o~.~. -:Lb~

DEC 3 0 1930 Received

THE SLIME SECRET I ON

of

illYXI 1ffi GLUT I~OSA

by

H~ RB B nT W. LEMON

THE S L:nr S S ~ Cn5TION O? 1VlYX I N~ G ~.:UTnJOSA

by

Herbert -:!. Lemon

University of Toronto.

The probl em inve st i gat ed Via s t he chemical nature of

the s I Lme s ecret ion of ~"yx i l1e Glut inasa. The work ';;a s done

at the Atlantic B~ o. l og i ca l Station, st. Andrews, N.B . ,

during t ile sumner of 1930.

l~xine Glutinosa, commonly cal18Q the Hag-fi sh , or

the 81ime 3e l , is a Cyclostome. i t derive u it s specific

-name f'r om it s habit of exuo i ng en ormou s quantities of slime.

According to J . E. Blomfield, two indiViduals thrown into

a bucket of wa t er are ca pa bl e of ge l a t inis i ng t ne whol e Wi t h

The se cre t i on i s derived from a double row of glands

or sl imn sacs on t he ventral surfa ce of t ne body, and extend­

ing a l :'-"ost the f'u i L length of t he b ocy , There are a hundred

or more s l ime sa CG in one an i ma l . They aT e not all equal ly

large. : a ch ha s 2 sepa raTe duct to t he surface. The s n sa cs

1 .are Virtually s i mp Le inv olutions of t he SRln, or epidermal

inpus hings into t he dermis. The se structures have been

de s cr ib e c1 by J . ri;u l l e r .

The slime itself as it is c7 cre t ed from t hS8S g l and s

presents a mi l ky appearance and is of extraordinary Viscidity.

ru en mixe d wi t h w a t er it forms a visci d ropy mas s. Mi cr o­

scopic exami na t i on has shown t llat t he milky a ppeara nc e i s

due to the presence in t h e sl ime o ~ a mu ~titude of ovoid

objec ts and f ine t hreads '.-.: ~l i ch are " in a Li qu i d me ddnm ,

2 .

Aol l i ke r ha s s~own that t he oval shape d objects or c orpus cles ,

are modi fi e d ce lls , an ~ t hat the t h r oa ds a re pr oduced by

t he s e ce l l s. Thu s t he corpus cle s a re cal l e d "th r ea d ce l ls".

He found a lso that t he t hr ea d ce l l s de vel0 0 from the epith­

e l ium of t he sl ime So. cs .

Acco rd ing t o 31omf ie ~ a , the c ontent s of t he sl ime S8C S

cons i s t of t wo di :ferent ce ll ty~e s, t he t hrea d cell s a l ready

desc r ibe d , and a 180 " s pider cells ll whi ch form a stroma f or

the t h r ea d cell s. He holds it probab l e t hat t ue spider ce l l s

y ield t he liq i d of t he s l i me sec r etion i n which t he thr ~a ds

an d t hread ce l l s are suspended .

E. ~ . Rei d wa s t he fi r s t to study t he mi cros co pic a ppear­

a n ce s of t he so cr ~tion in the f resh state , al l t he previous

work hav i ng been done on ~res e rv e d spe c imens . ue f ound t hat

a f r esh preparation t e ems wi th gr anules fil~ ing a lmost

c ompl et e l y t ''le .irrt e r spac ns b e t we en t he t hr ead cells. The s e

gr anules r epemble muc i n g ranule ~ . They d isi~ te £ rate in

wa t er a nd most r eagent s . ~eid states t 0at t he ETanule s

a r e pr oduc e d i n mucous cel ls , which are the 8p i do r ce l l s of

Blomfi e l d. He a I s o s t a t es t na t .81omf ie ' drs sugge st i on t hat

t he sp ider c e l l s co ntribute t he l iqU i d per t of t he s e cr et i on

i s und o bte '~, ly c or r e ct , but t na t t '.. i S pa r t of t he s ecret i on

i s fo rm Qd in t he cells in g ranules which ca n :o t be preserved

i n s i t u . .

3.

:PROCEDURE.

The only previ ous wo rk done on the chemical nature of

the slime secreti on of t he sl i~e eel was by E. W. Re i d .

Refe ren ce wi l l be made to h is work later.

COLliE C;l' I 021 OF 10iVl'ERI AL

luOs t of t he mat er i a l used in my expe r i ment s was collected

by put t ing some of t he fish in fresh wa t e r , into which t hey

exuce l a r Le quantiti es of sl i me. As s tated before, when

in contact wi th wat er t he slime form s a vi scid ro py ma ss ,

which can be ea s i ly collect ed. SOille material wa s collected

by Reid's me thod , that is by removing the skin with the

gl a nd s anu squee z ing cu t t ue s l ime . The r esults obtained

wi th t :lis mate r i al were t he same as those obtaine d wit h

material collecte d f rom ~Yste r, a nd as the f i sh wer e not

killed by the f irst met hod , it was t he one chie fly used.

S01131 L1:i.'Y Oi!' r.i.' .tI? SLU m

Te st s v.e r e mad e on t he s ol ubil i t ;'l of the s l ime in var­

i ou s r eagents. The r e sults were a s f ollows:­

1. O. OI}~ Sodirun hydr oxide. The g r eat mass of t he s La .ue

did n ot d i ssolve , but on f ilte r i ng a clear liqui d wh i ch was

ext r eme l y viscous was obt ained . ~v iden tl y s ome pa rt of t ile

sl i me had been d i ssolve d . This was prec i pitated by 'making

t he so l ut io r aci d wi t h ac etic acid.

2. I ii, S ad i urn hy dr O:.:ide. I n this cono errt r a't ion of

alkali, or i n g r eate r co ncenttat i ons, t he slime was complet ely

s ol uble. The s olut i on gave a l l the prot e i.n colour t ests.

3 . 1% Acetic a c id . The slime s eeme d to become more

stringy. On filt ering the so l nt i on a clear liquid \7aS ob­

t ained. On mak i ng it 81 ifht 1;7 alkal i ne a pre c i p i tate "va s

4.

forme d, s o l ub l e in ex ce s s of alkali.

4. Gonce ntrated hy drochlo ric acid. A purple coloured

solution wa s obtained. Thi s was similar in colour to that

produced in Liebermann's r ea ction for tryptophane in proteins.

Liebermann found that protein treat ed fir st with alcohol

and et her , a nd t hen with hydrochlo ric acid developed often

a voilet or bright blue colour. The r ~action is probab ly

due to the pr esenc e of a Ld ehyde s in the a l coh ol a nd ethe r

which combine W i t n the pr ot e i n , and on heet i ng ':1i th s t r ong

hydrochl or ic a c id dev e l op t he try pt ophane r eaction. If

t he pr otein contains both trypt ophane and sugar, it is n ot

ne ce s saz-y to treat it wi t h alcohol or ether f irat , since

by the action of t be strong acid on t he carbohydrate, ald­

ehydes are fo~ed which give a co loured r eaction pro Quct

Wi t h somo of the protein group s, a nd pr es~~ably with tryp­

to phane. ' This woul d auggost the presence of a carbohydrate

in the slime.

SEPARATION OF C ON STI TU ~ NTS

Attempts W8re now made t o fractionate the different

constituents of the slime. Reid found by microchemical ,

examination of fr esh slime, obtaine d OJ' squeezing it out

of t h e slime sacs, that with very dilute alkali {O.Ol%

potassiurn hyd r ox t ce .) t he gr anules we:ce completely d issolved

while the thre2.ds wero not diss olved. The granule substance

could be pr ecipit at ed from so lution by making faintly acid

with acetic aci d. He s eparat ed the rtgranulo substance rt and

the "thrcad sub stance" in t his way and stud Led s ome of the

propertios of each. He fo und that the granule substance

c

5.

r e s embled a mucin , ez ce pt that on h;y dr olys i s wi t h 2;; sUlph­

uric acid it di d not yie l d a substance which would r educe

Fehling's so lution. The thr ead substance he found t o be

somewhat simi l a r to t he g r anule substan ce , but of a mo r e

i n s oluble nature.

I s eparated the gr a nu l e a nd t hread substance s 1y

Re id ' s met h od . I also s eparated a thi rd sUbstance. It wa s

found t hat if the s l i me collect e d from water were allowed

to stana, much of t he wat er whi ch it cont ained s ett l e d out

and a t hick r opy mass float nd on t he top. The liquid was fil ­

tered clea r, and i t was f ound t hat on making i t slightly

alka l i ne, a pr e c ip itate ~as f ormed . Thi s substance was

f ound to be so l u bl e in wat er , and i n di l u t e ace t i c a c id . I t

coul d be aga in prec ip ita ted from su rh s ol uti ons by making

t hem s l ight l y a lkal ine . This sub st an ce appear s to be t he same

a s t hat d i e s ol ve d out of t he s l ime by I rE, acet ic a cid a s de s ­

cr i bp. d ea r l i e r i n t he r e por t. The pr ope r t i e s of t his sub ­

stance have be en l i t t le s tud ie d , but it may be a pr ot e i n

wi th i t s isoelectric po int on the a lka l ine s i de of neutrality.

Be s i de s t hes e t hre e substance s, it wac found t hat some

f atty or lipoid .na't e r f a l a c ou l d be extra ct e d fr-o m the s l i me

with hot a l coho l or ether.

PROPEHTIES OF GRA2:DLE SUBSTANCE

Mos t of my inve stigation of t he pr ope r t ie s of the

f r actions was done on t he granUle s ubsta nce s as it waG t hougtt

t o be a mucop rote i n . Before d iscus s ing t tli s par t of t he

wO: k i t is ne ces sa ry t o des cr ibe b r ie f ly t he natu r e of

mucoproteins .

6.

A mucoprotein is a conjugated protein. It consists

rof a protein molecule conjugat ed wi t h a carbob0 drate radicle.

The work of Levene has shovm that the carbohydrate group

is built up of fo ur component s in equimolecular pro portions.

The component s arG sulphuric acid, acetic acid. a hexosa­

mine, and glucuronic acid. There are two types of carbo­

hydrate groups, chondroitin sulphuric acid, a nd mucoitin

sulphuric acid. The diffe rence lie s in their hsxosamines.

In cho ndroitin sulphu~ic acid i t is chondrosamine--an

amino derivative of galactose. In mucoitin sulphuric acid

it is chitosami ne, an ami no de r i va t i ve of glucose. The

probable formula, according to Levene, of mucoitin sulphuric

acid is as fol lows:­

On pa r t i a l hy dr oly s i s the sulphuric acid radicle is

detache d, Leav t ng chondr oit in or mu c ort tn , On further' hydrolysis

t.e a ce t y L g r oups ar e lost and t he molecule splits forming

a disacchari de, chondrosin or muc os i n . Chondrosin Qr mucosin

will re duce -Feh l i ng f s solut ion.

7.

To s~para te t he granule subs tan ce from t he sli~e,

Reid 's me t hod was l argely u sed. The met h od of collecting

t he s l i me has a l ready been de scribe d. An amount of 0 .01%

s od ium hy dr oxide equal to t en t ime s t he volume of the slime

was a dde d t 0 it. Th is was aha ke n v r gor ou s I y f or some t i rne

t hen a Ll owed t o sta nd ove r n i ght , The granul e sub st ance

was dis s olved whi l e t he throuGs were not . The mixture wa s

filt ered t hr o ugh several thic ~ne 8 8 e s of cotton. The s olution

was v a ry v i sc ous. To t he so lution was now ad ded g l a c i a l

ac et i c a c id dro l"' by dr op urrt il no t'urt her pre cipitate f ormed .

The pre c ip i t a t e was v er y st rin~J, a nd adhe red to t he sides

of t he ves seL, Ln t h is way it r o s emb l e d mucin . I t v-a s

collect e d by f i l t e ring , or by ce nt r ifuging, and r edi s s olved

i n 0 . 1·,1j s od i um hy dr ox i de , f rom v; ': 1ch i t was preci p itated as

before . Now i t wa s wa s r.ed su c c -i es i ve Ly wi th 30%, 7O'}0, and

ab s o1ute ethyl a Lcoh o; , and f inal J.y wi t h et he r . I n orde r to

r eDove lipoid mate r ial , t he snbstapce wa s ex~r2ct e d wi th

bof l Lng al coho l fo r s i x hours , u sing a r e f l ux condens er . l"t was

' n ow dr i ed i n a va cuum dessi c8tor .

Now 0.1 gram aampLe s of t he s ubstance v.e.r e hy dr ol i s e d

wi th 10 o.c. of 5% SUlphuric a cid . This wa s ca rri ed on in

a wate r bath . At int erva l s Lc .c . sampl e s of t he mi.xt u r e

were taken , fi l tereo , neut ra l i ze d , a nd boi l ed wi th 1 c.c.

of Fehlin~· l s s ol ut i on. Und oubte d reduct ion was obtained

a f t cr t en mi nute s hydro lys is , and coul d s t i l l be obtained

a fter two h ou r s , Whi ch ra s long a G t no proce s s v.a .: C -rrt Lnue d ,

Th i s re sult i s contrar y to t hat of R ~i£ who obta i ne d no

r e du ct ion on ~y dro lysi 3 of t he g r anule SUb stance. Pr oof

8.

that t he gr a nule subst an ce contains a carbohy dr ate radi cle

was afford ed by t he fa ct t hat osazone cry st a l s wer e obt a i nec

aft er hy dr oI y s i s . The se crystals were e ;~tremely small but '

could be exami ned w it ':, a mt cr oecor-e of fairly h i gh power .

The ir s tructure r e s e mb l ~d t hat of glucosazone crystals.

The s e r esults s eem to s how t hat t he granule substance

is s, t ru« mu c in . To compl ete ly pr ove t his , it would iJ e

nece ssary to prepa re muco i t i n SUlphuric acid fr om it. 1 was

una ble to a t tempt thi s, as I ha d not suffic ient of t he substan ce

for a prAr aration.

Ni t rogen determinat i ons were made on t he granu l e s ubstance,

using t ne macro-i\..j elda hl met .iod , It wa s f ound t hat the

sub s tan ce contains 12.6); notrogen. Th is is f urt .ie r ev i dence

t ha t the subotance i s a mu copro te i n , a s simpl e pr ot e i n s

ha v e a n i t rogen cont ent of a out I G% Wh i l e in mu c opro te i ns

it i s usua lly between 10% a nd 14%.

PROPERT IES OF ,T,:ffiFAD SUBSTANCE

On hydrolysis of the t h r ea d subs t an ce , no substa nce re­

duc ing F r'h ling I s so l uti on was obta i ne d . )

CCPWLUSI ONS

The s l ime produce d by myxi ne Glut inosa consi st s o f the

f ollowi ng substRDces : ­

1. The Granule Substance . This has t he propertie s

of a mu copro te i n . The prese n ce of a car bohydr a te group

i s Bho~n by the f act t hat on hyd rolys is with 5% sulphuric acid,

a bod y which r i l l re duc e Fehli ng' s so l ution is obta ined , and

"""i ch g i ve s an osa z o'ne when t r na t e d 'w i th ph enyl hy dr-azLne ,

The nitr og~ n cont ent of t he granUl e substance is 12.6%.

9 •

2. The Thread Substance. Thas substance gives typical

protein r eactions. It does not yield a reducing substance

on hydrolysis.

3. A substance solubl o in water and dilute acetic . acid,

but precipitated by making the solution faintly alkaline.

Its properties have not been investigated. I '~ 4. Lipo id ;:a t erial. A fat l i ke substance can be ex­

tracted with hot alcohol.

It is 0vid ent that furt her re s oarbh should be done

on the slime secretio : of this fish, both on its chemical

natu~e and on its physiology of which nothing is known.

In conclusion I would l i ke to say that I am greatly

indebted to Dr . Koma r ov of the Department of Phy s i ology ,

McGill University, for valuab le a dvice.

)

.s

10.

1. L e vene, ~. A. " Hexosamines and iolUcopr ot e i n s "

2. J oh . Idil 1e r " Unt ers, uber die Ei ngeweid e der

Fi sche" Abh. Akad. Be r l i n , 1845 •

3 . Koll t .ke r ••fl U.d. Inbalt de r Schleimsacke

der Myx i noi den u. d. ~p id e rm i s

\ '-­ ' der Neunaugen" Wur zburge r ~ at urw .

Ze i t s ch . Ed . 1. 1860, p. 1-10.

4. Blomfie ld TT The Threa d Ce l ls and Epede rmi s ar i,:yx i ne . tT Quar t . Jo urn. of I\licr os .

Science . Vol . XXI I , 1882, p . 355.

5. Re id , .s . »s: "Mucin Gr anules of Llyx i ne lt

Jo ur n. of Phy s ±ol . Vol . XLV ,

18 93, p. 340.

6 • Re i d, E . Vi . tf Chemica l :Note on t"he Secre t i on of

Myx i ne Glutino sa fl J our n . of Phy s mol .

Vol. XV , 1894, p. 488.

I' i

I .