ANALYTICAL

BIOCHEMISTRY

Isoelectric

MICHELE Cattedra

88,

212-224 (1978)

Focusing and Electrophoresis Subzero Temperatures

PERRELLA, ADRIANA HEYDA, ANDREA AND LUIGI ROW-BERNARDI

di Enzimologia,

University

of Milan,

Via G. Celoria,

at

MOSCA,

2, 20133

Milan,

Italy

Received July 28, 1977: accepted January 26, 1978 Electrophoretic and isoelectric focusing separations have been achieved at -20 to -30°C i.e., at temperatures considerably lower than previously reported by using as supporting media gels of acrylamide-methylacrylate copolymers and dimethylsulfoxide-water mixtures. Hybrids of human and sickle cell hemoglobin and partially oxidized human carboxyhemoglobin have been separated in the temperature range -20 to -30°C both by a discontinuous buffer gel electrophoresis and by isoelectric focusing.

Short-lived intermediates of biochemical interest can be stabilized at subzero temperature and then analyzed by various physical and chemical techniques (1,2). A key issue in this approach is the development and adaptation of current biochemical methods of separation at low temperatures using suitable hydro-organic, antifreezing solvents. The application of ion exchange, gel filtration, and IEF’ methods at low temperature has already been reported (3-5). In this study we describe how electrophoretic and IEF separations can be achieved, at considerably lower temperatures than previously reported, by using as supporting media gels of acrylamide-methylacrylate copolymers and DMSO-water mixtures. MATERIALS

AND METHODS

HbA and HbS were prepared by the method of Adair and Adair (6), respectively, from the blood of a normal and a homozygous sickle cell donor. HbA, as the carbonmonoxy derivative, was partially oxidized by addition of stoichiometric amounts of potassium ferricyanide at pH 6.5, ’ Abbreviations used: IEF, isoelectric focusing; DMSO, dimethylsulfoxide; TEMED, N,N,N’,N’-tetramethylethylenediamine; AP, ammonium persulfate; EGOH, ethylene glycol: DMF, dimethylformamide; MeOH, methanol; HbA, (Y&&, normal human hemocarbonmonoxyglobin; HbS (Y&*, sickle cell hemoglobin; HbCO, (YIPS, hemoglobin; MetHb, ao+&+, methemoglobin; a,(CO)Pz+, hemoglobin with CO-liganded (Y chains and /3 chains in MetHb form; a,+&(CO), hemoglobin with u chains in MetHb form and CO-liganded p chains. 0003-2697/78/088 l-0212$02.00/0 Copyright All rights

0 1978 by Academic Press, Inc. of reproduction in any form reserved.

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R-

FIG. I. Lucite apparatus for IEF and electrophoresis at subzero temperatures. A, anodic compartment; B, refrigerated tube holder: C, cathodic compartment; D, cathode carrying cover: R, coolant inlet and outlet; S, glass coil for refrigeration of liquid in A; P, glass tubes for tilling A with a precooled liquid; T, gel containing glass tubes; G, rubber gaskets.

20°C. The reaction was stopped after 30 or 60 min by removing the ferricyanide on a Sephadex G25F column equilibrated with 30 mM phosphate, pH 6.5. Ampholine was obtained from LKB, Stockholm, Sweden; methylacrylate, from Eastman Organic Chemicals, Rochester, N. Y.; and ethyl, n-butyl, and 2-hydroxyethylacrylate. from Fluka AG, Buchs, Switzerland. Equipment for gel IEF and electrophoresis. Figure 1 shows the Lucite apparatus of Righetti and Drysdale (7) modified for tube electrophoresis at subzero temperatures. The tubes (T) containing the gel were fitted in the refrigerated tube holder (B) through the rubber gaskets (G). B was sealed to the anodic compartment (A) by means of a plaster strap. The whole apparatus, with the exception of the upper edge of the cathodic compartment (C), was enclosed in a polystyrene box provided with a Lucite window and containing bags of dry silica gel to minimize condensation. The gels and the liquid in the anodic compartment (A) were refrigerated by circulation of an antifreezing solution from a Lauda K4R thermostat. The liquid in A was precooled and introduced into this compartment through the glass tubes (P). The liquid contained in the cathodic compartment was precooled and poured directly in C. Heat exchange with the coolant circulating in B was usually sufficient to prevent the temperature of the liquid in C from rising significantly above the temperature of the refrigerating fluid. The upper part of the apparatus (C) that was not enclosed in the polystyrene box was also thermally insulated.

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ET AL.

Preparation of the gels. Gels were prepared in glass tubes 11 cm long, with an inner diameter of 2.5 mm and a wall thickness of 0.7 mm. The gels were made 8 cm long to ensure that their main part, and especially their tops, were efficiently cooled. Copolymerization was performed at 0°C by the addition of 0.2% (v/v) TEMED and 0.2% (w/v) AP to a solution of acrylamide, acrylic acid ester, and bisacrylamide in the hydro-organic solvent that also contained the electrophoretic buffer or Ampholine. This solution was deoxygenated and introduced into tubes T that were supported in a specially made holder surrounded by water refrigerated at 0°C. The filling of the tubes with the polymerizing solution was done using a l-ml plastic syringe provided with a l-mm-diameter silicone tubing attached to the syringe needle. The tubes were slowly filled from the bottom to eliminate entrapment of air bubbles. A few microliters of the same cold solvent used for IEF or electrophoresis, including TEMED and AP, were deposited on the polymerizing solution. The procedure described is essential for obtaining homogeneous gels with a flat top surface. The use of a larger than normal amount of AP (7) was necessary because of the presence in the acrylic acid esters of stabilizers that had not been removed. The polymerization time was 3 to 4 hr at 0°C as estimated by infrared spectroscopic examination of the copolymer. Preparation of samples for ZEF and electrophoresis. Samples for IEF in gels containing DMSO were prepared by mixing isoionic hemoglobin solutions with Ampholine (final concentration, 2% [w/v]; pH range, 5 to 7) and EGOH, 40% (v/v) at 0°C. EGOH was used in the samples instead of DMSO, since it never caused denaturation of the protein on mixing with hemoglobin. It also provided the high-density medium needed to avoid excessive convective disturbances during the initial stage of protein entrance into the gel. Samples for electrophoresis were prepared in the aqueous bufferDMSO mixture having the same DMSO composition as the gel phase. This was accomplished as follows. Hemoglobin was gel filtered at 4°C in aqueous buffer, and the solution was added to a precooled (- lO’C> DMSO-water mixture to obtain the final buffer and DMSO compositions. Although this procedure was not equivalent to that suggested by Douzou (8), namely, the synchronous addition of organic solvent and cooling, no significant protein denaturation was observed. Protein denaturation occurred only when hemoglobin solutions were mixed with pure DMSO. Sucrose (10%) was then added to the samples used for electrophoresis in order to increase the density and minimize the convective disturbances that would interfere with the stacking process (see later). Measurement of gel temperature. The difference in temperature (At) between the central region of a gel contained in a glass tube (5 mm id, 1 mm wall thickness) and the refrigerating fluid was measured as a function of the

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21.5

power (I x V) applied in an electrophoretic experiment at subzero temperatures. In this experiment, the gel phase and the electrodic liquids had the same buffer composition. The measurements were taken with a thermistor embedded in the gel. The thermistor diameter was about 2 mm, and its sensor was about 1 cm below the gel surface. A nearly linear relationship, independent of the subzero temperature of the experiment, was found between the power applied and At. As an example, At was less than O.YC at I x V = 0.1 W and about 2°C at I x V = 0.5 w. In IEF, local differences in ohmic heat are possible because of the pH gradient. The same heat exchange efficiency was also found in IEF experiments. Protein migration in our IEF experiments occurred about 2 cm below the gel surface, and the applied power was never greater than 0.1 W/tube. Thus it is likely that in both the electrophoresis and IEF experiments described in this paper the gel temperature did not rise higher than 1°C above the temperature of the refrigerating fluid. RESULTS

Choice of the Copolymer

and the Hydro-organic

Solvent

It has been shown by Park (5) that IEF of hemoglobin can be carried out at -5 to - 10°C in polyacrylamide gels (T% = 8%, C% = 1.2% bisacrylamide) containing 6% Ampholine (pH range, 7 to 9) plus 3% Ampholine (pH range, 6 to 8) in 20 to 25% (v/v) EGOH-water mixtures. Polyacrylamide gels in EGOH-water and other hydro-organic solvents become opaque and unsuitable for IEF or electrophoresis at a temperature near or below - 10°C. This condition is reversible and probably related to the attainment of the glass transition temperature of the polymer (10). For this reason, the experimental conditions described by Park cannot be used for experiments that require temperatures lower than - 10°C. Another difficulty that we encountered in our attempts to carry out electrophoretic experiments at subzero temperatures was that IEF in a water mixture that contains a high proportion of EGOH is very slow both at above zero and subzero temperatures. At -lo”C, using the conditions specified by Park, we found it extremely difficult to obtain satisfactory IEF of hemoglobin solutions. Park’s results were more easily reproduced by lowering the amount of EGOH to about 10% and by using only 2% Ampholine (pH range, 6 to 8). Under these conditions, however, the temperature could not be lowered below -5°C. DMSO-water mixtures seemed to be more suitable hydro-organic solvents. IEF on polyacrylamide gels prepared in DMSO-water mixtures containing up to 50% (v/v) DMSO (9) can be carried out satisfactorily at abovezero temperatures (11). A careful comparison of ion and protein mobilities at different

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ET AL.

temperatures in gels prepared in different hydro-organic solvents would require the investigation of a number of several of the parameters affected by the temperature and nature of the solvent, i.e., ion activities, pK values of the charged groups, and also the porosity of the gels. The latter varies because of the effect of the hydro-organic solvent on the degree of polymerization of the gel and because of the swelling effect produced on the gel by the different solvents. For an approximate comparison of hemoglobin mobilities, we observed that the migration rates of HbCO focused at 5°C on polyacrylamide gels (T% = 7.62%; C% = 1.57%; 2% Ampholine; pH range, 6 to 8) prepared in water, 25% DMSO, and 25% EGOH were, respectively, about 2.1, 2.1, and 0.9 cm/hr when the applied field was about 110 V/cm. Since the glass transition temperature of polymeric acrylic acid esters is depressed by increasing the length of the aliphatic chain of the alcohol (lo), we examined the behavior of the copolymers of the acrylamide with some acrylic acid esters in relation to (a) the nature of the ester, (b) TABLE

1

PROPERTIESOFGELSPREPAREDBY COPOLYMERIZATION OFACRYLAMIDE WITH SOME ACRYLIC ESTERS IN DIFFERENTHYDRO-ORGANICSOLVENTS

Hydro-organic solvent Acrylic ester Methylacrylate 0-CH, CH,=CH-CL0 Ethylacrylate O-CH,-CH CH,=CH-CL0 n-Butylacrylate

/

T’%”

C’%”

(%,

Approx. temperature limit in IEF or electrophoretic separations*

[V/VU

(“a

7.95 8.13 8.44 8.13 8.13

(0.2)’ (0.3) (0.5) (0.3) (0.3)

1.57 1.54 1.48 1.54 1.54

40% DMSO 40% DMSO 40% DMSO 40% MeOH SO% DMFd

-15 -25 -30 -8 -25

to -20 to -30 and lower to -12 10 -30

8.26 8.57 ‘8.88 8.57 8.57

(0.2) (0.3) (0.4) (0.3) (0.3)

1.51 1.46 1.41 1.46 1.46

40% 40% 50% 40% 50%

-20 -30 -30 -5 -25

to -25 and lower and lower

8.25

(0.1)

1.51

50% DMSO

DMSO DMSO DMSO MeOH DMF

to -30

-20 to -25

0-CH2-CH2-CH,-CH3 CH,=CH-CL0 p T’ and C’ are defined in the Results. * Estimated from the appearance of opacity of the gels. c Molar fraction of the acrylamide-ester mixture only. d DMF slowly reacts with the alkaline cathodic solution used for IEF. The hydrolysis products were found to interfere with IEF.

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the relative proportion of the two copolymerized monomers, and (c) the hydro-organic solvent employed. It was found that even in hydro-organic solvents some of the copolymers studied have physical properties, such as mechanical strength and transparency, that are very similar to those of polyacrylamide gels in acqueous solvents. The results are summarized in Table 1. In the table the usual T% and C% quantities defined by Hjerten (12) have been modified as follows: T,% = acrylamide c,~

0

+ ester + cross-linker 100 ml

= lOO.cross-linker

(g)

(g)

T’%

Proportions of the acrylate esters greater than those indicated in the table can be used, but polymerization must then be carried out at abovezero temperatures in order to solubilize the monomers. Gels prepared by copolymerization of acrylamide with 2-hydroxyethylacrylate were found unsuitable for gel electrophoresis. They were hard and brittle and not permeable to macromolecules. The gels prepared by the use of methylacrylate were found most satisfactory for their general properties, band resolution, and reproducibility of results. Gels made by using 50 to 60% methylacrylate are probably suitable for gel electrophoresis at temperatures lower than those reached in this study (-30°C). IEF at Subzero

Temperatures

It has been shown that partially oxidized HbCO is resolved by IEF at above zero temperatures into four components: a2+&+, a,(CO)p,(CO), and two hybrid forms, presumably a,+&(CO) and a,(CO)/32+ (5,13). If the same solution is focused at subzero temperatures, additional components are resolved; they presumably are the remaining five theoretically predictable, but temperature-labile, hybrid species (5). The components present in a sample of 60%-oxidized HbCO as separated by gel IEF at -20°C are shown in Fig. 2a. The color of the five main components on the gel varied gradually from the brown color of MetHb to the red color of HbCO. Two other minor colored components could be detected on the gels soon after focusing. If cyanide is added to the partially oxidized sample, a single red band is obtained, indicating that the pattern shown in Fig. 2a is not due to oxidation of side chain groups of the protein or reaction of the protein sulfhydryl and e-amino groups with traces of noncopolymerized methylacrylate (14). The same pattern was obtained by running the partially oxidized sample at -5°C on polyacrylamide gels in lO%EGOH as described by Park (5).

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1 mm

ET AL.

I

+

a

+

b

FIG. 2. (a) Microphotograph showing the pattern obtained by IEF at -20°C on a sample (about 120 pg) of 60%-oxidized HbCO. Gel contained acrylamide, methylacrylate, bisacrylamide (T’% = 8.44%; C’% = 1.48%: ester molar fraction, x = 0.5), and 1.6% (w/v) Ampholine (pH range, 6 to 8 ) in 35% (v/v) DMSO. The gels were prefocused for 2 hr at 350 V. After sample application, focusing was continued for 3 hr at 700 V. The current dropped from 0.07 to 0.04 mA/tube, and the power applied decreased similarly (0.05 to 0.03 W/tube). NaOH (20 mM) and HRPO, (10 mM) in 35% DMSO were used, respectively. as cathodic and anodic liquids. (b) Microphotograph showing the pattern obtained by IEF at -20°C of a 1: 1 mixture of CO-liganded HbA and HbS preincubated at 4°C in 0.1 M KC1 for 24 hr. Total hemoglobin was about 40 pg. Other conditions as in (a).In these experiments, true equilibrium in focusing was attained after longer times (about 5 hr). Under these conditions, a slight improvement in band spacing was observed without resolution into other components.

The second system examined by IEF at subzero temperatures was a mixture of HbA and HbS in their CO-liganded state. It has been shown that in this mixture hybrid molecules of the type, q@p” are formed (5,15). The relative concentrations of a,&, a&/P, and (YJ~~~should approximate a ratio of 1:2:1, respectively (15). At above-zero temperatures, however, the hybrid molecules in the liganded state cannot be separated by IEF or electrophoresis because the dissociation rate of the liganded tetramer into dimers (which can then recombine to give mixed tetramers or parent tetramers) is too fast compared with the separation rate (16). At subzero temperatures the dissociation rate is probably sufficiently slow to allow detection of the hybrid molecules in the liganded state (5). IEF of a mixture of HbA and HbS in the CO form at -20°C is shown in Fig. 2b. The mixture was preincubated in aqueous 0.1 M KC1 at 4°C for about 24 hr. The gel was not scanned for quantitation of the three components, but qualitatively the third middle component was present in a larger amount than in the parent species, as expected. Figure 3 shows the pattern obtained by focusing pure HbS (a), pure

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a

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C

b

219

SEPARATION

d

+ FIG 3. Microphotograph of a gel IEF of CO-liganded hemoglobins at -25°C. a, HbS; b, HbA; c, HbA and HbS incubated at -25°C on the gel top for 1 hr; d, mixture of HbA and HbS preincubated at 4°C in 0.1 M KCI for 1 or 24 hr. Samples contained a maximum of 20 pg of hemoglobin. Gels were prepared in 40% (v/v) DMSO. For other conditions see Fig. 2a.

HbA (b), and HbS and HbA mixed together at -25°C and left at this temperature for 1 hr before focusing (c). If the HbS and HbA are mixed together and left for 1 or 24 hr at 4°C the pattern in Fig. 3d is obtained, indicating the temperature-dependent formation of the hybrid. Electrophoresis at Subzero Temperatures

Hybrid molecules, such as those formed in a mixture of HbA and HbS, have been resolved by electrophoresis at above-zero temperatures only upon removal of the hemoglobin ligand (CO or 0,) (16). In the absence of the ligand, the dissociation rate of the hemoglobin tetramers is slow enough to allow detection of the hybrid species formed in the liganded state. As mentioned before, the dissociation rate of the liganded tetramers is strongly temperature dependent. We therefore used a mixture of COliganded HbA and HbS to test the feasibility of electrophoretic separation at low temperatures. The optimal conditions for electrophoretic separation were obtained by trial, since an estimate of the net charge of hemoglobin at the required temperature in the hydro-organic solvent used was not available. The most satisfactory method for electrophoretic separation on gels consisted of a modification of the discontinuous buffer electrophoresis, as originally described by Davis (17). Tris-glycine buffer in 40% DMSO-water was used as the electrode buffer, whereas Tris-HCl buffer in 40% DMSO-water was used in the gels. The stacking gel was omitted, since a very effective stacking of the proteins could be achieved as follows. Hemoglobin samples were

PERRELLA

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ET AL.

lmm I

a

b

1

+

G

d

FIG. 4. Microphotograph of an electrophoretic separation of CO-liganded hemoglobins at -30°C. a-d, as in Fig. 3. The total hemoglobin applied was no more than 40 ,ug/tube. One milliliter of the polymerizing solution contained: 0.4 ml of Tris-HC1 buffer (75 mM Tris, 13.4 mM HCl, pH 8.7, at 23°C) and 0.4 ml of DMSO; 0.2 ml of a water solution of acrylamide, methylacrylate. and bisacrylamide; TEMED; and AP (final volume, T’% = 8.44%, C’% = 1.82%: ester molar fraction, x = 0.5). The anodic and cathodic buffer was a mixture of 40% (v/v) DMSO-buffer (80 mM glycine, IO mM Tris, pH 8.4, at 23°C). Electrophoresis was carried out at 300 V, 0.03 mA/tube, during the stacking process (30 mitt) and then at 500 V, 0.06 mA/gel, for 2.5 hr.

equilibrated in Tris-HCl buffer and diluted with a DMSO-water mixture so to give a final DMSO concentration equal to that of the gels and a buffer concentration about half as large. Sucrose was added to the samples to increase the density. Stacking occurred in the liquid phase above the gel. Hemoglobins entered the gels as thin bands, and resolution of the mixture containing HbA, HbS, and the hybrid species (in the liganded state) was attained soon after entering the gel. HbA mobility under these conditions was about 1 cm/hr in a field of 100 V/cm. Figure 4 shows an electrophoretic run at -30°C on HbA and HbS solutions the same as those in Fig. 3. Figure 4a refers again to pure HbS, Fig. 4b, to pure HbA, and Figs. 4c and 4d show patterns similar to those of Figs. 3c and 3d. The measurements of the rate of hybrid formation at subzero temperatures in a mixture of HbA(C0) and HbS(C0) could be used to study the rate of dissociation of carbonmonoxytetramers into dimers in a manner similar to that employed by Bunn and McDonough (15), who obtained the dissociation rates of the deoxytetramers. As shown in Fig. 4c, no hybrid species were detected after 1 hr of incubation at -30°C of a one-toone mixture of HbA(C0) and HbS(C0). Therefore, such a mixture could be incubated at higher temperatures for varying times, the reaction could

SUBZERO TEMPERATURE

a

b

PROTEIN

221

SEPARATION

c

-

FIG. 5. Formation of the hybrid species in one-to-one mixtures of HbA(C0) and HbS(C0) after different incubation times at -5°C. which were terminated by lowering the temperature to -30°C. (a) 8, (b) 30, and (c) 60 min of incubation. The upper part of the figure shows microphotographs of the gel-containing tubes where the hemoglobins were separated at -30°C by electrophoresis. For additional details, see text. The lower part of the figure shows the corresponding microphotograph scans obtained by the use of a Joyce-Loeb densitometer.

be “stopped” by lowering the temperature to -3O”C, and the mixture could be analyzed by electrophoresis. Figure 5 shows microphotographs of HbA(C0) and HbS(C0) mixtures incubated for varying times at -5°C and separated by electrophoresis at -30°C in about 1 hr. Gel composition, gel buffer, and electrode buffer compositions were the same as those described for the experiment of Fig. 4. Samples of HbA(C0) and HbS(C0) containing about 2 pg/pl of protein were prepared in 10 mM Tris-HCl buffer that contained 50% DMSO. The pH of this buffer is about 8.5 at -5°C and 9.65 at -30°C (18). Samples also contained 10% sucrose. The experiment was carried out as follows. The gel-containing tubes were fitted in the electrophoretic apparatus and refrigerated at -5°C by the use of a Lauda cooler. All the liquid on the gel was replaced with n-hexane, which filled the tubes to the top. Five microliters of each hemoglobin sample was deposited on the gel top by the use of a Micropettor (Micropettor SMI, Emeryville, Calif.) and mixed there by sucking the

222

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ET AL.

liquid up and down gently 20 times. Hexane was used because (a) it allowed the samples to be cooled at -5°C by immersing the Micropettor in the supematant cold solvent for a few minutes before delivering the sample, (b) the hemoglobin mixture was rigorously kept at -5°C during the mixing procedure and incubation, (c) the hemoglobin samples were not diluted with other liquids and contamination with other buffer ions was minimal, and (d) hexane was well separated from the immiscible aqueous solution at the end of the mixing procedure in spite of the fact that some hexane droplets were sucked into the Micropettor with the hemoglobin solution. At elapsed times recorded from the beginning of the mixing, the temperature of the gels was brought down to -30°C in less than 1 min by switching the circulation of refrigerating liquid to a second Lauda cooler kept at -30°C. Ten microliters of gel buffer, diluted by half with a 40% DMSO-water mixture and containing 5% sucrose, was layered on the samples, and finally, all the hexane in the tubes was replaced with electrode buffer before starting the separation. The different sucrose concentrations were used to establish a density gradient that would minimize mixing while depositing the different liquid layers. Mixtures of HbA(C0) and HbS(CO), incubated at -30°C for 1 to 2 hr in the same solvent, pH 10.6 at -30°C did not show hybrid formation. Instead, a progressive formation of hybrid species at -5°C is evident from Fig. 5. Since the rate of dissociation of the hybrid tetramer is probably similar to that of the parent tetramer molecules (15), clearly this approach with some technical refinements could be exploited to study the dissociation rate of liganded tetramers of hemoglobin. DISCUSSION

AND CONCLUSIONS

The results obtained show that both IEF and electrophoresis can be carried out at subzero temperatures in hydro-organic solvents without a great loss of speed and resolution. Band patterns obtained in this study by IEF were quite sharp. However, in both Hb systems examined, the components that can be well resolved under ordinary conditions (such as HbA and HbS or MetHb and HbCO) were more closely spaced than usual. This was probably due to a shift in the pK values of all the charged groups involved, caused by the combined effect of the hydro-organic solvent employed and the low temperature. In addition, the lower the temperature, the lower was the migration rate of the proteins in focusing. High electrical fields were of no help, since the gels, starting from the gel top, became increasingly distorted. An additional problem in IEF was caused by protein precipitation during the stacking process that was due to the combination of high protein concentration, low temperature, and low ionic strength. To avoid this ef-

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223

feet, it was necessary to reduce protein concentration by trial and error so as to not overload the gels. Thus, whereas IEF at -20°C was usually accomplished with relative ease (Fig. 2b), focusing at -25°C was found to be a much more critical experiment (Fig. 3). Another complicating factor at this low temperature is due to Ampholine precipitation extending from the anodic region. Ampholine solubility is thus another limiting factor for IEF separations at very low temperatures. Electrophoretic separations, on the other hand, could be carried out more easily at the lowest temperatures attained in this study (-30°C). No problems were encountered with gel distortion, protein solubility, or macromolecular mobility. In some cases, better resolution than in IEF was obtained. For instance, the separation between HbA and HbS obtained by electrophoresis at -30°C after 180 min at 100 V/cm was about 10 mm, compared with a separation of 2 mm attained in IEF runs at -25°C. We have not attempted the separation of protein systems other than hemoglobins. However, the gels used here for IEF or electrophoresis showed, upon staining with Coomassie blue, other protein components in addition to hemoglobin, such as carbonic anhydrase, catalase, and other minor hemoglobins that are also seen in gel IEF or electrophoresis in normal conditions. This indicates that more complex protein systems differing in molecular size, as well as charge, can be separated at subzero temperatures following the procedures of the present study. The two hemoglobin systems examined to test the feasibility of subzero gel IEF or electrophoresis were selected because their behavior had been previously studied. Therefore, they seemed suitable for examination under the unusual conditions employed in this study. The subzero methodology described can probably be extended to the investigation of problems concerning hemoglobin and other proteins, either as is or after extensive and detailed studies of so far unknown parameters, such as the Ampholine pH gradients in focusing, buffer pH values, and protein titration curves in hydro-organic solvents at subzero temperatures. It should be stressed, however, that the development of suitable gel matrixes such as those described here represents an essential preliminary step in this kind of study. IEF or electrophoretic separations in free hydro-organic solvents at subzero temperatures encounter great difficulties due to convective disturbances. On the other hand, the method of IEF described by Park (5) has limitations with regard to its extension to temperatures lower than - 10°C. Some systems are unstable unless very low temperatures are attained. As for hemoglobin, for instance, any attempt to study mixtures of hybrid forms by resolving them into single components must consider the possibility that changes in the composition of the original hybrid species can be brought about through dimerization and reassociation reactions. We have shown here (Figs. 4 and 5) how this kind of problem may be circumvented by the use of a suitably low temperature.

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In conclusion, we have shown that electrophoretic separations can be carried out at temperatures as low as -30°C. It is likely that separations at temperatures lower than -30°C can be carried out by the use of gels prepared by copolymerization of acrylamide with higher percentages of methylacrylate or ethylacrylate than those used in this study. ACKNOWLEDGMENTS We thank Prof. P. G. Righetti for his helpful discussion. The study was supported in part by a grant from the Consiglio Nazionale delle Ricerche, Rome.

REFERENCES 1. Douzou, P. (1974) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 22. pp. 401-512, Wiley, New York. 2. Fink, A. L., and Ahmed, A. I. (1976) Nature (London) 263, 294-297. 3. Balny, C., Le Peuch, C., and Debey, P. (1975) Anal. Biochem. 63, 321-330. 4. Debey, P., Balny, C., and Douzou, P. (1976) FEBS Lett. 69, 231-235. 5. Park, C. M. (1973) Ann. N. Y. Acad. Sri. 209, 237-257. 6. Adair, G. S., and Adair, M. E. (1934) Biochem. J. 28, 1230-1258. 7. Righetti, P. G., and Drysdale, J. W. (1976) in Isoelectric Focusing (Work, T. J., and Work, E., eds.), North-Holland, Amsterdam. 8. Douzou, P. (1973) Mol. Cell, Biochem. 1, 15-27. 9. Douzou, P. (1975) in Proceedings of the 10th FEBS Meeting, Vol. 40, pp. 99-l 11. 10. Mark, H. F., Gaylord, N. G., and Bikales, N. M. (eds.) (1967) Encyclopedia of Polymer Science and Technology, Vol. 7, p. 461, Wiley, New York. 11. Righetti, P. G., Gianazza, E., Brenna, 0.. and Galante, E. (1977) J. Chromatogr. 137, 171-181. 12. Hjerten, S. (1962) Arch. Biochem. Biophys. Suppl. 1, 147-151. 13. Bunn, F., and Drysdale, J. W. (1971) Biochim. Biophys. Acta 229, 51-57. 14. Cavins, J. F., and Friedman, M. (1968) J. Biol. Chem. 243, 3357-3360. 15. Bunn, F., and McDonough, M. (1974) Biochemistry 13, 988-993. 16. Bernstein, S. C., and Bowman, J. E. (1976) Biochim. Biophys. Acta 427, 512-519. 17. Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404-427. 18. Maurel, P., Hui Bon Hoa, G., and Douzou, P. (1975) J. Biol. Chem. 250, 1376-1382.

Isoelectric focusing and electrophoresis at subzero temperatures.

ANALYTICAL BIOCHEMISTRY Isoelectric MICHELE Cattedra 88, 212-224 (1978) Focusing and Electrophoresis Subzero Temperatures PERRELLA, ADRIANA HEY...
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