On the stability of bovine gamma II crystallin

Vo1.187, No. 1,1992

BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS Pages 39-44

August 31,1992

ON THE STABILITY OF BOVINE GAMMA II C R Y S T A L L I N

F r e d e r i c k A. B e t t e l h e i m l * , M a r k B. Reid 2, Peter McPhie 3 and Donita Garland 2

iDept.

Chemistry,

Adelphi

University,

2National Eye Institute, and 3 N a t i o n a l D i g e s t i v e and Kidney Diseases, NIH,

Garden

City,

NY,

11530

Institute of Diabetes, Bethesda, MD, 20892

Received July 8, 1992 Bovine gamma II crystallin, undergoes structural changes when lyophilized. The lyophylized powder does not readily dissolve in buffer, although it can be taken up in distilled water. The lyophylized sample, as opposed to a sample concentrated by vacuum distillation at 30°C, does exhibit different migration on isoelectric focusing gels. The hydration and denaturation properties of the two preparations are different. The lyophylized sample possesses a higher non-freezable water content as a function of concentration than its counterpart. The lyophylized sample also shows less denaturation in differential scanning calorimetry scans, up to ll0°C, than its counterpart. This indicates that lyophylization may induce a slight denaturation, due to structural-conformational change. On the other hand, the CD spectra of lyophylized and non-lyophylized gamma II crystallins do not differ significantly. This implies that the conformational changes upon lyophylization do not involve the secondary structure of gamma II crystallin. © 1992 A c a d e m i c

Press,

Inc.

Gamma c r y s t a l l i n s are the monomeric, about 20kD, components of the

structural

crystallins largely that

concentrating

gamma

for

lenses,

crystallins

the

powder

lens

the

and

of the

eye,

commonly

called

We

lyophylization.

(2,3).

It has

been

alleged

cryoproteins

(4,5)

and

mainly

cataract

most from

formation

often

are

in

calf

observed lenses

lens h o m o g e n a t e s

ion-exchange

samples

does

nucleus

are

cold

are isolated

form.

crystallin

in the

reported

chromatography isolation

of the

crystallins

responsible animal

proteins

(i). They are u n e v e n l y d i s t r i b u t e d in m a m m a l i a n lenses,

often

made

the

not

dissolve

chance

and

observation

completely

young

(6,7).

Gamma

by gel p e r m e a t i o n

chromatography lyophylized

in

in

(8,9). stored that

After in

the

gamma

II

buffer

after

This would indicate that l y o p h y l i z a t i o n itself may

cause some c o n f o r m a t i o n a l change and thus influence the studies on

* To w h o m c o r r e s p o n d a n c e

should be addressed. 0006-291X/92 $4.00 39

Copyright © 1992 by Academic Press, Inc. All rights" of reproduction in any form reserved.

Vol. 187, No. 1, 1992

the p r o p e r t i e s

of g a m m a

The following induced

by

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

II c r y s t a l l i n s

study was designed

obtained

to s h e d some

by l y o p h y l i z a t i o n .

light

on the c h a n g e s

lyophylization.

MATERIALS

AND

METHODS

Frozen fetal bovine eyes were obtained from Pel-freez Biologicals, Rogers, AS. After the eyes were thawed, the lenses were removed, decapsulated and homogenized in 50 mM Hepes buffer, pH 7.6, containing 200 mM KC1. The homogenate was centrifuged at 27,000 x g for 30 min. and the pellet was discarded. The low molecular weight fraction containing the gamma crystallins was obtained by gel permeation chromatography using a Sephacryl 200 column (2.5 x 100 cm). The individual gamma crystallins were separated by ion exchange chromatography (10). A portion of the samples was dialyzed against distilled water and lyophylized. The remainder was stored at 4°C until used. Dilute solution of the isolated gamma II crystallin was dialyzed against three changes of distilled water at 4°C for 48 hours. The dilute solution was also concentrated to about 30 % (w/V) by vacuum distillation maintaining the temperature of the residue at 30°C. Lyophylized samples were prepared for isoelectric focusing by redissolving in distilled water or in 200 mM sodium acetate buffer, pH 5.0, or in 75 mM Tris-HC1 buffer, pH 9.0. The samples were centrifuged to remove protein that did not go into solution. Isoelectric focusing was done on a Pharmacia Fast Electrophoresis Apparatus with IEF gel, pH 3 to 9 for 500 aVh. Circular dichroism spectra were taken on a JASCO J-500C Spectropotarimeter. The pathlength was 1 cm for the near UV and 1 mm for the far UV range. Samples for hydration studies were prepared by diluting the stock solution (30 %) to the desired concentrations using distilled water at pH 7.0. Hydration studies were performed by a combination of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Five to ten mg samples were hermetically sealed into preweighed aluminum sample pans. An empty sealed pan served as reference. The freezable water content of the sample was obtained in a DSC apparatus (DuPont 990; DuPont, Wilmington,DE) first by cooling the sample to -30°C, maintaining it at this temperature for 30 minutes and finally heating it to 30°C at a programmed rate of 3°C/min. The DSC curves obtained, differential heat flow versus temperature, were integrated to obtain the heat used to melt the frozen water (11,12). From this the freezable water content of the sample was calculated. The total water of the sample was obtained in the TGA part of the instrument. The sample pan was punctured and the weight loss was recorded on a Cahn balance while the sample was heated from 30 to 105°C (12). The non-freezable water content was obtained from the difference between the total and freezable water content and recalculated as % of the total water. Denaturation studies were run in the DSC apparatus on hermetically sealed samples between 30°C and 100°C.

RESULTS

The and

results

lyophylized

preparations very basic of

of g a m m a

they

results

crystallin formation

is of

conformation

AND

DISCUSSION

isoelectric

II

II c r y s t a l l i n

are

Some

that

in

changed

after

several

discrete

lyophylized,

surface

aggregated

40

water

new species

i.

Fresh

regardless or

buffer,

are m o r e

and s o m e a r e m o r e charge

lyophylization.

states.

prepared

in F i g u r e

distilled

of the

the

freshly

focus on t h e s e g e l s as a s i n g l e

that was not lyophylized indicate

of

shown

that have been

redissolved

species.

focusing

crystallin

Samples

are

as s e v e r a l

than the sample These

gamma

species.

whether

appear

of the

of

This

forms,

or

the can to

basic

acidic. gamma

be

II

due

to

changes

in

Vol. 187, No. 1, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Basic

Acidic 1 Figure

The

2

3

4

I. Isoelectric focusing of gamma II crystallin (i) before and (2,3,4) after lyophylization. The lyophylized samples were r e d i s s o l v e d (i mg/mL) in (2) d i s t i l l e d water, (3) 200 aiM sodium acetate buffer, pH 5.0, and (4) 75 mM TrisHCI buffer, pH 9.0.

hydration

preparations calculated

are here

properties given

is

,g %

in

given

of

the

two

2.

Figure in

terms

II

crystallin

The

hydration

parameter

the

non-freezable

of

gamma

water

40t 30

•

/j~"

20 e..''~

-FJ

N

10

,"o

o~-~:~ ~ D ~ - - ~

Figure

2.

o

° o

,,+_

z

0

O[j 0

o

. 5

.

. . . 10 15 20 7o ConcentPation

25

30

N o n - f r e e z a b l e water content (as % of total water) of gamma II crystallin O .... O n o n - l y o p h y l i z e d and • ...... • p r e p a r e d by lyophylization as a function of concentration.

41

Vol. 187, No. 1, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

content p e r c e n t of the total water content. content that

can be identified

is incapable

with the bound

to undergo

The n o n - f r e e z a b l e or p a r t i a l l y

first order transition.

value d e p e n d s on the m e t h o d of i n v e s t i g a t i o n etc.)

(13).

However,

the

concentration

of the m e t h o d employed.

crystallin

shows

sample

not l y o p h y l i z e d experimental

much

sample.

points

higher

this

bound

sample

is

shows

near IR the

same

The l y o p h y l i z e d gamma II water

It is also apparent

in

bound water Its absolute

(NMR, DSC-TGA,

dependence

trend i r r e s p e c t i v e

water

content

than

the

that the spread of the

much

greater

than

in

its

counterpart. One could underwent

interpret

some

aggregates

can trap

non-freezable

more

result

process

water

(immobilized)

non-freezable properties

this

aggregation

that

in the

water.

hydration

the

behavior

The n o n - f r e e z a b l e higher

than

happened

gamma

We

that

inspite

of the

the

LMW p r e p a r a t i o n high m o l e c u l a r

gamma

of

the

the

endothermic exhibiting heat

range. slight

heat.

In

consumption

same

seems

that

the

the

self

gamma

The

II

sample.

was p r e p a r e d

presence

during

the

denaturation

in Fig.3

The

lower

lyophylized

DSC

scan

can be seen

in

consumes

is that

this

range

113

kcal/mol

sample,

although

under

the

by

Sen

et

al.

has a

curve)

These heats of d e n a t u r a t i o n

reported

(16)

that are of

albeit

d i f f e r e n t buffer and hence at somewhat d i f f e r e n t temperatures. decrease

in

the

endothermic

heat

42

of

lyophylization

sample

that

are

this

behavior

The main d i f f e r e n c e

integrated

by

other

of

in the DSC scan in this range,

only to 62 kcal/mol. as

of

aggregation

from

sample.

the

This

electrophoresis.

non-lyophylized

contrast,

(15).

easily and does not show

gained

denaturation

(the

magnitude

be

II sample.

II

from the s o l u b i l i t y

in buffer

similar

is only slightly

gamma

in SDS-gel

can

enhanced

shows

gamma

3. The upper DSC scan curve

a few f l u c t u a t i o n s

corresponds the

crystallin

the lyophylized

a

others,

of what may have h a p p e n e d

in Figure

80-95°C

undergoes

it

residues

non-lyophylized

represents in

beside

prevents

w h i c h dissolves weight

II

similar

of LMW c r y s t a l l i n

is also evident

An i n d i c a t i o n

scans given

II

form of

when the h y d r a t i o n

fact that the LMW c r y s t a l l i n

gamma

This

in the

the

(14). It is interesting

non-lyophylized

Therefore,

beside

cryoprotein.

seen

to that of the n o n - l y o p h y l i z e d water content

lyophilization.

the

have

and

(LMW) fraction of c r y s t a l l i n s

fractions

proteins

of

interstices

of G- and F-actins were compared

contains

II c r y s t a l l i n

lyophylization

water content upon p o l y m e r i z a t i o n

to note that the low m o l e c u l a r weight which

the gamma

during

implies

that

the

in The

lyophylized

Vol. 187, No. 1, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

o 1,.

~

-0.12-

5b

65

7~

s~

95

ioo

(°C)

Temperature

Figure 3. Differential calorimetric scans during denaturation of gamma II crystallin samples between 30 and 100°C. A. non-lyophylized sample;B, lyophylized sample.

sample

is

strength gamma DSC

already if

we

denatured

take

II c r y s t a l l i n the

into

account

samples

resultant

somewhat.

are

This

that

when

scanned

integrated

area

the

between

approximately

t h e same h e a t 80-97 k c a l / m o l

the partially

denatured

changes

that

lyophylized No

gamma

preparations. exhibited The implies

comparable

can

change

be

seen

in in

of t h e s e

that the more

suffered

magnitude

around

95°C

by

yields Thus,

conformational

to

the

secondary

the

CD

observations

purified

gradients;

in t h e l i t e r a t u r e been

denatured

that

of

the

structure

spectra

of

upon

the

two

of the t w o s a m p l e s

differences.

a gamma

it is to s u f f e r c o n f o r m a t i o n a l

have

80-and

T h e n e a r U V and the far U V s p e c t r a

importance

obtained

in

time

II c r y s t a l l i n .

no s i g n i f i c a n t

concentration

that

be

partially second

gains

for b o t h s a m p l e s . .

g a m m a II c r y s t a l l i n

conformational

lyophylization

prone

may

interpretation

second on g a m m a

lyophylized. 43

are two fold:

II p r e p a r a t i o n

changes one

must

For one it

is, t h e m o r e

due to t e m p e r a t u r e reevaluate

II c r y s t a l l i n s

the

especially

and data

those

Vol. 187, No. 1, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGMENTS This

research

was

partially

supported

F.A.B) f~m the National Eye Institute,

by

a

grant

EY-02751

(to

N.I.H.

REFERENCES

i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16.

Bjork, I. (1964) Exp. Eye Res. 3, 254-261. Papaconstantinou, J. (1965) Biochim. Biophys. Acta 107, 81-90. Slingsby, C. and Croft, L.R. (1973) Exp. Eye Res. 17, 369-376. Zigman, S. and Lerman, S. (1964) Nature 203, 662-665. Siezen, R.J., Fisch, M.R., Slingsby, C. and Benedek, G.B. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1701-1705. Tanaka, T., Ishimoto, C. and Chylack, L.T., Jr. (1977) Science 197, 1010-1012. Clark, J.I., Biblin, F.J., Reddy, V.N. and Benedek, G.B. (1982) Invest. Ophthalmol. vis. Sci. 22, 186- 190. Slingsby, C. and Miller, L. (1985) Biochem. J. 230, 143-150. Mandal, K., Chakrabarti, B., Thomson, J. and Siezen, R.J. (1987) J. Biol. Chem. 262, 8096- 8102. Slingsby, C. and Miller, L. (1983) Exp. Eye Res. 37, 517-530. Bettelheim, F.A., Ali, S. White, O. and Chylack, L.T., Jr. (1986) Invest. Ophthalmol. Vis. Sci. 27, 122-125. Castoro, J.A. and Bettelheim, F.A. (1987) Exp. Eye Res. 45, 191-195. Ali, S. and Bettelheim, F.A. (1985) Colloid & Polymer sci. 263, 396-398. Kakar, S.K. and Bettelheim, F.A. (1991) Invest. Ophthalmol. Vis. Sci. 32, 562-566. Bettelheim, F.A. and Popdimitrova, N. (1990) Exp. Eye Res. 50, 715-718. Sen,A.C., Walsh, M.T. and Chakrabarti, B. (1992) J. Biol. Chem. 267, 11898-11907.

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