BIOTECHNOLOGY TECHNIQUES Volume 7 No.1 (January 1993) pp.9-14 Received as revised 27th September

FACILE TECHNIQUE OF PROTEIN PRECIPITATION BY .APPLICATION OF ELECTRIC CURRENT

Anita Sharma I, R.P. Sinha 2, Minni Srivastava I and Ashok Kumar 2.

iCentre of Advanced Study in Botany 2School of Biotechnology, Banaras Hindu University,

Varanasi 221 005, INDIA.

suMMARY

Precipitation of proteins has been achieved following passage of direc£ electric current in various protein solutions. Application of as low as 3 V of electric current showed precipitation but the rate increased with increase in electric current. With 9 V there was more than 85% precipitation of protein within 15 min. Precipitation occurred at a wide range of pH and temperature. Electrophoretic analysis of precipitated proteins show that they are not denatured by application of electric current. Proteins thus precipitated can be easily recovered by centrifugation.

INTRODUCTION

Protein is the most important nitrogenous organic matter and

constitute about half of the dry weight of all the living cells

(Dabah, 1970). Like amino acids, proteins are ampholytes, i.e.,

they act both as an acid and a base. As electrolytes, they

migrate in an electric field, and the direction of migration is

determined by the net charge of the molecule (Dickerson and Geis,

1969). The net change is influenced by pH and for each protein,

there is a pH value at which it will not move in an electric

field; this pH value is the isoelectric point (PI).

Proteins are precipitated from aqueous solution by high

concentration of neutral salts (Bailey, 1967; Ng et al., 1991).

Commonly used salts are ammonium sulphate, sodium sulphate,

magnesium salts and phosphates. The most effective region of the

salting out is at the isoelectric point of the protein. Although

this method is routinely used for fractionation/purification of

proteins, it cannot be employed for large scale precipitation of

proteins. Attempt has also been made to recover protein from

reversed micellar solutions through contact with a pressurized gas

phase (Phillips e_~t al., 1991). Currently, the commercial

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exploitation of biotechnology is centered around the production of

various proteins, enzymes and vaccines especially required for

human health care (Geisow, 1991). Therefore, a real revolution in

process technology involving massive efforts to apply novel and

cost saving techniques to processing will be required (Bowden,

1985; Phillips et al., 1991).

Electrophoretic dewatering, electroosmotic dewatering, forced flow

electrophoresis, electrodecantation and electrofiltration are

processes which are used in downstream processing (Bowden, 1985;

Grau and Bisang, 1992). In the present investigation, we have

made an attempt to develop a technique for protein precipitation

by simply passing electric current in the protein solution.

MATERIALS AND METHODS

Sources and preparation of protein samples. Proteins used for electrical precipitation test were water soluble, extracted from two species of cyanobacteria viz. Nostoc and Anabaena sps. and a higher plant (Bean seed's extract). Nostoc and Anabaena sps. are our own laboratory cultures and Bean seed was purchased from the local market. Protein extraction was done in 100 ml of phosphate buffer (2.5 mM, pH 7.0) containing 2mM NaCl. Suspension was sonicated for 2-3 min in Branson Sonifier 450. After complete extraction the resulting suspension was centrifuged at 20,000 rpm for 10 min. Desired pH of protein solutions was obtained by the use of suitable buffer and NaOH/HCI. Protein precipitation test at desired temperature was conducted in a temperature controlled mobile cold chamber. 'Pest samples were precooled to 4 or 25°C before~placing in the cold chamber. Lysozyme (Sigma Chemical Co., St. Louis, MO, USA) was used as standard proetin in precipitation test.

Application of electric current. For experimentation, 50 ml protein suspension was taken in a beaker containing two aluminium electrodes (12 cm x 3 cm), kept 8 mm apart and attached to a voltage eliminator supplying 3-9 volts of direct current. Depending on types of experiment, voltage was regulated from the eliminator itself.

EStimation of protein. Protein was estimated by the method of Lowry et al. (1951). Estimations were made before and after passage of electric current in the solution.

Gel electrophoresis. Protein samples were subjected to disc gel electrophoresis (Davis, 1964) and/or SDS-PAGE by the procedures of Laemmli (1970).

I@

RESULTS AND DISCUSSION

The effects of changing of voltage on protein precipitation are

shown in Fig. i. It is evident that after 15 min at 3 V, ca. 25%

precipitation was achieved. An increase in voltage increased the

precipitation rate and at 9 V, there was more than 85%

precipitation of proteins. The degree of precipitation was almost

identical for purified single protein (lysozyme) or heterogenous

crude proteins. From the findings it seems that application of

electric fields cause precipitation of all types of protein

present in the solution. It is well known that application of

electric fields into the protein solution results into the

mobility of individual class of protein but as yet little, if, any

attempt has been made to apply this phenomenon for protein

precipitation (Dickerson and Geis, 1969; Laemmli, 1970). However,

the flocculation of the unicellular green alga Chlorella Vul~aris

by electric fields has been reported (Kumar et al., 1981).

Similarly application of electric field in whole broth has been

reported to cause movement of the negatively charged bacterial

cells towards the anode (Bowden, 1985).

Once it became apparent that application of electric field indeed

causes precipitation of protein, we bacame interested to study

various factors involved in the above process. Knowing that Bean

seed extract proteins showed the highest degree of precipitation,

unless otherwise stated, all other experiments were done solely

with this extract. Fig. 2 shows the effect of varying time period

but constant voltage (3 V) on the rate of precipitation. An

increase in time period showed almost linear increase in the rate

of precipitation even at a voltage of as low as 3.

With a view to test the practical applicability of electrical

precipitation of proteins, we tested the response of electric

field in solutions containing varying concentration of proteins.

As application of 6 V or 9 V for 15 min did not elicit much

difference in the precipitation rate, 6 V was routinely applied in

all the experiments. With increase in the concentration of

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proteins, there was increase in the rate of precipitation.

Solution containing 1 mg/ml showed only 50% precipitation whereas

about 85% precipitation was achieved in solution containing 20

mg/ml or above concentrations of protein (Fig. 3). Nevertheless,

precipitation did occur in solution containing as low as 5 ~g/ml

of protein. Mostly proteins are labile to temperature and thus

electrical precipitation test was performed at 4 and 25°C. There

was insignificant difference in the rate of protein precipitation

at both temperatures.

Table 1 shows the effect of pH of the protein solutions on the

degree of precipitation. Although precipitation did occur in a

wide range of pH (5 to 9), maximum precipitation was obtained at

pH 5. There was insignificant change in pH value near the

electrodes however prolonged application of the electric current

caused a buffering effect on the suspension, causing a rise in the

pH of the initially acidic medium and a fall if the initial pH was

above 7.5-8.0.

100

~ 8 0 a

D.

6 g O e L

~ c ~, &O

o

2 0

(15 nnin )

3 B g

Votts

~oo~

o [ / / / __i . . . . c gO

._~ u

a. " 0 _=

o

0. 2O

Z ! L ..... ~_-

15 30 d5 GO

Time [~,n)

Fig. 1 Fig. 2

Fig. i. Effect of different voltages on protein precipitation. Precipitation was tested in water soluble protein of Bean seed extract e--e; Anabaenaouo; Nostoc ~-0 and lysozyme "u-. Concentration of each test protein solution was 10 mg/ml. The results are representative of three separate experiments. The S.D.'S were consistently less than 10% of means.

Fig. 2. Protein precipitation at different duration of time. Bean seed extract (10 mg/ml) was employed in this experiment. Other conditions as in Fig. i.

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The solubility of most proteins in aqueous solutions is mainly due

to the hydrophilic interaction between the polar molecules of

water and the ionized groups of the protein molecules (Dickerson

and Geis, 1969). It is known that chemicals that change the

dielectric constant or the ionic strength of an aqueous solution

therefore would be expected to influence the stability of

proteins. That is why the addition of ethanol or acetone or

certain salts such as ammonium sulphate results in precipitation

of proteins. The above precipitations result when the attractive

forces among protein molecules exceed those between the protein

molecules and water. In general the net charge on any give n

protein molecule and hence its attraction to another protein

molecule are functions of the pH and ionic strength of the medium

(Bailey, 1967; Dickerson and Geis, 1969). Precipitation observed

by electric field is not due to pH because precipitation was

observed at a wide range of pH. Most probably net charge present

in proteins is directly affected by passage of electric field

irrespective of pH of the solution. The notion that the

application of electric field in the protein solution may cause

denaturation of many proteins prompted us to check the native

structure of these proteins. Electrophoretic characteristics of

original unprecipitated protein and proteins precipitated by

passage of electrical field showed identical bands on PAGE/SDS-

PAGE gels (Fig. 4).

TABLE i. Effect of pH of protein solution on the rate of

precipitation a.

Sample Bean seed pH extract

4 5 6 7 8 9

% protein b

Precipitation

75 85 81 78 74 70

a. Protein concentration 10 mg/ml. b. % precipitation is the recovery of protein in the precipitate

after application of electric field in the solution. Results are based on three separate experiments.

From our findings it is evident that the native structure of

proteins is maintained even after passage of electric field.

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~cJo

60 .o

O 60

.E

?

I I u

ol ; • I | O =

Ol 'I, Io |

Ol m • i |a

. ~ |= / i

I 10 IS 20 100 C o n c e n l r orion ( rng / n~()

~V( 15 m~n)

7 /

/ , i " l

/

c

Fig. 3 Fig. 3. Role of varying concentration of protein on the rate of precipitation.

Fig. 4. SDS-PAGE protein profile of original crude extract (A) and after precipitation (B). Equal concentration of both the protein was loaded in each well.

Application of electric field leads to total precipitation of

protein or individual protein is precipitated at specific time

needs thorough and critical investigation. Based on our findings

we suggest that proteinspresent in solutions of larger volume may

be recovered by application of electric fields. This method seems

more convenient and rapid than the conventional ammonium sulphate

induced precipitation of proteins. Precipitated proteins form

compact pellet upon centrifugation. Further work is under

progress for revealing the mechanism of precipitation and ensuring

the applicability of this method at larger scale.

REFERENCES

i. Bailey, J.L. (1967). Techniques in protein chemistry. Elsevier, New York.

2. Bowden, C.P. (1985). J. Chem. Tech. Biotechnol. 35, 253-265. 3. Dabah, R. (1970). Food Tech. 29, 659-662. 4. Davis, B.J. (1964). Ann. N.Y. Acad. Sci. 121, 404-427. 5. Dickerson, R.E. and Geis, I. (1969), The structure and action

of proteins. Harper and Row, New York. 6. Geisow, M.J. (1991). Bio/technology 9, 921-924. 7. Grau, J.M. and Bisang, J.M. (1992). J. Chem. Tech. Biotechnol.

53, 105-110. 8. Kumar, H.D.; Yadava, P.K. and Gaur, J.P. (1981). Aquatic

Botany. ii, 187-195. 9. Laemmli, U.K. (1970). Nature. 227, 680-685.

i0. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L. and Randall, R.J (1951). J. Biol. Chem. 193, 265-275.

ii. Ng, P.K.; Figueroa, C. and Mitra, G. (1991). Biotechnol. Lett. 13, 261-264.

12. Phillips, J.B.; Nguyen, H. and John, V.T. (1991). Biotechnol. Prog. 7, 43-48.

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