14 lowered content of low nlolecular weight sulphy,dryl compounds has been demonstrated in human cataractous lenses. (Consul and Nagpal, 1968; Harding,.
Exp. Eye Res. (1977)
25, 139-148
The State of Sulphydryl Groups in Normal and Cataractous Human Lenses* R. J. W. TRUSCOTT
AND
R. C. AWWSTEYN
RussellGrimwade Xchool of Biochemistry, lImiversity Parhille, Victoria 3052. Australia
of
Melbowxe,
(Received30 November 1976, Yew Yo7k) The levels of non-protein sulphydryls, protein sulphydryls and protein-bound sulphydryls were measured in the separated nuclei and co&ices from individual normal and cataractous lenses. As the colour of the lens nucleus increases, there is a progressive decrease in the levels of non-protein sulphydryl and protein sulphydryl. In the nuclei of advanced cataractous lenses, these decrease to 10% and 30,(,, respectively, of the levels found in the normal nucleus. Similar, but smaller, changes take place in the cortex. The level of protein-bound sulphydryl increases in the early stages of cataract formation but, thereafter remains constant in both the cortex and nucleus. In the advanced cataractous lens. the protein bound sulphydryl accounts for only about, Z”,, of the loss of protein sulphydryl. There appear to be no intermolecular disulphide bonds in the urea-soluble proteins of the lens but some may be present in the urea-insoluble proteins. Key words: human lens; cortical cataract; nuclear cataract; protein sulphydryl; proteinbound sulphydryl; non-protein sulphydryl.
1. Introduction Senile nuclear cataract formation in man is characterised by a brown pigmentation of the lens and an accumulation of insoluble proteins (Pirie, 1968; Truscott and Augusteyn, 1976). Little is known about the processes which result in these changes. The role of sulphydryl containing compounds in the maintenance of lens transparency. has been of interest for many years. 14 lowered content of low nlolecular weight sulphy,dryl compounds has been demonstrated in human cataractous lenses (Consul and Nagpal, 1968; Harding, 1970; Mach, 1966) and it has also been shown that cataractous lens proteins contain higher disulphide and lower sulphydryl contents than normal :lenses (Auricchio and Testa, 1972; Testa, Fiore, Bocci and Calabro, 196X). It has been proposed that a function of the glutathione present in normal lenses might be to protect protein sulphydryl groups from oxidation (Kinoshita and Merola, 1973). It has been suggested that oxidation of protein sulphyclryls to disulphide bonds during aerobic extraction is largely responsible for the increased amount of insoluble protein isolated from cataractous lenses (Harding, 1972a). This observation could also account for t,he higher levels of disulphide bonds in cataractous lenses. However, oxidation of protein sulphydryls does not necessarily produce insoluble protein, antl, also. it has recently been shown that the insoluble proteins of the cataractous lens are not artefack of the exkaction methods (Kramps, Hoenders and Wollensak. 1976; Truscott and ,4ugusteyn, 1976). * Reprint
quests
to: Dr K. C. Augusteyn
I:.
110
.I.
\\‘.
‘I’ler‘s(‘o’l”l’
.\SI)
1:.
I’.
.\I~(:~s’I’lc:~s
111 orcler t’o clarify t,he role of sulphydryl oxidation in sctiilf~ (:ilmt;tr;tCt lilr.trldt lol. we have conducted a systematic investigation of normal and caataractoris l(~li,~c~sii t The levels of fret, low moltcula~~ \vcaigllt progressive stages of cataract formation. sulpllydryls. protein-bound sulphydryls and prot,cin sulph.vdryls in the wpara t wl nuclei and cortices from individual lenses are presented iti this coilinwi icatioil.
2. Materials and Methods Cataractous lenses were obtained from operations performed at the Royal Victorian Eye and Ear Hospital and were classified into types I- TV on the basis of increasing nuclear colour as described by Pirie (1968). Lenses were processed within 3 hr of removal from 1he eye. Normal lenses were obtained at post mortem within 24 hr of death. Whole lenses were separated into cortex ;mtl nucleus using a cork borer (internal diameter 5 mm) followed by slicing off the ends of the core. These operations were performed 011 the pestle of an homogenizer to reduce the loss of protein. Spproximat,ely equal amount~s of protein were obtained from the separated nucleus or cortex.
Each cortex and nucleus was assayed for noll-protein sulphydryl (NP-SH), protein sulphydryl (P-SH) and protein-bound sulphydryl (PB-SH). The methods used for determining sulphydryl contents are mo&ficatioIls of those described by Sedlak and Lindsay (1968). All solutions used in these determinations were flushed with Nz and urea solutions were passed through a mixed-bed ion exchanger, immediately before use, to remove ammonium cyanate. Each cortex and nucleus was homogenized in 1 ml of l(, “’;{I trichloroacetic acitl ~OIItaining (1.02 M-EDTA (TCA/EDTA solution). The homogenate w-as transferred to a capped centrifuge tube, flushed with N,, and centrifuged at 3000 ~g for 10 min. The pellet was washed with a further 1 ml of the TCA/EDTA solution and the combined supernatants were used for assay of NP-SH. The pellet was washed with 5 ml (Jf 10”; trichloroncetic acid (which was discarded) and was then dissolved in 2 ml of 04 M ammonium bicarbonate containing 8 M urea, to produce solution A which was usetl for the determination of P-SH, PB-SH and protein concentr;rtion.
Non-proteLl
sulphydryl
(NP-XH)
1.0 ml of the combined TCA/EDTA supernatants was mixed with 2.0 ml of 1.0 MTris-HCl, pH 9.0 and the A412 was noted. 50 ~1 of DTNB (0.01 M in methanol) was added; the solution was mixed, and the AdIz was read again within 60 sec. Hulphydryl levels were calculated from the increase in A,,, by reference to a standard curve covering the range 0-l ,X 1()~4nl-cysteine. The curve was Identical with that obtained by Sediak a.1~1 Lindsay (1968). Quantitative recovery of cysteine added to the supernatants was obtained with this method.
Protein
sulphydryl
(PSH)
Solution A (250 ~1) was mixed with 3.95 ml of 0.5’?~ SDS and 0.75 ml of 0.2 nl-Tris--HCl, p-H 8.2; A,,, was noted and then 50 ~1 of DTNB (0.01 M in methanol) was added. Solutions were allowed to stand for 30 min, with occasional shaking, and the absorbance at 412 nm was determined. P-SH was calculated from the increase in d,,, by reference to a standard curve covering the range O-l x 10e4 M-BSA.
SULPHYDRYL
Protviwbownd
sztlphydryl
GROVPS
l?;
(~‘ATARACTS
141
(PB-SH)
Holution A (500 pl) was mixed with 5 mg of KaBH, and reaction was allowed to proceed at room temperature. At the end of 25 n1in, protein was precipitated and excess NaBH, was destroyed by the addition of 1.0 ml of 20 yb trichloroacetic acid containing 0.2 M-EDT.4. Pentanol (20~1) was added to prevent frothing. The solution was allowed to stand fol 10 min ; then centrifuged and 1.0 ml was a.ssayed for sulphydryl content as deacrib~~ll for SP--SH.
Protein was determined using a modification of the Biuret metl1od (Gornall, Barcl;~~~ill a,nrl David, 1949). Solution A (3~0 y 1) was incubated at 20°C for 30 min with 60 ~1 of 10 .\I-?\‘aOH. Water (700 ~1) was then added followed by tj ml of the Biuret reagent. Aftetrr 30 GO min, djsO was measured and protein contents were calculatetl hp reference tcb a st,antlard curve constructed using BSA (GlO mg/ml). Incubation with SaOH was included in order to solubilize those prot#eins from catarac,tous lenses which remain insoluble in 8 &I-urea. Omission of this step resulted in erroneousI> high readings due to light scattering. The incubation did not affect the subsequent cc)10111 drvrlopment.
Nuclei were honlogenized with 3-O ml of 0.05 nr-Tris HCI, pH 8.5, containing 8 RI-UK:I! and were then dialyzed against the same buffer. Insoluble material was removed by cerl-trifugation and samples of the soluble proteins were chromatographed on a 1.3 x 55 (~11 column of Biogel A-15111 equilibrated in the above buffer. The column was operated at room tempemture at 3.0 ml/hr and the efflueut was monitoretl continuously at 280 WI. Samples of t,he proteins were also reduced with (b.1 M mercaptoethanol and c:hrornat~c)graphed on the sa,n1e colum11 with 1 x 10-4 &I-dithiothreitol added to the buffer. *ill the other methods used have been described previously (Truscott) anal Augustcayrl. 197(i).
3. Results Table I lists the values obtained for non-protein sulyhydryl (NP-SH). protein sulphydryl (Pm SH) and protein-bound sulphydryl (PB-SH) in the cortices and nuclt>i of 38 normal and cataractous lenses. Whenever possible the clinical description of the opacity is included. These results will I)e discussed in the categories listed abovtl.
Amino acid analysis indicated that glutathione was the major (>90%) sulpltytlryl compound in the TCA/EDTA supernatants. The NP-XH results are summarizecl in Table II. Wide variations were observed in the NP-SH detected in both the cortex and nucleus of lenses of the same type (e.g. patients 10 and 11 in Table I). This is illu+ &rated by the large values obtained for the standard deviations in Table II. However, in all 54 lenses examined, the cortex contained considerably more NP SH than the nucleus. Similar results were obtained by Kinoshita and Merola (1958) using bovine lenses. Most nuclear cataractous lenses, especially types III and IV, contained very small
Sulphydryl
contents
SP-SH (n1n01) Patient Age
Type*
50 45 62 51 52 79
N N s s I 1
10 11 1” 13 14
.54 69 61 50 5.5 tin 71 74
1 1 1 1 1 1 II II
15
73
II
I6
72
11
17 18 19
84 83 66
II II II
“0
74
II
40 76 79 69 69 72 68 69 58 6X
11 III 111 III III III Ill III III 1v
31 3”
76 x5
IV IV
33 34
62 59
IV IV
35 36 37 38
83 84 76
IV IV IV IV
7 8 9
Clinical
Lens descriptionf
Post. cortical wsicles (diahctic) _.-Pmt. suhcapsalal Post. suhcapsulal Adv. cortical C’nrticsl (diahctic -Suclear cat. -Post. snhcapsulal Nuciear cat. + Post. suhcapsulat Nuclear cat. Post suhcapsular
Suclear cat. 7 Post. suhcapsulal Nuclear rat. + Post,. suhcapsulal
Nucleus
Cortex
129 115 110 15i 26 34
389 359 435 372 11’ 562
I .X9 1.84 1.48 I .92 1.1’1 .. 2.16
9 104 16 3 1L’.’ 6; Ii 13
19“ “89 144 Ii3 363 1% 2.57 1’”
1.59 l.il 1.oFi I .44 I .i!l 1.75 0.52 0.1:!
15
111
lb;5 1
I’
81
w.53
1.5 1 33
2 t:? (57 21x
lb36 0.11 0.6X
I6
I15
1 ,o:i
165 39 41 53 39 43 1X 216 56 51
Bilateral Nuclear sclerosis+ Cortical Nuclear sclerosis Nuclear sclerosis+ Adr. Cortical Nuclear sclerosis Nuclear sclerosisiAnt. Cuneiform
Ad\-.
nuclear
sclerosis -.
Cl.15 0.16 0.20 oal O.Oi
wo:1 lb37 047 04XI
I1 0
663 4”
0 (I.13
8 53
515 1s4
0.1” 0
i 0 31 5
163 81 46 ‘5
0.05 0.01 lJ,O:! 04x
* Based on increasing nuclear colour as described by Pirie (1968). 1’ Taken from ophthalmologists’ records. Post., posterior; Ant., cataract. $ Calculated assuming an average subunit mol. wt. of 20 000.
antorior:
-MY.
advanced;
(~‘at..
SULPHYDRYL
GROUPS
IN
(‘STARACTS
143
amounts of free sulphydryl in the nucleus, and lowered levels in the cortex. This is reflected in the average values listed in Table II. However, this is not a universal phenomenon associated with cataract formation since two of the nine type IV lenses examined (patients 34 and 37) had levels of nuclear NP-SH close to the average value obta,ined for type I nuclei (Table II). TABLE
II
T?w low moleculu~ weight sulphydryl (NP--SH) conterlt itl the nuclei and co&ices of normal and cataractous lenses
LtWs typt.~~
Average age
s I 11 111 IL.
5” 65 71 70 74
* SP-SH was determined of proteins witch trichloroacet,ic The figures shown represent
by
SPmSH* (nmol)
hrumber of determinations 4 16 12 13 9 measuring acid. the range
the
( ‘ort’ex
SUCteUS
110-1.X (127) S122 (41) 1- 55 (16) I- 11 (9) o- 63 (13) snlphydryl
of values
obserrrd.
content The
-‘7’-436 fx-363 5-2.57 18-216 ‘%663
of homogenates average
values
(364) (191) ( 150) (74) (189) after
a~
shown
prccipitatiotl irl brarkcts.
In several casesof advanced nuclear cataract (notably patients 31. 33 and 34). the total non-protein sulphydryl content of the lens was not markedly clrcreast~cl. These results do not, agree with those presented by van Heyningen (1972). Prot&~
sulphydryl
(P-SH)
In contrast to the NP-SH levels, the P-SH values for lenseswkhin each group fall into a fairly narrow range (Table I). The normal values are similar to those obtained 1)~ Kinoshita and Merola (1973) but are almost double t’hose reported bp Harding (197%). There is a decreasein the P-SH levels of both cortex and nucleus with the tlevt~lol~lllent of senile nuclear cataract. This is illustrated in Fig. 1 where the average values of P SH in thrb cortex and nucleus of each group of lensesis present)Pd.
NOVIlOl (52)
Type 1 (61)
TYP~~ (71)
Type m (701
Type= (741
FIG. 1. The protein sulphydryl content in the nuclei and cortices of normal and cataractous Values shown represent the averages of the values listed in Table I. Standard deviations are on each column and the nrerage age of each group of lenses is shown below. Hatched columns. open columns. nuclei.
lensca. indicated co&ices:
proteins are the same. With the onPet< of nuclear cata.ract (tvpe 11) thtl sull)ttylir>~I content of the nuclear prot,eins decreased to 0.49 ~nol~uwl protein. This ~~IY~II th a decrease of 67q;, relative to t,vpe I proteins. 117 these lellws the WrtiCitI I)r.otc~in sulphydryl level decreased by only about 20:,,. There is a further and progressive decrease in the sulptlydryl content of tllcb nut:l+~~I proteins in the more advanced stages of nuclear cataract. In the t)ype IV n11(:1(~11~. the average protein sulphydryl content is only 0.05 11101/mol protein con11~ r(~l wit,11 1.60 and l.T9 for the type I and normal nuclei respectively. The cortical l)rottaitl levels show a pronounced decrease between the type II and t,ype III ICWSW I jut (IO not change significantly in the type IV. The level of P -SH in the type IV c:or.tic:a1 proteins is about 0.55 ~nol/~nol protein. considerably higher than the valuc~ of 0.05 obtained for the nuclear proteins. It. might be argued that the decreases shown in Fig. 1 could be ascribed t,o ngtxing since lenses removed from older patients are more likely to have dark nuclei (t,yl”B III and IV) than those from young patients. However. all the normal arJtl t,ype 1 nuclei (age range. 45-78) have P-XH values above I.05 mol/~uol protein. \+~h(~r(~a~ t,he type III and IV nuclei (age range. 48-85) all have P--SH values below 0.4 anti lllost are below 0.1. Thus. lenses with very low nuclear P-SH contents can be found at ztl 1’ Sfl early age (e.g. patients 29 and 34) and conversely lenses with high nuclrar values can be found at later ages (e.g. patients 6 and 8).
Protein-bound
sulphydlclryl
(PB-NH)
Investigations into the conditions required for the liberation of prOt~ill-~JouJl~~ sulphydryls, indicated that the results obtained depended on the time of retluct,ion with NaBH,. This may be seen in Fig. 2 in which the reduction of cystine was studied. Although the absorbance reaches a value corresponding exactly to the theoretical
Time
(mind
FIG. 2. Time course study on the reduction of cystine by NaBH,. Cystitw was reduced with NaBH, (10 ma/ml). Samples (1 ml) were remored at various times into 3 ml of IO”,, TCB containing 0.02 x-EDTA. After 10 min at room temperature. 1 ml samples were assayed fol, solphydryl content as outlined in Materials and Methods (NP-SH).
absorbance after 24 min, there is a rapid decrease thereafter. Thus control over the reaction time is critical if an accurate measure of PB-SH is to be obtained. Part of
YULPHYDRYL
GROVE’S
IN
115
CATARACTS
this loss of sulphydryl was found to be due to reoxidation : the remainder had presumably reacted with cyanate generated in the urea. The averages obtained for the PB-SH levels in various lenses are summarized in Table III. There appears to be very little PB-SH in either the cortex or the nucleus of the normal lens. The values obtained correspond to about 0.27; of the cy&inc content of the proteins. TABLE
LII
Protein-bow& sulpl~yd~ryl cordeat (PB-SH) ,irl the m&i cortices of normal 0 rrd ctrtnrwtous hu~rr~~nlensrs
* PH-SH was dekrmined by measuring after reduction of t.he prooteins with ?JaBH,.
the amount
of low mr~lerular
wc,ight
rr ml
sulphytl~~~l
liberntd
In the cataractous lenses the levels of PB-SH in the nuclei were about double thohtb found in the cortex. Since the levels in types II-IV lenses were approximately douljle tho;;e in the type I lens it would appear that the development of nuclear cat,aract ii: accompanied by an increase in the protein-bound sulphydryl. However. the levc~l~~ found are insignificant, in terms of the total decrease in P-SH. In addition: the lt~~ls of PB-SH remain constant in type II&IV lenses whereas the P -SH continues to fall with the development of cataract. Amino acid analysis of the liberated thiols indicated that, glutathione was the majo (‘>900/,) protein-bound sulphydryl.
The mixed water-soluble and urea soluble proteins from normal and type 1~ IF nuclei were chromatographed on Biogel A-15m (in 8 al-urea) in the presence and absence of dithiothreitol. No differences could he detected between the profile obtained. This is not consistent with high levels of inter-molecular clisulphide I)ontlh since reduct’ion of such bonds would be expected to alter t’he elution profi1t.s. 4. Discussion In st,udies rising whole cataractous lenses, most workers have described a decreasta in the non-protein sulphydryl content (Consul and Nagpal. 1968; Harding. 19iO: Mach. 1966) although others (Barber, 1968; Dickenson, Durham and Hamilton. 1968)have reported levels varying both above and below normal. The results presented in this paper indicate that most nuclear cataractous lenses do have non-protein sulphydryl contents well below normal. In t,he advanced cataractous lens the average level in the nucleus is about 10% of that in t.he normal nucleus.
146
I!.
J.
\\‘.
‘I’l~I’St’O’l”l’
;\KLJ
JL.
c’.
-AkIt:C:S’I’E\~S
Nevertheless, some lenses with advanced nuclear cataract contain ;ituouut.s (Jf' SIII~L~~ Iv-eight sulphytlr~~l conq~ounds well in excess of t)hc averegc ~>orllli~l vii luc! (e.g. patients 31 and 33 in Table I). However. in these cases, less tha.n 2”,, of ttlca totirl non-protein sulphydryl is found in t,he nucleus lvhereas in tlw awragc Ilorlrlal 1t.w more than 257; is found in the nucleus. This serves t’o emphasixc! t trc, inlport;inc.c* cbf separating the nucleus and cortex of the lens in studies such as t)hcst>. The importance of such demarcation is also illustratetl t)y t,tw tlwxtaw in pr.c,t.t~il~ sulphydrylobservecl in type II lenses. In this init,ial stage of nuclear cataract format km, the sulphydryl content of the nuclear prot’eins has dropped to 33”;, of that fount1 ill the type I lens. This large decrease would have been masked in whole lrns stlulic~.s ;I.S the cortical proteins sho& only a small reduction in sulphydryt cont.ent. Pirie (1973) has reviewed the problem of defining the r~uclc~~s and corttls in the human lens. In this work. the inner 5074 of the lens was considered to reprclsent the nucleus. This corresponds roughly to the weight of the lens at birth (Ehlrrs. Mahhiessen and Anderson, 1968). Ideally. concentric layers from the lens shoultl lw sturtiwl. However, not enough nlaterial would he present in such a layer for thtb stutliw described in this paper and the risk of oxidation of sulphydryl group< during r~, of the total protein is insoluble in urea. This protein is localized in the nucle~~s of tltt lens and has been called the yellow protein fraction (Truscott and dugusteyn. 1Siti). It seems probable that this fraction contains inter-chain disulphide l)ontls sinccs it becomes soluble on addition of a reducing agent. It is likely that there is some mechanism in the normal lens which functions to keep the proteins in a reduced state. Failure of such a system may allow tht, INIS proteins to oxidize and thus to become crosslinked and insoluble. Glutat,hione reductasc may serve this purpose in the normal lens by maintaining a high level of reducetl glutathione. However, the results obtained in t,his work indicated that the level of 1~~01ec~l~~r
SULPHYDRYL
reduced proteins.
glutathione
does not
GROUPS
always
correlate
IS
CATARA(“I’S
with
the state
I47
of oxidation
of the
There may be other systems which act to maintain the lens protein in the form in which they were synthesized. It is also possible that no such protective mechanism exists in the nucleus of the lens, but that the conformation of individual polypeptides as well as the tight packing of proteins in the fibre cells are alone sufficient to prevent,
oxidation of protein sulphydryl groups. Over a period of many years. conformational changes in these proteins, due perhaps to continual stress during accommodation (Patnik, 1967) may alter this protective structure and thus make the proteins susctptihle
to oxitlation. SCKNOWLEDGMESTS
This work w‘~s supported, in part, by grants from the Sational Health and Medical Resear(:h Council of Australia and from the National Eye Institute, N.I.H.. li.S.;\. (Grant number 1 RO 1 EY 09525-01). The assistanceof ProfessorG. W. Crock and his staff, at the Royal Victorian Eye and Ear Hospital, in obtaining the lenses used in this work, is gratefully acknowledged. Thr competent, technical assistance of Xs S. Jorgenson is also acknowledged. REFERENCES
G. and Testa, M. (1972). Some biochemical differences het,neen cortical (pale) and nuclear (brown) cataracts. Ophthalmologica 164, 228-35. Barber. G. W. (1968). Free amino acids in senile cataractous lenses: possible osmotic Ptiology. Auricchio,
Invest.
Ophth.almol.
7, 564-83.
Consul. R. N. and Kagpal, P. N. (1968). Q uantitative study of t,he variations in the levels of glutathione and ascorbic acids in human lenses with senile catarart. Eye, Kor. .Vose. Thr.
Mon. 47, 33G-9. Dickmson,
J. C.. Durham, D. G. and Hamilton, P. B. (1968). Ion exchange chromatography oi’ free amino acids in aqueous fluid and lens of the human eye. Incest. Ophthdmol. 7, 551-W. Ehlers, ?u’., Matheissen. M. E. and Andersen, H. (1968). The prenatal growth of the human ey(*. Acts
Ophthalmol.
46,
329-49.
Gornall. A. G.. Bardawill, G. S. and David, M. RI. (1959). Determination of serum proteins I)>means of the biuret reaction. J. BioZ. Chem. 177,75L66. Harding, J. J. (1970). Free and protein bound glutathione in normal and cataractous human lenses. Biochem. J. 117,157-60. Harding, J. J. (1972a). The nature and origin of the urea-insoluble protein of human lens. /‘:.rl,, Eye Res. 31, 33-40. Harding, J. J. (197213). Conformational changes in human lens prot*eins in cataract. Riochem. ./. 129,97-100. Harding, J. J. (1973). Disulphide cross-linked protein of high molecular wright, in human wt,ar;tc.. tous lens. E.rp. Eye Res. 17, 377-83. Kinoshita. J. H. and Merola, L. 0. (1958). The distribution of glutathione and protein slllphydryl groups in calf and cattle lenses. Am. J. Ophthalmol. 46, 36-42. Kinoshita. J. H. and Merola, L. 0. (1973). Oxidation of thiol groups of the human lens. In Thu Human Lens in Relation to Cataract. Cibn Foundation Symposium 19(h'ewSeries). Pp. 178-84. Elsevier. Amsterdam. Kramps, H. A., Hoenders, H. J. and Wollensak, J. (1976). Protein changes in the human lens during development of senile nuclear cataract. Biochim. Biophys. Actn 434, 3243. Mach, H. (1966). Untersuchungen von Linseneiweiss und Mikroelektrophorese von wasserlijslirhem Eiweiss im Altersstar. KZin. MbZ. Augenheilk. 143,689-710. Maraini, G. and Mangili, R. (1973). Differences in proteins and in water balance of the lens in nuclear and cortical types of senile cataract. In The Huma,n Lens in Relation to Cntnract. !‘ibn Foundation Symposium 19 (New Series). Pp. 79-95. Elsevier, Amsterdam. Patnik, B. (1967). Photographic study of accommodative mechanism: changes in t,he lens nucleus during accommodation. Invest. OphthnZmoZ. 6, 601-l 1.
11s Pirie.
I:. .I. \v. ‘1’I~I’(‘SO’l’T
.\Sl) 1:. (‘. Al’(1rs’l’15\‘s
A. (1968). t’olor and soluhility of tlw proteins of human c3taract. f rcwsf. I)~,hlhrt/wr4. 7, W- 54 ). l’irie, A. (1973). Chairman’s introduction. In 7’8~ lf~wnflt~ L~rrs i/l. /klrrtio)/ lo (‘crtrr/,fwl. ( ‘i/w Founrhfion ~Yymposiunl 19 (Xew Series). Pp. 1~ 3. IClsevier. iZmsterdam. Sedlak. .J. and Lindsay. R,. H. (1968). Estimation of total. protein-bound. and non-protein sulphpdryl groups in tissue with Ellman’s reagent. Analyt. Rioche~. 25, IW-2OS. Texta. M.. Fiore, C.. Bocci, N. and Calabro, S. (1968). Eflkt of the oxidation of’sulpl~ytlryl groups on lens proteins. EJ.rp. Eye Res. 7, “76-90. Trnscott, R. J. W’. and Augnskyn, Ii. (1. (1977). Changes in human lens prokins during mrckat cataract formation. Exp. Eye Res. 24, l:i997(l. ran Heyningcn, R. (1972). The human lens. 11. Some observations on catarx~ts removed in (lxfortl. England. fi.qj. E?/e RPS. 13, 14&X.
