, after anincrease in temperature of 2 °C from 20 °C.

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