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Hadidian, Z. and Pirie, N. W. 1948. The effects of serum and of
hyaluronic acid derivatives on the action of hyaluronidase. Biochemical
Journal. 42 (2), pp. 266-274.
The publisher's version can be accessed at:
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https://dx.doi.org/10.1042/bj0420266
The output can be accessed at:
https://repository.rothamsted.ac.uk/item/8wywz
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© 1 January 1948, Portland Press Ltd.
I948
The Effects of Serum
and of Hyaluronic Acid Derivatives
onthe
Action
of
Hyaluronidase
By Z. HADIDIAN AND N. W. PIRIE*, The WorcesterFoundation for Experimental Biology, Shrew8bury, Massachusetts, and the Department of Physiology, Tufts MedicalSchool
(Received 26 June 1947)
In thepreceding paper(Hadidian&Pirie, 1948) the viscosity of hyaluronic acidpreparations inadefined environment was used as a criterion in selecting standard preparations. In this paper the loss of viscosity by such a preparation has been used to follow the course of hyaluronidase action. This method has the primary advantage that loss of viscosity appearstobe the firstchange that can be recognized during the enzymic decomposition of hyaluronic acid. Factors influencing this in vitro changearemorelikely,therefore,toaffect the action ofhyaluronidase in vivo than those influencing the later stages of the decomposition of the molecule. It is clear that severalenzymesareinvolved in the hydrolysis of hyaluronic acid to reducing sugars. ThusHahn (1945)haseffectedapartial separation of the enzyme responsible foroligosaccharide pro-duction from thatresponsible for monosaccharide production; and Rogers (1946b) finds differences between theproducts of the reactioncatalyzed by enzymes fromdifferentsources. Heinterprets this
asevidence that the enzymes studied by Hahn are present indifferentratios in thesesourcesand points
outthatit isimprobablethateither enzymebrings about the reduction inviscosity.
Several inhibitors are known for the action of hyaluronidase on hyaluronic acid, whether this is measuredbyfall inviscosity,mucin-clot prevention
or appearance of acetylglucosamine. McClean & Hale(1941) andHobby, Dawson, Meyer& Chaffee (1941)found that theseraofanimals, that had been injected withhyaluronidase from different sources, inhibited the action of the enzyme used for immuni-zation butdidnotinhibit the action of other hyaluro-nidases. Later,however, McClean(1942)mentioned briefly an inhibitor present in normal serum, and this substance has also beeninvestigated by Haas (1946). Heparin, gastric mucin, chondroitin sul-phuric acid and partly depolymerized hyaluronic
acidalso inhibit theenzyme. McClean (1942) sug-gests that theseglucosamine-containingsubstances
act competitively because of their structural similarity tohyaluronic acid. Workingonthis sug-gestionwehave madesomeinhibitorsby modifying hyaluronic acid. We also record some observations
onthe inhibitorpresentinserum.
* Onleave ofabsence from Rothamsted Experimental Station,Harpenden,Herts.
EXPERIMENTAL
General
Viscositymeasurements weremadebythetechnique used in the preceding paper(Hadidian&Pirie, 1948). The time taken for the viscosityto fall half way from the initial to thepresumed final value has been takenas ameasureof the rateof enzyme action.This measurement of'half-time' is conventional in studies on this enzyme and has been adopted forconvenience, although it isclear that it has
noparticular merit. It does not, forexample, correspond to theloss of viscosityby half of thehyaluronicacidpresent butto asmallerproportionthanthis; andto aproportion that varies inversely with the original viscosity of the hyaluronic acid. This is due to the non-linearity of the relation between the concentration and the viscosity of
ahyaluronic acidsolution,andtothenon-linearityof the relation between the axial ratios ofelongatedparticles and the viscosftyof their solutions. The enzymeaction weare studyingcanbepicturedin two extremeways.The enzyme may attack aparticle ofhyaluronic acid andproceed to
breaklinkagesinthatparticleuntiltheproductshavelittle
or noeffectonthe viscosityof the solution;thehalf-time wouldcorrespondtoastatewhere less than halfthe particles have beenaffected because of the firstnon-linearity. On the otherhand, itmay breakall theparticlestosomeextent before anyoneparticlehas beensofarreducedastohave littleeffectontheviscosity of thesolution;because of the secondnon-linearity the half-time wouldcorrespondtothe breaking of less than half the relevantlinkages.
Some confusionis alsointroduced into the use of
half-time measurements instudyinginhibitionreactionsbythe viscosityof the inhibitors. In manycasestheviscosity of theinhibitorisunaffectedbytheenzyme; the finalvaluefor the endof theaction cantherefore be takenastheviscosity of the inhibitor solutioninthe saltmixtureused. Insome
cases, however, the sum of the increments in relative viscosity, duetohyaluronicacid and inhibitorseparately,is
less than theobservedincrements when theyarepresent
together.Thiseffect,whenitoccurs,issmall andaslongas
no significance isattachedtodifferencesin half-time less than30%,itisunlikelytoalter theconclusions.
Measurementsof therate ofloss ofviscosityhave been
madeon anextensiverange ofhyaluronicacidpreparations
underidenticalconditionsandwiththesameenzyme pre-paration. The half-times have varied by afactor often.
Part of this variation is due to differences in the initial
viscositiesof thepreparations. Ifdifferencesinviscosityare
due todifferencesintheaveragelengthof theparticlesin
a preparation,thehalf-timecorrespondstothebreakingof a smaller number oflinkageswhen theparticlesare long
usthat umbilical cord may contain substances that activate or inhibit the action of hyaluronidase on hyaluronic acid, and that these may have been removed to varying degrees during the purification. The fact that viscous preparations are the most uniform in their half-timeis in favour of this interpretation. We have tested many of the products
dis-cardedduring the purification and have found, in agreement with other workers, that fractions of very low viscosity are inhibitors. No fraction giving activation has been con-sistently preparable.
Preparation of
inhibitor8
Nitration of hyaluronic
acid
and8ubstances
related
to it.Dry hyaluronic acid, with the loose, open structure it has
afterdrying by sublimation, is cooled in a freezing mixture to about -100. Sixty parts of fuming
HNO3
(sp.gr. 1.59), also cooled to the same temperature, are added and the mixture is maintained at -100 for 15 min. with occasionalstirring. All the hyaluronic acid dissolves in a few minutes. Ice (300-400 parts) is now added with assiduous stirring. The temperature falls still further and a white curd separates from abrilliantblue fluid. As soon as most of the ice has
meltedand all pockets of undiluted
HNO3
in the curd have been broken up with a rod, the mixture is centrifuged for afew minutes and thesolid
is washed 3-4 times on the centrifuge with ice water. It is kneaded carefully each time toget rid of as muchHNO3
as possible, and the washing is continued until the wash water can be neutralized by a drop or two of 0- 1 -NaOH. Thesolid
is now suspended indis-tilledwater and brought into solution by adding sufficient NaOH to bringthe pH to 6-7. This pale yellow, viscous fluid will be referred to as acid-insoluble nitrate.
All the supernatant fluids are mixed and kept cold. They are neutralized with 5N-NaOH with vigorous stirring and
coolingsothat the temperature never rises above 200 during the neutralization. The fluid, at pH 6, is
concentrated
on a hot plate in a draught of air untilNaNO3
begins tocrystallize out. It isthen dialyzed for 10-20 hr. to remove most of the nitrate, concentratedagain,and redialyzed. At this stage thesolutionfrom 100 mg. of hyaluronic acid has avolume of 2-3 ml. The solution remaining after thorough
dialysiswill be referred to as acid-soluble nitrate. The ratio of acid-soluble to acid-insoluble nitrate varies from preparation to preparation. When hyaluronic acid of low viscosity (e.g. with a relative viscosity of 1-4 for a1 g./l.
solution measured in the presence of salt) is used as the starting material there is
little
acid-insoluble product. When even less viscous fractions are used there is no acid-insoluble material at all. It seemed possible, therefore, thatall
the acid-soluble material was derived from the less viscous components of aninitially
non-homogeneous hyaluronic acid. It isunlikely that this is the sole origin; for if the acid-insoluble nitrate is dried bysublimation
and nitrated again by themethoddescribed, a further quantity of soluble nitrate is formed. Some degradation of the molecule during nitration is to be expected, and this explanation of the origin of some of the soluble nitrate is borne out by the increased proportion of product in that form if the nitration is continued for longer than 15 min. The sum of the weights of nitrate in both forms is about equal to the starting weight of hyaluronic acid and, when highly viscous hyaluronic acid is used, 85%is in the acid-insoluble form.Nitration always leads to some fall in the viscosity of hyaluronic acid. The extent of this fall is
variable,
but inisabout half that of the
parent
hyaluronic
acid.
Theacid-soluble
nitrateis
muchless
viscous.
Nitrated
hyaluronic
acid,
of bothtypes,
can besaltedout from neutralsolutionby
(NH4)2SO4.
With theacid-insoluble
product,
pre-cipitation
begins
at about one tenthofsaturation
and iscomplete
at one third of saturation. Themorereadilypre-cipitated
fractions
are the moreviscous,
and they areprecipitated
fully
by
lowerconcentrations
of(NH4)2SO4
than the lessreadily
precipitated
fractions.
It is clear,therefore,
thatthenitrateis anon-homogeneous mixture, butit is not certain whether thisreflectsan
originalnon-homogeneity
of theparent
hyaluronic
acid
oris
due to destructionduring
thenitration.Precipitation
ofthe acid-soluble nitrates and of nitratesderivedfrom
hyaluronicacidpreparations
ofvery
lowinitialviscosity
does notstartuntilone third saturation
by
(NH4)2SO4.
Salting out
does not therefore seem to be a usefulalternative
toneutralization,
evaporation
anddialysis
in thepreparation
of acid-soluble nitratesbecause,
although
acid-insoluble nitrate ispre-cipitated
by
saturatedNaNO,,,
the solublenitrates
are not, and thehigh
nitrate concentration confuses(NH4)2SO4
precipitation.
Hyaluronic
acid is not salted outby
(NH4)2SO4
fromsolutions
containing
no othercomponent,
but,
asone of the methodssuggested
for thepurification
shows,
it can besalted out if
pyridine
or some forms ofprotein
are alsopresent.
The nitrates behavesimilarly,
for,
althoughesterifi-cation of
thehydroxyl groups
hasmadethe
nitratessuffi-ciently hydrophobic
to be saltedout,
they'an
besalted
outat lower concentrations of
(NH4)2SO4
in the presence ofpyridine.
There
is no reason to doubt that thehyaluronic
acidhasnitrated as other
polysaccharides
do(cf.
Speiser & Eddy,1946);
for thedry product
burnsin aflashandthe nitrogencontent
(Dumas)
hasrisenfrom3-4
to8-6 %.
Noestimateother
than the measurement ofnitrogen
contenthas been made of the number of nitrategroups
introduced. Ifhyaluronic
acid has the structuregenerally
assigned
toit, achain of
alternate
glucuronic
acid andacetylglucosamine
residues
connected by glucoside
linkages,
there arefourfreehydroxyl
groups
for eachrepeating
disaccharideunit. Thesodium
salt of the tetranitrate that could be madefromsucha
substance wouldcontain12%
ofnitrogen
andthatofthedinitrate
8-55%.
Esterification of thehydroxyl
groupsinthis
way
does
notapparently
affecttheN-acetylgroup,forthe
acetyl
contentfalls
from
10-11
to7-8%.Thevalue tobe
expected
for thedinitrate is 8-7%.
Controlexperiments show that neithernitrous nornitricacids, inamountscom-parable
to thosepresent
inahydrolysate,
areestimated asvolatile acid after
hydrolysis
anddistillation
by thepro-cedures used
on thesesamples.
On the other handdirectestimation,
by
the Elson &Morgan
(1933)
method, on thenitrated
material showsanapparent
disappearance oftheglucosamine.
This result is due to the action of the HNO8liberated
during hydrolysis.
The samedisappearancecan bebrought
aboutby hydrolyzing
normal hyaluronic acid inthe
presence
of
aquarter
ofitsweight
ofNaNO3,
anamountcorresponding
to four nitrategroups.
Furthermore,de-esterification,
by
the method to bedescribedlater,restoresthe
apparent
glucosamine
content,
and hydrolysis in thepresence
of
areducing
agent
suchas HIprotects
theglucos-amine to
agreat
extent from destruction. FumingHNO3
probably
acts to some extent as an oxidizing as well as28Z.
HADIDIAN
AND N. W.PIRIE
the glucosamineisgoingon to anextent atall comparableto thatfound by Skanse & Sandblad (1943)toaccompany peroxideoxidation.
Acetylation ofhyaluronicacid
Like some other acidpolysaccharides (e.g. alginicacid, pectinand agar)hyaluronicacid doesnotacetylatereadily. Aceticanhydrideinneutraloralkaline aqueoussolution,or
in the presence ofpyridine under anhydrous conditions,
failed to bringaboutsignificantacetylationin afew hours at room temperature or on slight warming. Two recent
methods are,however, effective;inonethe acidisswollen withaceticacid andH2S04isusedascatalyst(Chamberlain,
Cunningham&Speakman, 1946),and in the other the acid is swollen with formamide andacetylated withacetic
an-hydride andpyridine(Carson&Maclay,1946).Theproducts
of these two acetylations differ somewhat in their pro-perties.
(1) Hyaluronic acidisswollen withaceticacidby adding
glacial acetic acidinathinstreamto a3-10g./l. solution. Mixingiscompleteuntil about 2 vol. have beenadded,after which precipitation begins and is substantially complete
with4 or5 vol. Themixture iscentrifugedandthesolid
washed with acetic acid and usedimmediately. This pre-cipitation doesnotleadtoanysignificantfractionationof the hyaluronic acid, for only about 3% of the starting
materialisfoundinthe solution when this isevaporatedto
dryness. This soluble fraction has the normalnitrogenand glucosamine content, -but its viscosity is low. The pre-cipitate,onthe otherhand,ifdialyzed immediately, gives
asolution with theoriginalviscosity.We donot,therefore,
agree with the statement made by many workers on
hyaluronic acid thatprecipitationwithacetic.acidleadsto
afallinviscosity; butourobservations arein agreement
with thoseofMeyer,Smyth&Dawson(1939)whoprepared
arelativelyviscousproduct fromsynovialfluid
by
acetic acidprecipitation.The fibrous clot ofhyaluronicacid ispressedasfree from acetic acidaspossibleandaddedto200 times itsweightof
a mixture of 1 part of conc. H2S04, 90 parts of acetic
anhydride and 120iparts of benzene. There islittle or no
solution of the hyaluronic acid, and, after a suitable
interval,themixtureiscentrifugedand the solidsuspended
in wateranddialyzed. Ifacetylationisallowedto
proceed
forafewminutesonly,theproducthasanacetylcontent of 16-20%;after40 min.at220 this risesto25-33%and isnotincreasedbylonger acetylation atthat temperature.
By
following the method of Chamberlain et al. (1946) and raising thetemperatureto600 for 15min.,afteracetylation
hasproceeded for1hr.at 250, the acetylcontent can be raisedto36%. Acetyl determinationsweremadeby
the method usedinthepreceding
paper(Hadidian
&Pirie,1948)
and include both0-acetyl and N-acetyl. The theoretical value for the tri-O-acetyl derivative ofapolysaccharide
built up from equal quantitiesofglucuronic acid and
N-acetyl glucosamine is 34% and for the tetra-0-acetyl
derivative 394%. It would therefore appearthat,asin the
case ofnitration, esterification remainsincomplete.
Pro-ductsthat have been
acetylated
forafew hoursonlyarestillsolublein wateroverthe whole
pH
range, but those that have beenacetylatedatroomtemperaturefor manyhours, orhave beenheatedto 600during acetylation, are notsoluble onthe acid side ofneutrality. There is a
pro-gressive fall in viscosity during acetylation. Products
acetylated for a few minutes only retain 70-80% of the original viscosity increment, whereas the tri-O-acetates retain only10%or less.
(2) Hyaluronicacid that has been dried bysublimation
does not change its appearance when left for a few hours at
200in formamide, but at400itswellssomewhat and at500
turns in 1 hr. to a viscous dough when suspended in ten times its weight of formamide. This dough can be mixed with an equal volume ofpyridine by stirring for a few minutes. The plastic, rather than fluid, mixture is now mixed with half its volume of acetic anhydride, which causes an initial shrinkage, but is incorporated into the mixture in a few minutes. As in the other. method of acetylation the properties ofthe final product depend on the duration of acetylation and the temperature at which it proceeds. The mixture is stirred with three times its weight of ice; when it is brought to pH 2 with 5w-HCI
a soft curd separates. Thefluid is dialyzed. If acetylation has proceeded for 1 hr. at
200
this acid-soluble product has an acetyl content of14-16%, while after 3 hr. at300 it rises to 30%. In neither case has it any significant viscosity. The curd is not soluble at neutrality in the absence of salt and is only partly soluble in the presence of salt, but it swells slowly to give a suspension of gelatinous particles, which dissolve- at pH 9-10. The solutions have only one fifth of the viscosity of the parent hyaluronic acid. All these acetylated fractions coagulate with strong acid and, like the nitrates, can be precipitated with(NH4)2S04.
De-esterification. Thenitrates and acetates of hyaluronic
acid can be partly esterified without extensive de-gradation by exposure to alkali in the presence of ethanol. Solutions of either ester containing 3-10 g./l. are mixed with 10 vol. of ethanol. With the esters, unlike with hyaluronic acid itself,noprecipitate separates even on the addition of a salt such aspotassium acetate. The addition of ethanolic KOH at 220 leads to the appearance of an opalescence, and later, afteratimeinterval depending on theamount of alkali added, ofaprecipitate. For example,
an ethanolic solution containing 0-8 g./l. of a moderately viscous nitrate (rel. visc. 2-3 at 1 g./l. in salt) gave an immediate precipitatewith 7 g./l. KOH; but with 2-5 g./l. precipitation took 2hr., and with 0 7 g./l. precipitation occurredonly on the addition of some salt. Similarly with theacetatethereisimmediate precipitation with 1-2 g./l. KOH, but with less than this amount precipitation is delayed. Under these conditions de-esterification remains incomplete as determined from the nitrogen and acetyl content of these products. The immediate purpose of the work, the making of substances that inhibit hyaluronidase is, however, satisfied; and conditions that would lead to complete de-esterification have not been investigated.
Factors
affectinq
thehyaluronidase-hyaluronic
acid reaction
Effectofsaltconcentrationonthe rate ofreaction. In studying the effect of possible inhibitors and
acti-vators on the hyaluronidase-hyaluronic acid re-action, it would first be necessary to establish con-ditions of saltconcentration under which such work
can be carried out with the least possible inter-ference from salt effects. Results of Robertson, Ropes&Bauer(1940), McClean&Hale (1941), and
optimmum salt concentration for thereduction of the viscosity of hyaluronic acid by hyaluronidase, but the optima theyfind vary with their different
sub-strateand enzyme preparations. We have tried the effect on this reaction of several salts, previously studied for their viscosity-reducing activity, with results shownin Fig. 1. These reactions were carried
oNaCl
1-4 oKCI
oNH4C1
1-2 0
/M0CI2
QCaCI2
1-0 0
SodiuIm phosphIate
eNa2SO4
0-8
0.6
0-4
0-2
0-1 0-2 0-3
Molarityof salt
Fig. 1. Variationsintherateof hydrolysis ofhyaluronic
acid by hyaluronidase with varying concentrations of differentsalts. Temp. 25°; pH 6-9±0-10; 12-5mg./l. bull-testis enzyme; 0-3g./l. solution of a medium viscous
hyaluronic acid preparation. In the experiments with
NaCl, KCI,NH4C1and
Na1SO4
thepHwasmaintained withphosphatebuffer, and in those withMgCl1 andCaCl2with borate buffer. Inallcasesthemolar ratio of saltto
buffer was 100. In expressing the relative rates, the
reciprocal ofthe half-time in 0-125M-NaCl is taken as
unity.
outatpH 7-0,using salt solutions buffered with 1 % of phosphate or borate buffer. 'Of necessity, pH control in low salt concentrations was not quite adequate, but frequent checks showed that variation
was not morethan 0-2 unit; this mayproduce as
much as 20% variation in the rate of reaction. From theresults shown and from othersondifferent substrates, three groups are distinguishable:
CaCl2
and MgCl2, effective in very low concentrations; NaCl,KC1 and
NH4CL,
justaseffective but inhigher concentrations; Na2SO4 and Na2HPO4, relatively ineffective. Sodiumacetateis intermediate between the chloride andphosphate. Lineweaver & Ballou (1945), in their study ofthe effect of saltconcen-trationontheactivity ofpectinesterase,find similar variations in the effectiveness of various salts in activating the enzymic reaction, but with that enzyme there is the samemaximum activation at
with McClean & Hale (1941) that the chloride ion is an important activating agent. However, the difference in the effectiveness of the chlorides cannot be attributed to Cl- alone. Expressed as normality of C1-, assuming complete ionization, the optima with Mg++, Ca++,
Na4,
NH+ and K+ are at 0-05, 0-06, 0-1 and 0-15 respectively. Furthermore, in the presence of0-05M-phosphate, maximumactivationis obtained with 0-05m-NaCl. Two factors seem to be involved; general salt concentration and the presence of an activating ion, such asCl-.ThepH optimum in the presence of0-05m-NaCl and0-05M-phosphate is at 6-3; this value is intermediate between the optima of 6-8 and 5-0 found by McClean (1943) in 0 017M-and 0-167M-NaCl respectively. Our experiments have been carried out at pH 7 as a compromise between the pH optimum and the pHatwhich the enzymeand inhibitors areused in in vivo tests that willbe describedelsewhere.Estimationof inhibitoryactivity. Sincenomethod exists for direct estimation ofhyaluronidase, the course of the reactionbetweenit andaninhibitor has to befollowed by the indirect methodof determining theamount ofhyaluroni-dase that is made unavailable for the hyaluronic acid-hyaluronidase reactioninthepresenceof the inhibitor or afterthe hyaluronidase has beenexposed to the action of theinhibitor foragivenperiod of time. If, then,oneworks in a region where there is a linear relationship between hyaluronidase concentration and therateof loss of viscosity by hyaluronic acid, one can justifiably consider the rate as adirectmeasureof theamountof activehyaluronidase
inthe system.The rangeoverwhich sucharelationexists
varieswithdifferent preparations ofhyaluronicacid tested
atthesameconcentration.Withhighlyviscouspreparations andwith thepartiallypurified enzymepreparationthatwe
use,deviation fromlinearity isslightupto anenzyme
con-centration of 8mg./l. With less viscous preparations this range may extend upto50mg./l. Thefollowingmethods
areusedinestimatinginhibitory action: in instances where thelengthof exposure of the enzymetothe inhibitorisof little or no consequence, the enzyme is introduced into amixturecontaining both inhibitor andsubstrate,and the subsequent viscosity changesfollowed;where theextentof
inhibitionisdependentontheduration of exposure of the enzyme to the inhibitor, as in the case of inhibitionby
serum,the enzyme and the inhibitorareincubatedtogether
underthe proper salt andpH conditions foragivenlength
of time, then the substrateisintroducedinto themixture,
and the course of the reaction followed. The extent of inhibitionisdeterminedfrom theratioofthehalf-timeof the reactionwith inhibitortothat without. Thusathreefold inhibition would be obtained ifahalf-time of600sec. is
obtainedwith inhibitorascompared with200 sec.without. Thereciprocal of this ratio is the fraction of the initial enzyme concentration thatisactive. Partiallypurified
bull-testisenzyme(Schering)wasusedinalltheseexperiments. Inhibitionbyhyaluronicacidesters. Nitrates and
acetates that have beenmade from viscous
hyal-uronic acid retain muchof their initial
viscosity.
TheZ.
HADIDIAN AND N. W. PIRIE
tested in the usual way. The viscosity of nitrates is notreduced by hyaluronidase and the viscosity of acetatesoftenfalls at first and then stays constant. We look on acetates behaving in this way as
mixtures of material that can be attacked and material that is not attacked and doesnotinhibit thereaction. The stability of these hyaluronic acid esters towards the enzyme contrasts with the sus-ceptibility to attack of the p-aminobenzyl ether (Humphrey, 1943). In Humphrey's preparations, however, less than one ether linkage seems to be present for each glucosamine in the hyaluronic acid, and no indication is given of the extent to which hydrolysis, which was recognized by the appearance ofN-acetylglucosamine, took place.
withnitric esters aremoresystematic; all of them inhibit. Attempts to get less nitration by using
asmaller ratioofnitric acidtohyaluronicacidorby allowingthenitration toproceedforashortertime have led to less active products. The acid-soluble fractionsfrom the nitration of viscous materialand theacid-soluble nitrates of non-viscous materialare
strongerthan theacid insoluble nitrates. By suit-ably controlled removal of nitrate groups with alkali and ethanol the inhibitory power is sub-stantially increased; but it is clear from the table that the intensity of the alkalitreatmentis of im-portanceand that toodrastic a treatment leadsto aweaker inhibitor. Forcomparison the results of experiments withnon-viscous hyaluronic acidand
Table 1. Effects of various inhibitors on the hyaluronidase-hyaluronic acid reaction
Descriptionofinhibitor
Viscoushyaluronic acid acetylated by sulphuric method for 60 min.; 33 % acetyl Above product deacetylated for 15 min. with 2 g. KOH/I.; 14% acetyl
Viscoushyaluronic acid acetylated by sulphuric method for 10 min.; 18 % acetyl Viscous hyaluronic acid acetylated by formamide method for various times;
15,25 or30% acetyl
Acid-solubleproduct from nitration of viscous hyaluronic acid
Acid-insolublepart of the samenitration
Acid-soluble product from nitration of viscous hyaluronic acid Acid-insolublepart of the same nitration
Aboveinsoluble product denitrated for 2 hr. with: 0 7 g. KOH/I. 2-5 g.KOH/L.
7-5g.KOH/I. Nitrated non-viscoushyaluronic acid
Thesamenon-viscoushyaluronic acid before nitration Heparin
Concentration
(g./l.)
0.10.1 0-1 0-1
0-01 0-1 003
0*03
00050004 003
0-01
0*1
0-1
Inhibition
1-5 6 4 Inactive
3-5
7-3
30 2-2 5 7-5 2-2 5 2 2
Inthese experiments2ml. of a0-6 g.l. solution of hyaluronic acid in 0IM-NaCland0-Im-phosphatebuffer pH 7 were mixed with 1ml. ofasolution of theinhibitor atfour times the concentration specified in the second column. 1 ml. ofa50mg./l.solution of hyaluronidase was then added and the flow time in an Ostwald viscosimeter was measured at
variousintervals. Thehalf-time was foundinthe usual way, and the amount of inhibition set out in the third column
isderivedby dividing the half-time foundinthe presence of the inhibitor by that found in a control experiment with enzyme andsubstrate alone.
The inhibition is measured by mixing suitable
amountsof inhibitor withhyaluronic acid and then addingtheenzyme. There isaslight increase in the inhibition if the enzyme and inhibitor are mixed first, butin this instance theextentof inhibition is
notincreased by allowing the enzyme and ester to bein contactformorethan the time necessary for mixing. Some comparable results are collected in
Table 1. Valuesare givenforonly oneconcentration of each inhibitor because the inhibition is pro-portionaltotheconcentration.Thetableshowsthat
acetatesinhibit weakly,ornot atall, and the most active are partially acetylated products the pre-paration of which isamatter ofsomeuncertainty. On partial de-acetylation the inhibition always increases, but conditionsthatwouldlead toregularly repeatableresults havenotbeen found. The results
heparin are also included in the table. Many
non-viscous preparations of varied origin have been tested andnonehas been found withasignificantly greater inhibitory power than the one cited. Only
one sample of heparin was tried; the inhibition found hereiscomparabletothatfoundby McClean (1942) and Rogers (1946a) using different experi-mentalprocedures. Ourtests weremadeat250with hyaluronicacidhavingarelativeviscosityof7-8for
a1 g./l. solution in005M-NaCl and
0.05M-phosphate
bufferpH7,althoughexperiments described inthe next paragraph show thatthere is more complete inhibitionwithsmallerconcentrations ofphosphate. The same sample of dried, partially purified hyaluronidase from bulltestes (Schering) wasused throughoutat12-5mg./l. Similarexperimentshavebeen made withlargerand smallerconcentrations of
having a relativeviscosity as low as 2 for the 1 g./l. solution. These variations in the conditions have not significantly affected the amount ofinhibition observed.
Variations in phosphate and NaCl concentration, however, have a marked effect on inhibition by the esters. Several of these, including an acid-soluble nitrate, an acetate, a denitrated nitrate and a de-acetylated acetate,-were tested in0-05M-NaClwith phosphate concentrations varying between 0-013 and024M; and in 0 05m-phosphate with NaCl con-centration varying within thesame range. Within this range there is an increasing inhibitory activity with decreasing salt concentration. None shows a significant inhibitory activity in 005M-NaCl and
024M-phosphate and all show maximum activity in.
005M-NaCl and 0 013M-phosphate; but they all follow different courses between these extremes. Products with identical inhibitory activity under
our standard testing conditions may differ by a factor of 2, at lower phosphate concentrations. Thus the figures given in Table 1 would not necessarily indicate the relative effectiveness of these substances underphysiological conditions.
For convenience of writing, the operations that have been performed on hyaluronic acid in making theseinhibitors have been called acetylation, nitra-tion and de-esterificanitra-tion. It is certain that the bulk of the material is undergoing these changes, for the observed acetyl content can be increased and diminishedas canthe Dumasnitrogencontentand theinflammability of the nitrates. It is less certain that these are the changesprimarily responsible for the inhibition. All of them are associated with changes in viscosity andsopresumably in particle size, and it is possible that this is the significant change rather than theextentof esterification. The difficulty found in getting reproducible results from experiments on acetylation and de-acetylation slightly favours this interpretation. On the other hand, the stabilityof these inhibitors in the presence ofhyaluronidase suggeststhat agraded breakdown of the molecule isnotalonesufficienttomakeagood inhibitor. Furthermore, our attempts to degrade the molecule by othermeans, e.g. acid, alkali and partial enzyme digestion, have resulted in only feebly active products. It is probable that in-hibitors have both anoptimum particle size andan
optimurit degree of substitution. Thehigh activity foundwithnitrated non-viscous products suggests thatthissize may befairlysmall; but active material doesnotdiffuse through cellophan.
Serum as an inhibitor. McClean (1942), in the
course of studying the in vivo decapsulation of streptococci, discovered a factor inthe seraofseveral
specieswhichinhibitsenzyme from different sources, andnoted that they variedgreatlyin theirinhibitory
inhibition is due to the presence in the serum of a polysaccharide which acts by competing with the substrate for the enzyme. Haas (1946), on the basis of some by no means conclusive evidence, has claimed that this inhibitory action of serum is due
tothe presencein it of an enzyme which inactivates hyaluronidase.
Theinhibition of hyaluronidase by serum at 250 proceeds at a measurable rate, as shownby Haas (1946). The method used for itsstudy by Haas, and with somemodification by us, has beentoexposethe enzyme to theaction ofserumforagivenlength of time, then mix it withhyaluronic acid solution con-taining enough phosphate buffer to obtain a final concentrationof 0 05-0-1M-phosphate. The validity of this method restsupon the fact, discovered by Haas andconfirmed by us, that in the presence of such concentrations of phosphate (the exact
con-centration varieswith differentserumsamples) the activity of hyaluronidase in reducing the viscosity ofhyaluronic acid is unaltered byincubationwith
serum. One cannot, however, assume on such
evidence that interaction between hyaluronidase and the serum factorceasesin the presence of phos-phate.
If one introduces phosphate into a mixture of hyaluronidase andserum that hasbeen incubated for a time sufficient to inactivate a part of the hyaluronidase, and thenremovessamplesatvarious intervals for testing their hyaluronidase content, one finds that in low phosphate concentrations (001-0.05M) the inhibition of hyaluronidase will continue, and in high phosphate concentrations
(005-0.1
M)itwill bereversed. However, atabout 0-05Mtherateofchangeineitherdirection is very slow. Since the process ofestimatingthe hyaluroni-dase after the inactivation reaction seldom lasts longer than 30min., this method of studying the inhibitory action ofserum seems valid so long assignificance is not attached to small changes in activity.
Like the reaction between hyaluronidase and hyaluronicacid,the reaction ofdialyzedserumwith hyaluronidase does not proceed in the absence of salt. It is activated by small amounts, with an
optimum between 0-03 and 01m-NaCl. The exact
optimum varies somewhat with the amount of
serumused, but variations within the rangespecified
areslight. In0-2M-NaCltheinactivation of hyaluro-nidasebyserumattheendof 10min. ofincubation is aboutone fifth ofthemaximum. Phosphatewill
Z.
HADIDIAN
AND N.
W. PIRIE
the serum-hyaluronidase reaction is apparently in-hibited, 0*075M-phosphate giving full inhibition. The serum used in these experiments was dialyzed for 4-5 days against several changes of distilled water. For the most part we have used pig serum in this study, but most of the experiments described
Scrtim(nil.)
o004
o0o10
* 0-20
10 20
Timeofincubation (min.)
Fig. 2. Rate ofinactivationof hyaluronidase byvarying
amounts ofserum. 0 04, 01 and 0-2ml. dialyzed pig
serum, pH 7; incubation at 25° with 50 /Ag. oftestis
enzymein2mLof 0-05N-NaClcontaining0.3ml. borate
buffer pH 6-9. 2ml. of 0-6g./l. solution of a -high
viscosity hyaluronic acid in01IM-phosphate buffer and
005M-NaCl added tothe serum-hyaluronidase mixture attheendofincubation and theresidual hyaluronidase
activity determined viscosimetrically. Hyaluronidase half-timewithout inhibition
activity
= half-time with inhibition .Owing
to thenon-linearity ofthe relationship between enzyme
con-centration and therateof hydrolysis of this particular
hyaluronic acid preparation the actual amount of
hyaluronidase, expressed as fraction of the original
amountremaining active after incubationis aboutathird
morethan theactivityindicated.
._
'.
-5 4a
Ca ._
Go
0
'0
S'4
have been repeated with human, rabbit, chicken or bovine serum. In view ofthestatementofHaas (1946) that serum obtained by clotting is less active than that obtained from blood defibrinated by stirring, all sera used were obtained in the latter manner.
However, in subsequent experiments with sera
obtainedby clotting and by stirring from thesame
specimen of blood,wehave beenrepeatedly unable
to demonstrate under optimal salt conditions any significantdifference between thetwo.
As aresult of these findings, the following pro-cedure was adopted for testing the inhibitory activity of serum, with such modifications aswere
required by varying conditions. The enzyme (50
pg.)
isincubated with 0-05-0 3 ml. ofdialyzed,
neutra-lized serum for 10 min. in 2 ml. of 0.1M-NaCl buffered with0*2 ml. of 0 2M-borate bufferatpH6-9. To this is then added 2 ml. of hyaluronic acid solution, 0 6g./l. in 0OIM-phosphate, and the course of the depolymerization of hyaluronic acid followed vis-cosimetrically.
Having established beforehand the range of enzymeconcentration over which therelationship between hyaluronidase concentration and therate
ofloss of viscosity by thehyaluronicacid prepara-tiontobe used doesnotdeviatetoomarkedlyfrom linearity, one can follow the course of apparent inactivation of the enzyme by the serum. At 25° with 50 ug. of enzyme and 0 10 ml. ofdialyzed pig
serumin 2 ml. ofbuffered 005M-NaCl, onlyabout 50
%
of the enzyme remains active 30 sec. after mixing, and 25%
after 1 min. (Fig. 2). Maximum inhibition is reached within 10-500 min.depending upon the serum concentration. With high concen-trations ofserumforthenext5-10 hr.noapparent change occurs,buteventually,in everyinstance the hyaluronidase activity begins to return (Fig. 3).Sertim(ml.)
0-00
O004
o010
*020
5000
Timeofincubation(min.)
Fig.3. Recovery of hyaluronidaseactivityafteritsinhibition by varyingamountsofserum. Conditionsofexperiment
arethesame asspecifiedunder Fig.2, where thecourseof the reaction for the first 35 min. isshown indetail. Serum
incubated under thesameconditions, but without hyaluronidase, had lostnoneofits inhibitoryactivityattheernd
ofthe experiment when testedon afreshenzymesolution.
O.1
.,
I948
recovery of hyaluronidase activity immediately follows itsmaximum inhibition. When there is an
excess, apparently a condition of equilibrium is
reached and is maintained as long as there is an excessofthe serumfactor available. The recovery
process in every instance is slow, and is complete
within72 hr.only withlowconcentrations ofserum.
Ahyaluronidase solution kept under thesame tem-perature,salt and pHconditionsmayloseaquarter
of its activityduringthis period. Serum kept under identical conditions losesvery little, ifany, of its
original activity. In repeated experiments, the final hyaluronidase activitywasnever morethanthatat
the beginning of the experiment, thus making it unlikely that this phenomenon is due to the
pro-duction ofhyaluronidase bycontaminatingbacteria. Experimentswerealso carried outin the presence
of 005M-NaF and 005M-benzoateto prevent any
possibilityofbacterial contamination. Both
inter-fere tosomeextentwithhyaluronidase activity but
thereappearance ofhyaluronidase after inhibition
canbedemonstrated in theirpresence.
In these experiments inactivation and
re-acti-vation proceed inasolution that is not otherwise
altered. By changing the enviroument the
re-activationprocesscanbehastened. Dilution of the mixturewithoutchangein saltconcentrationcauses a15%increasein theratefor fourfold dilution.The samedilutiondoubles the rate ofre-activationif it
is madewithNaClcontaining003M-phosphateand
quadruplesit if made with NaCl containing 0
1M-phosphate.
We find thereactionbetweenserumand
hyaluro-nidasemuch faster than is manifest from the data of Haas(1946).This inpartis due to the fact that Haas
usesno salt inhisreaction mixture exceptborate
buffer and whatever salt there is present in the
serum,inpart to the fact that he hasno determina-tions at less than 5 min., and in part to his
pre-sentation of thecourseof the reactionas anincrease
in the 'activity' ofserum. Thus, according to his
data,0 1ml.chickenserumhasan'activity'of 2 at
the end of 5 min., and 4 at 20 min. The former
correspondstoaninhibition of 65%of the
hyaluro-nidase, andthelatterto80%,i.e.onlyanadditional 15% of the original hyaluronidaseis inhibited be-tween 5 and 20min. How much of the 65% in-hibition at the end of 5min.takesplaceinthefirst
few seconds there isno way ofdetermining from
Haas'sdata,butfromour owndata it is evidentthat
thereaction is slowedabruptly after about 1 min. whenan excessofserumispresent.
Theinhibition ofhyaluronidase byderivatives of
hyaluronicacid resembles theserum inhibition, in
ageneral way, in its dependence on the ionic
en-vironment, although the serum inhibition is
sup-pressed byconcentrations ofphosphateat whichall
Biochem.1948,42
actions differ in two significant respects; ester in-hibition reaches its maximum more quickly than serum inhibition and there is no recovery on pro-longed incubation. It is obvious that the hyaluronic acid esters cannot becalledenzymes, but the ter-mninology most suitable for the serum inhibitor is uncertain. Haas (1946) has called it an enzyme because the reactionproceedsatameasurablerate
andbecause the inhibitor is destroyed at
550,
but these properties, which we confirm, are notex-clusively the properties of enzymes and the fact that theinhibited hyaluronidase can recover its activity isastrong argumentagainst theapplicability of the word enzyme to the inhibitor. Essentially, an enzyme is a substancethat combineswithits sub-stratein such a way that, whendissociation occurs, the substrate is altered; the serum inhibitor
com-bines with hyaluronidase and dissociates from it withoutaffecting the only property ofhyaluronidase ofwhich anything is known. Under these circum-stances the
anqlogy
would seem to be with the antigen: antibodyand toxin: antitoxin reactions ratherthan with theenzyme: substrate reactions. The indiffusibility of the inhibitor and its inacti-vation by heating suggest that it is, or that it is associatedwith,aproteinand this isbore
outby McClean's observation, which we confirm, that it precipitates in theglobulin fraction ofserum. The destruction of the inhibitor on incubation with ahyaluronidasepreparationcould be dueto aprotease present in the enzyme, but it is alsopossible that it isanothermanifestation ofhyaluronidaseactivity. On this view the inhibitor isasubstrate for hyaluro-nidaseon which the enzymeactsslowly; it is this that distinguishes it from the esters for they are
apparentlynot attackedat all bythe enzyme. In
anattempttoget evidence for thispointof viewwe
have estimated theglucosamine inhydrolysatesof theultrafiltrate fromdialyzedserumthathadbeen incubated with hyaluronidase for sufficiently long
todestroyapartof itsinhibitorypower. No glucos-amine could be detected. This suggests that the inhibitor doesnotcontainglucosamine,thatthis is
not liberated during its destruction, or that the quantityistoosmall for detectionbyourmethods. There is therefore stillnoevidenceonthenatureof theinhibitororthemechanism of its action.
% SUMMARY
1. The effect of various salts on the action of hyaluronidaseonhyaluronicacid has been studied viscosimetrically. There isanoptimum molarityfor eachsalt,but itsvalue and therateof the reactionat
theoptimum vary with different salts.
2. Methods ofpreparingnitratesandacetatesof hyaluronic acidaregiven. These estersinhibit the
274
Z.
IIADIDIAN
AND N.
W. PIRIE
1948
enzymeaction and thisinhibition is compared with that brought about by a constituent of normal serum.
3. The extent of the inhibition by these agents is dependent on the saltconcentration.
4. The 'seruminhibitor, unlike the inhibitors made byesterifying hyaluronic acid, is destroyed by the testicular hyaluronidase used.
Thisinvestigation was aided by a grant from G. D.Searle
andCo.
REFERENCES
Carson,J. F. & Maclay, W. D. (1946). J. Amer. chem. Soc. 68, 1015.
Chamberlain, N. H., Cunningham, G. E. & Speakman, J. B. (1946). Nature,Lond., 158, 553.
Elson, L. A. & Morgan, W. T. J. (1933). Biochem. J. 27, 1824.
Haas, E. (1946). J. biol.Chem. 163, 63.
Hadidian, Z. & Pirie, N. W. (1948). Biochem. J. 42, 260. Hahn, L. (1945). Ark.Kemi Min.Geol.19A, no. 33. Hobby, G. L., Dawson, M. H., Meyer, K. & Chaffee, E.
(1941). J. exp. Med.73, 109.
Humphrey, J. H. (1943). Biochem. J. 37, 460.
Lineweaver, H. &Ballou, G. A. (1945). Arch. Biochem. 6, 273.
McClean, D. (1942). J. Path. Bact. 54, 284. McClean, D. (1943). Biochem. J. 37, 169.
McClean, D. & Hale, C. W. (1941). Biochem. J. 35, 159. Madinaveitia, J. & Quibell, T. H. H. (1941). Biochem. J.
35,456.
Meyer,K.,Smyth, E. M. & Dawson, M. H. (1939). J. biol. Chem. 128, 319.
Robertson, W. B., Ropes, M.W. &]Bauer, W. (1940). J. biol.Chem. 133, 261.
Rogers,H. J. (1946a). Biochem. J. 40, 583. Rogers,H. J. (1946b). Biochem. J. 40, 782.
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The Fate
of Certain
Organic
Acids and Amides in the Rabbit
3. HYDROLYSIS OF AMIDES BY ENZYMES IN VITRO
BY H. G. BRAY, SYBIL P. JAMES, BRENDA E. RYMAN AND W. V. THORPE Departmentof Phy8iology, Medical School, Univer8ity of Birmingham
(Received4July 1947)
In the first two papers of this series itwasshown that the rabbit is able tohydrolyze the carbamyl group in benzamide and phenylacetamide (Bray, Neale & Thorpe, 1946), but that the introduction of a
hydroxyl group in thepositionpara to thecarbamyl renders the latterstable, less than 7
%
hydrolysis occurring in vivo (Bray, Ryman & Thorpe, 1947). Furtherinvestigations are inprogress of theeffect ofhydroxyl groups in other positions and also of amino and nitro groups. This paper reportsaparallel study in which we have examined the activity of various enzymepreparations, especiallythosefrom rabbit liver, in hydrolyzing the carbamyl group.Similar investigations are those of Gonnermann (1902, 1903), in which the action of several enzyme preparations, including pepsin, trypsin and sheep's liver andkidney, on a number of amides and anilides was studied, of Geddes & Hunter (1928) and of Grassmann&Mayr (1933) who examined the action ofyeastasparaginase on several amides. Thecriterion usedby Gonnermann asanindication ofhydrolysis
was the isolation of the unchanged amide or of productsof its hydrolysis, whilst Geddes & Hunter
estimated the ammonia liberated. Relevant results from theseinvestigations are summarized in Table 1. Geddes & Hunter claim that asparaginase is present in calf liver and also that yeast asparaginase will hydrolyze glutamine. Glutaminases hydrolyzing thisamide but not asparagine have been shown to be present in various tissues, including liver and kidney (Krebs, 1935).
Inthe present work we have determined quan-titatively the degree of hydrolysis by a formol titration method based on that used by Balls & Lineweaver (1939) for assessing the activity of papain with hippurylamide as substrate. We have also compared the stability of the amides used to acidandalkaline hydrolysis.
Material8 METHODS
Amide8. Theamideswhich couldnotbepurchasedwere
prepared by the action of aqueous ammonia upon the
correspoadingacidchloride or uponthemethylesterofthe parentacid. Aminobenzamides wereprepared by the