Variations in the makeup of lens insoluble proteins

Exptl Bye Res. (1970) 18, 5 8 - 6 3

Variations in the M a k e u p of Lens Insoluble Proteins* S. Zm,~t_~N, J. Scnuy, Tz A N D T. YuLo

DeT~a.rt.me~t of ,S'~.rge~nj: Ophthalznology, Un.iveran:ty of Rochester School of Mediaine and. Dentist~j, Roct~,ster, New Yceck, U.S.A. Received 8 October 1969, a~d in rcv£~ed form. 10 February 1970, Bostort) n this study dkfference~ in the cOlnloosit,ioll of lert~ water-lnsoluble protein of s~veral species were sought. Lenses of young and old rat,s, X-irradiated rats, Noting and old dogfish and c~ws, and nornm] and nuclear cat~ractous humar~ were ~eparated into eortr~x a~.d nuvleus fr~ml which toNI iztsoluble prot~i~ was isolatad by eentrifugation. Urea soluble (US) and ~ e a insoluble (UI) portio~ts were separated, aRd tile amounts of protein r e -

presented by these fractions were estirnttted. The soluble c o m p o n e n t ; s eomprisinE %hese fraet~ions ~ere observed by ~crylamido gel eteetrophoresis. I~ z ~ and dogfish te~ses, UI levels increa~d with aging in the nucleus of the Iens, ~hile in bovine a~d ~tor-mM human lenses US predominated in both cortex and n~teteus. t~tuma~ nuclear eataractous ictus xmclei, Ilowe~er, co~tained much Mgher Ievels of U I t.han of ?US rnateriaJ. Possible raod~fiers o£ the formation of ir~soluble protein leading to tko diversity of 'eomtyosieio~s obs0rved are discussed. I. Introduction

W h e n t h e lenses of r a t s a n d h u m a n s a r e h o m o g e n i z e d in w a t e r r,he r e s u l e i n g w a t e r i n s o l u b l e p r o t e i n s s e d i m e n t e d a t low s p e e d m m be s e p a r a t e d i n t o t w o f r a c t i o n s based u p o n ,solubility i n 7 ~ u r e a (Pirie, 1968; Z i g m a n . Sehulgz a n d Y u l e , 1969; ]{arding. 1969). Since it, dissolves c o m p l e t e l y in urea, t h e e x t r a c t e d u r e a - s o l u b l e (US) mat, erial m u s t be i n s o l u b l e d u e to n o n - c o v a l e n t forces, w h e r e a s t h e e x t r a c t e d urea.-in.~oluble mat, eriM is b o u n d -~ogether p a r t l y b y disulfide b e a d s a n d parbty b y h y d r o g e n b o n d s a n d h y d r o p h o b i c a t t r a c t i o n s (Zigm.an eb el., 1969; Ha.rdi1~g. 1969). E a c h fl'aetion c o n t a i n s d i f f e r e n t p r o p o r t i o n s o f eomponeat~s s i m i l a r t o a- a n d ~,-crystallins bug l o w Ie~:els of c o m p o n e n t s s i m i l a r t o fi-e.~ystaltins w e r e Mso d e t e c t e d . T h e p r e s e n c e of b o t h 3 , - a n d .~-crystallin i n U S a n d U I of ra~ l e n s e s h a s b e e n s h o w n i m m u n o e h e m i e a l | v ( Z i g m a n s t el., 1969). M a n s k i (1968), h a s s h o w n b y i m m u n o e h e r n i c a l m e a n s t h a t pre-~ is also p r e s e n t , lV.hether the~e t w o f r a c t i o n s are a c t u a I t y present., as such in t h e lens or if t h e y r e s u l t f r o m c h a n g e s in ~oluble p r o t e i n e n v i r o n m e n t d u r i n g t h e i r (c~tract.ion we.?3 p a r t i a l l y a n s w e r e d b y H a r d i n g (1969), w h o s e resu!~s s t r o n g l y i n d i c a t e d that. s o m e of t h e SS b o n d s presen~ m t-he U I f r a c t i o n are f o r m e d b y air o x i d a t i o n d u r i n g t h e i r w o r k u p trader a e r o b i c c o n d i t i o n s . L e n s e s h o m o g e n i z e d d~reotly i n 6 ~ guaniclinimm h y d r o chloride: e v e n u n d e r a e r o b i c co_~itioiu% were dim~olved e n t i r e l y , i n d i c a t i n g t h a t all prot.eins presen~ i n t h e s e !crises w e r e d i s s o i v e d b y rup~atring n o n - t o r M e n t b o n d s only. B u t t h e t i m e r e q u i r e d for t o t a l d i s s o l u t i o n o f lenses ltomogeai:.~-cI i n g u a n i d i n i u m h y d r o e h t o r i d e was g r e a ~ r (18 hr) for t h e lenses o f o l d t h a n for y o u n g rai~s (immeclJate). w h i c h m a y be a f u n c t i o n o f s t r o n g e r b i n d i n g forces presen~ in t h e i n s o l u b l e p r o t e i n f r a c t i o n s of o l d e r ra~ Ienses. * Tbi~ ~n~-¢~tigat~on w a s s~tpported in p~rt b y a gr~n~ ft'ov~ t h e ~ k e ~ b e r E y e a n d H u m a n P a r t s B ~ k , arid a r ~ r c h g r a n t fr~r~ the Natlon~M E.3'~ Isis~itu~e o f T h e PtlbIio H e a l t h ~ r v i ~ (q~Y0(OSO-~0I).

58

V A I ~ I . A T I O N S OaF LENS INNOLUiBL:E I'I~OT/z]INS

89

D i f f e r e n t levels of n a t u r a l p r e c u r s o r s of t h e a t l e a s t p a r t l y artificial U S a n d U1 f r a c t i o n s m u s t be p r e s e n t i n t h e n u c l e u s a n d c o r t e x o£ t h e lens, since d i f f e r e n c e s in t.he d i s t r i b u t i o n of U S a n d U I in n u c l e u s a n d c o r t e x or w i t h degree of opaeific'afiou h a v e been o b s e r v e d ( Z i g m a n , 1969a). A I / I precuxsor s e e m s to lu~ve a g r e a t e r prop e n s i t y t o b e air o x k S z e d v i ~ SS b o n d s d u r i n g i s o l a t i o n p r o e e d m ' e s b e c a u s e of its h i g h y-erystallh~ cont.en~. ( 8 I i l e v e l = 150/z_~,]g), a n d since a h i g h e r level of U t is e x t r a c t e d f r o m t h e n u c l e u s t h a n f r o m t h e c o r t e x , i t s precm'sor m u s t be present, M, a h i g h e r l e v e l in t h e nucleus. T h e v~ri~.tions in e x t r a c t e d U S ~md U I le:~eIs t o be r e p o r t e d beIow a r e a c t u a l l y reflections of e x i s t i n g precuxsor l e v e l s f o r ~ U S a n d U I w i t h i n t h e lcnses s t u d i e d . C h a n g e s i n U S a n d U I levels of nuclei a n d Cortices o f t h e ~,,,~s,~ ~. . . . . . of several species a t different, ages a n d s t a g e s of opacities.ties, a n d fm a~te.mpt t o f u r t h e r c h a r a c t e r i z e t h e soluble p r o t e i n s c o m p r i s i n g i n s o l u b l e fra.ctions will be described. 2. M a t e r i a l s a n d M e t h o d s

R.a% lenses were obtMned from t h e eyes of 5- and 52-week-old }Ioltzman white males. For X-irradiation studies, 5-week-old rats were exposed to 1500 r of irradiation to the !mad only and wore killed 1 a n d 4 weeks later..I)ogfish lenses were obtMned fresh from y o u n g (2"4" in length; lens weigh~ o£ 100 rag) and old (about, 48-60" in |ength; lens weigh~ e l i g) fish eyes, a n d were im'.nediately frozen. Bovine eyes wer6 o b t a i n e d front 6-month-okl (calves) a n d 7-8-year-old {cows) ¢~ni:mals a t a loeM abb,utoir w i t h i n ½ h~ of dearth, and they were dissected ~thiit 1 hr of death. Non-eataraetous h u m a n eyes were supphed by the l~oahester ]~ye Bank, and the lenses were removed within .12 hr of the dea.gh of tim individffajs a n d frozen. Nuclear non-diabetic }]llman Gatzl,Facto[18 lenses were obtain0d directly J>om the operating r o o m at Strong Memorial Hospital and were also kept frozen until use. Only non-br,J.neseen~ nuolea.r cataracts .were used. ]fresh or freshly frozen decapsltlated lenses were stirred slowly b y lneans of soft)plaatioeot~tex{ m~'Lguetie stirring bars ~tg 5°C in a beaker cont)aining 4. ml of clisgilled w a t e r for approximately 1 g of lens (wet weight) for 1 hr. B y this time, the soft ea.sily r e m o v e d cortical portion was suspended bl the liquid phase, and the he;~vy t.ough nuclei r e m a i n e d at t.he b o t t o m of the beaker, Havh~g observed t h a t the nuclei h a d m a i n t a i n e d a. toe,stunt size a n d shape for about, 15 rain, t.he cortical suspension was deeant:ed off, a n d t h e nuclei and cortices were eo.eh homogenized separately in 5 ~1 of water usbag ]3ounce glass homogenizers. :['he total insoluble protein ('£IP) was s e d i m e n t e d ag 2500 ray/ntis (900 g) a t I0°C for 20 min, a n d t h e n re-suspended a n d reeentrifuged 10 times, or until t h e super~atant showed no ultraviolet absorption a.t 280ju/z. The resultan~ residue was suspended in neutral aqueous 7 ~ urea solutions (1 ml/5 rag) at room t e m p e r a t u r e for ½-hr, and the r e m a i n i n g insoluble materiat was sedimenv~ed at 10°C~ and 105,000 g for ..~ hx. The u~easolubilized material was rep~eeipita~ed by dialysis against multiple changes of wa.ter a t 10°C for 3 d~ys. E:~;h precipitate [the vires sohabte (US) ~nd urea-knsoluble (IJ]')] w,-~ suspended in iBaile,y's r e a g e n t (.~,Os ----; S,O, =- ; p H 9.9 ;7 ~ urea), in which the US dissolved completely. The tJI dissolved nearly entirely: but_, a slight opalescence was presen~ which was removed by centri£ugat,ion ag 105,000 g for ½ hr a t 10°C. F~aeh snlfonated solubilized fraction was dialyzed for 1 hr agains~ p t I 9-9 bicarbonate buffer (0-0t .~) a n d ~hen a g a i n s t vca~er a t 10°0 overnight, D i a l y z e d solu.tiona were t h e n tyopbilized to dryness a.nd sCored in a freezer (--20°('/,) for lager use'. The phenol p r o t e i n analysis of Lowry, t~osebrough Narr a n d R a n d a l l (1951) was used to d e t e r m i n e ~he amotmt~ of p r o t e i a pre~eut. 8ubeomponent~s of ~tflfo.oated US ~rnd LTI f,'action~ and soluble p r o t e i n s~andarcla were separated and ~isualized b y using vhe polyacrylamide gel electrophoresis m e , h o d of Ornstein a n d Davis (1960). Samples of 50-100 ~,g wore applied to the top of columns of 7-5~/o gels La p]=[ 8'8, 0-4 ~ Tris-glyoiue buffer eon~Mning 7 N ure~ a.~ a current of 4 n ~ p e r

60

S. Z I G M A N ,

J. SCHULTZ

AND

T. YUL0

tube for 1 hr. Amido Schwartz 1 ~/o was used to stain the bands, and excess dye was laterally removed from the column of gel by electrophoresis in 7~/o acetic acid. In some cases, a modified Beckman photo-electric densitome~er (Analytrol) was employed to trace the hands in the gel columns. 3. Results

Table I summarizes the results of p r o t e i n m e a s u r e m e n t s of the :PIP (nag per lens) a n d of the US a n d U I fractions (per cent of T I P ) of t h e lens cortex a n d nucleus of ~'ABLE ][

Variations i n tens cm'tical and n u d e a r insoheble 2protdns T I P pet" leas (rag)

Lenses o f

N o r m a l r~t~

ll-week-old 52.week-old

RatB irradiat~.d a t 5 weeks w i t h 1500 r to t,he head N o r m a l rats

6-week-old (1 week p o s t - i r r a d i a t i o n ) 9-week-old (4 weeks pos~-irradiation) 9-week-old (control)

Dogfish

y o u n g (2 f t length) old (5 ft length)

Calve~ Cows Hu.r~ans

Cortex Nucleus US UI US UI T o t a l insoluble protein (%)

1-2 8-4

3 7

2 3

85 25

10 65

1.3

4

5

85

6

2-3 2.2

3 4

4 3

33 44

G1 49

1.O 238-8

9 21

26 4

20 6

46 70

B.month.ol4 7--8-year- old

5.4 57- 7

66 .26

5 1

28 60

I 3

normal (50-60-year-old) oabarao~ou8 (65-65-year-old)

10.5

36

2

6t

I

97-0

10

9

I5

67

All e s t i m a t i o n s o f US, U I a n d T I P were d o n e in d u p l i c a t e on i n d e p e n d e n t l y prepat'ezt samples. 1)iffereneea botweert d u p l i c a t e s were n o t g r e a t e r t h a n 6 % .

t h e species indicated. I n all species except the dogfish, t h e level of T I P increased a p p r o x i m a t e l y t e n f o l d when t h e lenses oi" y o u n g a n d oid animMs or n o r m a l a n d c a t a r a e t o u a h u m a n lenses were com.p~red. T I P increased some 200-fo!d d a r i n g ~ e * ;.... ~ pet-~,a:~ o£ v.ae'"dogfish study. I n the r a t lens cortex, lit~le change in the percent of T I P in US a n d U I fxactions was observed with increasing age. I n t h e nucleus, US d r o p p e d from 85 to 25~/o of T I P between 5 a n d 52 weeks of age, wbiie U I increased f r o m 10 ~ 65~/o. W h e n rags were i r r a d i a t e d w i t h 1500 r of X - r a y s a t 5 weeks of age, t h e i r lens nuciei were no different in t h e levels of the insoluble f r a c t i o n s t h a n t h e n o r m a l at, 1 week later. A t 9 weeks of age, or 4 weeks after irradiat.ion, t h e US of t h e nuclei has d r o p p e d b y 5 2 % while the U I has hmreased b y 55~/o of bhe T I P . Note t h a t t h e insoluble p r o t e i n ~ f t h e nuclei of the 9tweak-old controls changed in t h e same direction (US d r o p p e d b y 41~/o, U I increased by 43~/o of T I P ) b u t b y a b o u t 11 to 12°/o tess. The values for 9-week-old i r r a d i a t e d r a t lena nuclear US a n d U I are n e a r l y $he same as those for 52-week-old normMs. I n t h e dogfish lens cortex, US increased

VARIATIONS

OF

LENS

INSOLUBLE

PIAOTEINS

61

by 12°/o, while U I fell b y 22°~), in the y o u n g a n d old animals, respectively. The largest aging change noted was again in tbe nucleus, where US d r o p p e d b y 14°¢o c o n c o m i t a n t with an increase of U I of 14~/o. A t o t a l l y different s i t u a t i o n was found in b o v i n e lenses, in which US of the cortex of t h e calf drops b y 30% d u r i n g m a t u r a t i o n , while nuclear US increases b y 32% of T I P . Insignificant changes are noted in the O I fractions w i t h aging. The US and U I values for n o r m a l h u m a n lens nuclei a n d cortices are v e r y n e a r l y i d e n t i c a l to those o f the cow, b u t for c a t a r a c t o u s h u m a n lenses, t h e values of US a n d U I ~l'e in t h e same range as these of old r a t a n d old dogfish ~erses. As c o m p a r e d to n o r m a l h u m a n lens nuclei, U S is 4 6 ~ lower and U I is 66% higher in c a t a r a c t o u s lenses. Taur, E II

Alterations in rat lens cortical a,td. nuclear soluble Tcroteins ,

,

,

_

_

,

.

_

_

_

5-week-o|d rats C o t t~.x Nucleus

:Fraet, i o n

,

,

±L

,

,,

_

,,,

52-wct~k-old r a t s Cortex k'quoleu.~

mg of soluble protein per lens t.0

0.13

2.8

0"97

1-1

0.07

2-3

051

2-2

0,80

1.0

0-4,'2_

A t l e s t i m a t i o n s w e r e d o n e in d u p l i c a t e o n I ) E A E - c e t l u l o s c s e p a r a t e d l e n s p r o t e i n s ( S p e c t e r , us(ng the Lowry ctal. (195t) protein determination. D i f f e r e n c e b e t w e e n ~atues o f d u p l i c a t e r u n s we,.e n o t g r e a t e r t h a n 50/o .

1964)

Measurements of the soluble protehls of the r a t lenses used to o b t a i n t h e d a t a in Table I are shown in Table II. Nuclear y - c r y s t a l l i n level decreases f r o m 0.8 to 0-4 rag/lens, w]file cortical y - c r y s t a l l i n decreases from 2.2 to 1-0 rag]lens beVween 5 ~nd 52 weeks of age. Cortical v. increases f r o m ]-0 t o 2.8; nuclear ~ i n , e a s e s from

:f 'b' 5

~0o

tO

t5

~

'

0

~e

1 5

iO

15

20

(d)

7 5

3 ! 0

.5

IO

15

20 0 Cer~fime~eo~

5

I0

15

2o

~7@. 1~ DensUvome~rio t r a c i n g s o f a e r y l ~ m i d e g e l p r o f i l e s o f P l a t e 1. (a) 0~.e~mlmSlin; (b) 7 - o r ~ g t ~ l U n ; ra~ cort.ioa! U S ; 6 . w e e k - o l d r a ~ n u c l e a r U L

(c) 52-w~k-oid

{5~

S. Z I G M A N .

J. S C H U L T Z

AND

T. Y U L O

0-].3 to 0'97; cortical fl increases from I-i tm 2"S; and nuc]ea~/~ increases from 0'07 to 0-51 mg]|ens during the same tinle. The extant of decrease of y~erystallJn level in the nucleus is m u c h lower than the increase of Ul. Acrylanlide gel profiles of the snlfonated ]nso]nb|e fraot{ons of the nuclei and co,rices of the lenses of young and old Tats are sho~vn in Plate I. Also pictured are the profiles for sulfo-~ (S-~) and su[fo-z (8-:y) crysba|lin. ][t ]s [mfortunate thal~ there Js overlapping of some oomponenf~s of S-~ and 8-y, but major differences ~re still discernible by viewing the banding patterns. Su]fonated young rat n~mlear U l gels exhibit banding patterns similar to S-~, crystalHn. ]Emphasis of this finding Js m a d e }n I~ig. 1, which is a densitometric t r a c i n g of some of the gels pictured in l~lal:e 1. Old r a t nuclear U I contains m a n y b a n d s other t h a n those of S-y crystallin. I n old r a t cortical US, bands simiIar to those of S-y are deficient. Y o u n g r a t corticM US, y o u n g a n d old ray nuolear US, a n d y o u n g r a t cortical U I contain m a n y b a n d s which were difficult to identify. A similar set of d a t a for dogfish tens sulfo-proteins is sho~,n~ in Plate 2. Again, aging is accompanied by an increase of bands other t h a n 8-y in nuclear U I , while in cortical US, b~nds in the S-Z region decrease in intensity. 4. Discussion

Tkis c o m m u n i c a t i o n shows t h a t interacblons a m o n g the soluble i;roteins in the leases of several species can result in t h e i'orn~ation of different levels of two insoluble protein fractions [the urea soluble (US) a n d t h e ure~ insoluble (UI) fractions]. \Vhen t h e distribution of the weakly associated (US) and the ~trongly associated (UI) insoluble fractions e x t r a c t e d from w a t e r homogenized lens cortices and nuclei is examined, t w o different situations a p p e a r . I n r~ts, dogfish a n d ca~aractous h u m a n lenses, a d r a m a t i c shift in the nucleus t o w a r d p r e d o m i n a n t l y U I mateJ~al o c c u r s during aging (rats, dogfu~h) or with c a t a r a c t o u s changes (human, X - i r r a d i a t e d rat), whereas no such shift occurs in boviue or normal h u n m n lenses with aging. I t is convenient to consider t h a t the two insoluble fractions obtained b y aqueous homogenization a n d extraction of lenses as described here~n a.re the result of the final insolubiliz~tion of pre-existing soluble precursors of the US ~nd U I material. These precursors could be t h o u g h t of as w e a ! d y bound aggregates of the soluble proteins present. Since t h e distribution of soluble protehas capable of interacting varies in the nuclei and cortices of the lenses of cl/fi~rent species a t different ages of stages o£ c a t a r a c t f o r m a t i o n , these precuxsors (and therefore the r e s u l t a n t U S and U I fractions) v a r y ~n level a n d conlposition, as is shown. Conversion of t h e soluble precursors :into t h e US and 73I fractions m a y be depend e n t on two sets of f~-ctors. The first set, active in the living i n t a c t lens would include physical factors (such as pressure, d e h y d r a t i o n , and fiber disruption) and chemical changes in the lens fluid (such as a d i m i n u t i o n of reducing agents like aseorbate and glutathione), changes in ionic strength, changes in the level of the lens soluble proteins, a n d changes in the concentrations of m a n y other non-protein constituents. The second set of fac,toxs, ~mposed a f t e r removal of t h e lens from t h e eye, would include changes due to homogenization in aqueotu~ solvents a n d expostu~e to the air, which procedures w ~ ~ d then p r e c i p i t a t e these precursors i n t o t h e final form of US a n d U I obtained. A t this point, e.q)osed proteiu-SH groups could, be oxidized to {ntermolecu/ar SS | i n k s as shown b y Pixie (1968), Zigman 1969~b), and H a r d i n g (1969). The m o s t a t.Hking example of the relationship between soluble proteins a n d a specific fraction of the insoluble protein is provided by d a t a for n o r m a l r a t lens

/'r,,~'r~: 1. Vertical acrylamidc gel elcctrophoretic profiles of rat, lens S.protc.ins. Ilight to left, arc showtl bauds of: ~-¢rys.tallin, cortical US of 52-week.old rats; nuclear U[ of 52.week-old l ' a t s ; cortical US of 6.week-old rats: nuclear* !'[ of 6.wcck.~ld rats: y.cI'.vstallitl. (tcls were ~.a ,~.)[)olyacrylamidc contai,~ing 7 ~[ urea in Tris-glvc, iuc buffer at. I)H. 8.8. t(uns wet'c for 1 hr ;~t 4 mA p(,r tube.

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V A R I A T I O N S OF LENS I N S O L U B L E P R O T E I N S

63

nuclei. An increase between 5 and 52 weeks of ago in nuclear UI of 5 rag/lens, a decrease in ~,-crystallin of only 0.4 rag/lens, and an increase of bands other than those of S-y in electrophoresis show that the colltent of other crystallins than y in nuclear UI increases witix aging. That appreciable amounts of proteins oflmr than },-crystallin may be present in the UI of She whole mature rat ]ens as shown by ~he data of Harding (1969). 0nly trace ammmts of ottmr protein species than 7-crystallin could be detected in young rat nuclear UI by acrylamide gel electrophoresis, by amino ~cid composition [including the diflbrence il:dices described by Harding (1969), and by ultraviolet light extinction (Zigman, Seku.!tz, and Yule, 1969)]. The other US and UI fract.ions from rat leas nuclei and cortic¢s ,:ontaia ~ppreciable quantities of protein components similar in eleotrophoretJc mobility to those of other crystallins. No attempt to quan~itate fl-crystallba levels in the insoluble fractions was made~ since fl crystallins always contain appreciable :¢- and 7-~rystallin contaminants which are difficult to remove withou~ extensive purification. :It appears likely that in human cataractous leases: the increased UI level found in the nucleus is ~tso related to the decreased level of soluble ~,-crystallin in cataractous human lenses ~s shown by Clark, Zigznan ~nd Lerman (t969), ami o~hers (Francois, Rabaey and Stockma ns; 1965; Pirie, 1968~ fiharleton and van Heyningen, 1968). I¢~EFERENCES Gharleton, J. M. and van Ho3mingen, It. (1968). Ex,ptl Eye Rea. 7, 47. Clark. R., Zjgman, S. and L~rman S. (1969). Ezptl Eye Res. 8, 172. Francois, J., Rabaey, J. F. and Stockman.s, L, (1965). Ez~tl Eye Re~, 4, 3[2. Harding, J. J. (1969). ]~JxptlEye Res. 8, 147. Lowry, O. ~., Rosebrough, N. J., l~'arr, A. f~. arid I~andall, R.. J. (1959). J. Biol. Chem. 193, 265. 5[anski. iV. (1968}. Preser~t~(t in ~l'urLeat The Conferer~ce mt tim Biochemistry of the Leas. Abstract in Exptt Eye R~s. (1969) 8, 229. Or~Lst(,in, L. and Davis, B. J. (].960). D i~c Elearophoresia, Distillation Products ]ndust,riea, Rochester. ~ w York. [?irie, A, (1068). ln~est. Op~lmlmaI. 7, 874. ~W,,ct.or, A. (1964). Zn~e~t, Ophthalnml. 3, 18"2. Zigman, S. ([969o,). Annals Ophtha].moI. 1, 206. Zignmn, S. (1969b). Biochim. Biophys. Acta 181,319. Zigman, S., Schuitz, J. and YuIo, T. (1969). Biachem. Biophya. Res. Commun. 35, 93].