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Holden, M. 1946. Studies on pectase. Biochemical Journal. 40 (1), pp.
103-108.
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Vol.
40Studies
on
Pectase
By MARGARET HOLDEN,
Rothamsted
ExperimentalStation, Harpenden, Herts(Received
18October 1945) A previous paper (Holden, 1945) described a pHdrifttowardstheacidsidein minced tobacco leaves
when the pH was raised to pH 8 by addition of
NaOH, due mainly to the enzymic demethylation ofpectin by pectase. The present paper concerns the extraction, some properties and the partial purification oftobaccoleafpectaseand givessome
accountofthepectasedistribution in the leaves of
other species. Recent papers by Lineweaver & Ballou (1945) and MacDonnell, Jansen & Line-weaver (1945) havedealtwith the effect of cations ontheactivity ofalfalfapectaseand with the pro-perties oforangepectase.Theresultsreportedhere ontheextraction oftobaccopectasearesimilar to
those obtained by the other workers for orange pectase, but the properties of the enzyme from
varioussources appearto differtosomeextent.
MATERIAL AND METHODS
Leavesof Nicotiana tabacumvar.White Burley from
glass-houseplantswereusedformostoftheworkand leavesof wild woody nightshade (Solanumdulcamara) when tobacco
wasnotavailable. Other plants usedarelisted below. The
leavesweremincedtwiceinadomesticmincer and thesap
squeezed out byhandthrough madapollam. The residue, referred to asfibre, was washed twice witha volume of
distilledwaterapproximatelythesame asthat ofthesap,
andsqueezed dry. Fibre that had been groundina
triple-roller millasdescribed by Bawden& Pirie(1944) iscalled milledfibre.Therecorded weights of both thesefibresare
thewetweightsaftersqueezing by hand,thematerialthen containing about 75%ofwater. Measurements ofpHwere
made withaglass electrode.
Measurement ofactivity. The breakdown of pectin to
pecticacidandmethanol bypectasewasfollowedby
deter-mining the amount ofmethanol liberated. The substrate usedwas a solution ofcitruspectin(British Drug Houses
Ltd., 100grade) with amoisture contentof10% andan
ash contentof 1-4%. Themethoxylcontent, on amoisture
andash-free basis, as determined by Zeisel's methodwas
5-4%. Thepectin solution had apH of 3-1 and wasnot
neutralizeduntil just beforeactivitytestswerecarriedout.
The tests were made inphosphate buffer solution which
minimized any pH drift. A sample, 0-1-1-0ml. of the
solution to be tested was added to the buffer-substrate
which consistedof 5ml.pectinsolution(8mg./ml.)+5 ml. 0-2M-Na2HPO4 previously adjusted to pH 8-0 with HCI.
After 10 min., 1ml. 2N-HCI was added to lower the pH
and stopthereaction. Methanolwasdeterminedon a5 ml. sample bythe methodpreviouslydescribed(Holden, 1945). Itwasnecessaryfirsttodistil thesampleinthe Markham
steam distillation apparatus (Markham, 1942), for unless this was done the red colourdeveloped differed from that of the standards; moreover, many of the samples to be tested were coloured. If there was any gel formation on theaddition of acid the test wasrepeated using a smaller amountof enzyme solution as thisindicated such extensive demethylation that the assay of the enzyme was no longer precise. The amount of methanol due to non-enzymic de-methylation at pH 8 in 10 min. wasbarely detectable.
One unit of enzymeisdefinedastheamountwhich will
liberate 32 mg. methanol from pectinin 1 min. in 0-1 M-phosphate solutionat pH8 and200. Exceptfor the tem-perature, the unit isthesameasthat usedby Lineweaver etal. The purity ofpreparations isexpressedas units per mg.dryweight of materialprecipitablewith trichloroacetic acid.
Preliminaryobservations onthepreparation of the enzyme
Extraction. It had been found previously that pectase could be obtained from the washed milled fibreoftobacco leavesbyextractionatpH8either with NaOH or with phosphate solution, but that
waterextractsatpH 6,the natural
pH
of the fibre suspension,had littleornoactivity. Aquantitative
comparisonwasmade of theactivityof water, 5
%
NaClextracts at pH 6, and of
phosphate
extractsatpH 6 and 8using 10 g.portions ofmilled fibre. Table 1 shows that increase in theconcentrationof salt bringsabout extraction at
pH
6, but thatex-tractionatpH 8ismoreeffective.
Table 1. Influence
of
saltandpH onextraction ofpectase
fromtobaccofibre(Portions of milled fibre (10g.) extracted with 80 ml.
solutionat200 for2hr.)
pH Extracting solution Activityunits
6 Water 0-015
6 5% NaCl 0-60
6 0-2M-Na2HPO4 0 55
8 0-2M-Na2HPO4 0-78
A comparison was made of the activity of suc-cessive extracts at pH 8 from 9-3 g.
portions
of milledfibre made both withNaOHand with0-2M-Na2HPO4
extractants and from 15 g. minced fibre inphosphate solution (9-3g. milled fibre obtained from 15 g. minced fibre). The fibre was soaked in the extracting solution for 2 hr.; the NaOHM. HOLDEN
ofalkaliat intervals. The results in Table 2 show
that extraction with phosphate is more efficient than that with NaOH, due to the increased salt
concentration. With milled fibre the first extract
Table 2. Extraction ofpectasefrommilled and
minced fibreatpH8
(Portions of milled fibre (9-3 g.) and the equivalent amount in minced fibre (15 g.) extracted with 80 ml. solutionfor 2hr.)
Activityunits
Milled fibre Minced
, ^-- A fibre
Extract NaOH Phosphate Phosphate
1 0-14 0-58 0-29
2 0-07 0-12 0-17
3 0-03 0-07 0-07
0-24 0-77 0-53
hasbyfar thehighest activity, whereas with minced
fibre the extraction is slower. About 70% of the amount ofenzymethatcanbeobtained from milled fibre is extractable frommincedfibre. Itwas there-fore foundunnecessarytomillthe fibre, whichwas
anadvantagesince less timewasneeded for making
apreparation. Minced-fibre extractscontainmuch less totalproteinthanmilled-fibre extractssothat the formerenzymepreparations hadahigher initial
specific activity.
Sap-soluble enzyme. The distribution ofpectase between thesapandfibrewasnextinvestigated. It
was found that in most batches of leaves only a
smallproportion (from5to10%of the totalamount ofenzyme)waspresentinthesap,but intwobatches
ofleaves,tobe consideredlater,over25% was sap-soluble. The comparativelylowactivity of thesap, combined with itshighsolubleproteincontentmake
it unsuitableas a sourceofenzyme. Sap that had beencentrifuged toremovecell debris and
chloro-plastmaterial hadan activity of0-00016unit/mg.
dry weight, whereas a phosphate extract of fibre,
before purification, had an activity more than
twentytimes this value.
Ethanol and ammonium .sulphate precipitation. Neuberg & Kobel (1927) and Mehlitz (1930)
pre-cipitatedpectase fromthesapof tobacco andlucerne
respectively by the addition of 2 vol. of ethanol. Ethanolprecipitationoftheenzymefrom phosphate extracts resulted in complete loss of activity,
al-thoughactivepreparations could be obtained from
the sapinthisway.
Thebufferingpower ofNaOH extracts is lowso
that when ammoniumsulphate wasadded to
pre-cipitatetheproteinthe pectasewasinactivated,due
probably to the fall in pH. It was necessary to
carryoutammoniumsulphate precipitationsatpH
values above 7 in awell-buffered solution, and in
this respect phosphate extracts were satisfactory. Precipitation then causednoloss ofactivity. Frac-tionalprecipitationshowed that all theactivitywas
not precipitated until about 65
%
saturation wasreached. When fractionation was attempted on a,
diluteextract(notmorethan5
mg./ml.
dry matter) less than 10%
of theactivitywasprecipitatedbelow 45%saturation
with ammonium sulphate. How-ever, withmoreconcentrated solutions the enzyme was adsorbed on material coming out at a lower saturationandasmuchas30%
of theactivity
was precipitatedbelow 35%
saturation withammoniurn sulphate. Activitytestscouldnotbe carriedoutin the presence oflarge amounts of ammonium sul-phate asit hadaninhibitoryeffectonthereaction,
but theamountpresentincentrifuged
precipitates
from ammonium sulphate solutions was not suffi-cienttointerfereseriouslywith thedeterminations.
Methodof preparationandpartial
purification
Well-washed minced fibre was extracted three times with 02M-phosphate solution atpH 8. The volumes ofphosphate solution usedwere approxi-mately 3, 2 and 1 ml./g. fibre for the successive
extracts. The fibre was soaked for about 15 min. for each extract, as it was found that less
con-taminatingmaterialwasextracted with short soak-ing. The pH drift due to demethylation ofpectin
was not prevented altogether with the volume of phosphatesolution used and thecombinedextracts
had a pH of about 7. These were centrifuged at
3000 r.p.m. (1400xgravity) for 15 min. and the deposit of starch andchloroplastmaterialdiscarded. At this stage theactivityvaried between 0 003 and 0-006
unit/mg.
dry weight. The supernatant wasusuallyaclearred-brown,but sometimes there was
a considerable amount of green chromoprotein material that could not be spun out particularly when nightshade leaves were used. When this occurred, ammonium sulphate wasadded to 30
o/%
saturation and theprecipitate obtained on centri-fuging discarded. Ammonium sulphate was then addedto65%
saturation andthe solution with pre-cipitated protein was filtered with slight suction throughdiatomaceous earth(Hyflo supercel, Johns-Manville Co. Ltd., Artillery Row, London, S.W.1)
on a Buchner funnel. Filtration was more satis-factoryatthis stage thancentrifugationasthe pre-cipitatetendedtofloat. Theprecipitate wastaken upin 02M-phosphatesolutionatpH 6inavolume about one-tenth that of the original extract. The activitywasfrom 0-019to0-028unit/mg. dryweight and the solution dark brown dueto adsorption of coloured substances ontheprotein precipitate.
Whenthe
pH
was nowloweredto about 4-6 by adding dilute HCIthere was precipitation of some oftheproteinanf1
theactivity of the supernatant fluidwasincreasedto over 0-062unit/mg. ThepHSTUDIES
ON
PECTASE
of the solution was then raised to above 7 by theaddition of Na2HPO4 solution and saturated am-monium sulphate solution was added to 65
%
satu-ration. The precipitate was filtered off, and taken upinphosphate solution at pH 6. The precipitationatpH 4-6 and subsequent salting-out was repeated twice. In the final ammonium sulphate precipitation the fraction below 45
%
saturation wasdiscarded; thatbetween45 and 65%
saturation was taken up in phosphate solution at pH 7 and kept in the refrigerator. The activity had increased to 0-109-0-125 unit/mg. dry weight (or 0-78-0-90 unit/mg. protein-N) and the yieldwas about 60%
of theunits originally extracted. Much of the colour had been removed by repeated precipitation but the final solution wasstilllight brown in colour. Phosphateextracts from tobacco and nightshade differed, in that the former had a larger amount of acid-pre-cipitablematerial and the latter more protein pre-cipitable at a lower concentration of ammonium sulphate.
Ad8orption and elution. Lineweaver & Ballou (1945) and MacDonnell et al. (1945) have reported thatthe pectase from alfalfa leaves and citrus peel was adsorbed from salt-free solutions on 'Celite' analytical filter-aid (Johns-Manville Co. Ltd.) and could beeluted with dilute salt solution. Phosphate
extracts from fibre and purified concentrates of pectase were dialyzed for 18 hr. in cellophan sacs with frequent changes of water. When the pH value didnotfalllower than 6 during dialysis therewas
only asmall amount ofprecipitate in thesac and the loss ofactivity was less than 10%; but when the pH fell to about 5 there was then a larger
amount of precipitate on which the enzyme was
adsorbed andresultingin alarge apparent loss of activity. Adsorption on and elution from 'Celite' analyticalfilter-aidwas testedusing both tobacco leaf and orange-peelpectase preparations. Unless adsorption was carried out at a slightly acid pH valueonlyasmallamountof enzymewasadsorbed. AtpH4-8apurifieddialyzed preparationof tobacco pectase gaveabulky precipitate containingmostof theactivity,althoughbeforedialysisthe
precipitate
atthispHwasinsignificant. AdsorptionatpH 5-3, which is higher than that at which
precipitation
takesplacewas,however, foundtobe
possible.
Itwas stated by Lineweaver & Ballou (1945) that pectase couldbe eluted with 0-2M-NaCl. Thiswas
tested, using apreparation oforange-peel pectase and the results ofan adsorption-elution
cycle
aregiven in Table 3. Elution with NaCl was not as effective as found
by
Lineweaver &Ballou;
Na2HPO4, on the other
hand,
wasconsiderably
better, but one elution removed
only
50%
of the adsorbed enzyme. Theadsorptionandelution ofanenzyme
preparation
fromwoody
nightshade
leaves which had not beenpreviously
acidprecipitated
Table 3. Adsorption andelution of peotase
(150 ml. dialyzed solution of orange-peel pectase at pH 4-8 + 500 mg. Celite. Washed with distilled water beforeelution.)
Solution Dialyzed enzymesolution Filtrate from Celite Eluate 1: 10 ml. 0-9 %NaCl Eluate 2: 10 ml.0-9 % NaCl Eluate 3: 10ml.
0-2M-Na2HPO4
Eluate4:10 ml.0-2M-Na2HPO4 Eluate 5: 10 ml.
0-2M-Na2HPO4
Activity units 0-49
0-067 0-033
0-026 0-22 0-07 0-035
'/'
activity
onCelite
86
92
86 34 18 9
resulted in an increase of activity from 0-026 to 0-113 unit/mg. dry weight. A preparation which had been purified by acid precipitation had the activity increasedfrom 0-055 to 0-122 unit/mg. by oneadsorption and elution. Most of the colourwas
removedfrom enzymesolutions byadsorptionand elutionasit wasnotadsorbedonthe Celite.
Properties of the enzyme
Fig. 1 shows the variation ofactivity with pH. Activity tests were carried out by adding buffer solutions ofapproximately thepHrequiredtothe
pH
Fig.1. Variation of pectase activity with pH. 0-0
citrate buffers; 0 0E3 phosphate buffers; + + non-enzymicdemethylation; 0 0 values after
sub-tractionofnon-enzymic values.
pectinsolution and
measuring
thefinalpH
values. ForpH values 4-3 to 6-2 citrate bufferswereused andphosphatebuffers for 6-2 and above. The final concentration of Na+ in the solutionwas0-09Mfor thephosphatebuffers and varied between 0-08 and 0-13M for the citrate. 1 ml. dialyzedenzyme solu-tionwasaddedtoeach and the testcarriedoutin theusual way. Values fornon-enzymic
demethyla-tion were obtained
by carrying
outractivity
tests withoutaddition of enzyme. The whole of the solu-tion was distilled for methanol determination in-stead ofa5ml.sample. BelowpH4- 5no demethyla-tion occurred and theoptimum
forpectaseactivitywas nearpH 8.
M.
HOLDEN
The course of the demethylating reaction was followed by adding 1ml. enzyme solution to each ofaseries of flaskscontainingbuffer-substrate and stopping the reaction with HCI atdefinite intervals up to 18 min. For at least the first 10 min. the production of methanol followed a straight line relationship. In this time about
40%
of the methanolhadbeen split off, reducing the methoxyl content of thepectinto 3-2%.
Thevariation of activity with substrate concen-tration is shown in Fig. 2. The tests were made in the usual way except that the more concentrated
400 X 3300
O
' 200
n Xl
100
0 0-l 0-2 03 0-4 0o5 0-6 0-7 0-8
Pectin(g./100 ml.) Fig. 2. Variation of pectase activity
withsubstrateconcentration.
pectin solutionswereneutralizedwithNaOH before addition of the 0-2M-phosphate solution. The per-centage concentration of pectin which gives half the maximumvelocityof hydrolysis was found to be0-09.
Alinearrelationship (Fig. 3) was found between the amount of methanol liberated and amount of enzyme solution. Fig. 4 gives the results of an experiment onthe variation ofactivitywith tem-perature. Substrate and buffer in separate flasks werebroughttothe temperatureatwhich the test
was to be carried out,mixed, and enzyme solution added. Controls without enzymewere setupatthe higher temperatures to correct for non-enzymic demethylation. Theoptimumtemperaturefor
pec-taseactivityisat55°. Non-enzymic demethylation was not appreciable until 50°. The temperature coefficient
Qlo
between 20 and300
is 1-41; 30to40°, 1-50; and 40to500,
1-41.Stability.
The effect of.pH on the stability of pectaseis shown inFig. 5. Adialyzedenzymepre-8S
7(
-561
0
5C
20
10 00
-DO - /
DO_
)O
-)O- x
o0
0-1
0-2 0-3 0-4 0-5 0-6 0-7 0-8Enzyme
solution(ml.)Fig.3. Variation of activity with pectase concentration.
300k
0
._4
2
100
0
P-
7-ZL
,~~~~~~~~xZ
60 65
i
L ' 1
15 20 25 30 35 40 45 50 55
Temperature 0 C.
Fig.4. Variation ofactivity with temperature. 0- i
total methanol; x - x non-enzymic demethylation; (D Ovalues after subtraction of
non-enzymic
values.500- + +
400,
o
I; 300
-i200
-100
A 100_.x
o
/I
I
I
I
2 3 4 5 6 7 8
pH
Fig.5. Effect of pH on stability of pectase. x x
citratebuffers; + + phosphatebuffers. Activityat pH 8 determined after5hr.atthegivenpH.
106
I946
9(
STUDIES
ON PECTASE
paration was diluted tenfold with citrate and phos-phatebuffers at various pH values and the activity atpH 8determined after 5 hr. Between pH 5 and 3
some activity, and at pH 2 all activity was lost. There was precipitation of protein between pH 3 and 5, though not at pH 2, and solutions were centrifuged before determining the activity. Some activity was found to be associated with the pre-cipitatesothe loss is not as great as would at first appear. Whenplotting the curve this activity has beenadded to that of the solution.
The effect of temperature on the stability was determined by keeping enzyme preparations in phosphate buffer of pH 8 at 70, 80 and910for 5 min., cooling and determining the activity at 200. At 700, 30
%
of theactivity was lost and at 800 therewascompleteinactivation.
Enzyme preparations kept at 40 in phosphate solutions with pH values from 6 to 8gradually lost activity.With some concentrates the loss was much
more rapid than with others, but one concentrate afterasmallinitial loss in activityremained stable for 3 months. Loss of activity was much morerapid
at roomtemperature (200),50
%
or morebeinglost in 1 week. Dialyzed solutions lost activity at room temperature more rapidly than when salt waspresent. It was not possibletoincrease thespecific activity ofpreparations that had lost activity on
keeping by refractionation with ammonium sul-phate.
High-speed centrifugation. When purified enzyme preparationswith activities ofmorethan 0-1 unit/ mg. were spun at 40,000 r.p.m. (90,000 gravity) therewas nopelletand thebottomlayerofsolution hadan,activityperml.only about 20
%
higherthan therestof the solution. Partially purified prepara-tions with activities 0-025-0-04unit/mg.when spunatthis speed gaveapellet consisting partlyof in-soluble material andsomesoluble which had pectase activity. Above the pellet was a layer with an
activity per ml. twice as high as the rest of the solution above, but the specific activities did not
differgreatly. In the less purifiedpreparations the enzyme wasundoubtedly being partially sedimented due toadsorption on material of higher molecular weight.
Pectase distribution in various species
Table 4 gives values for the pectase activity of the leaves of a number of plants, and also of citrus peel forcomparison. There is considerable variation in the total pectase content and in the relative amountspresent in the sap and fibre. Members of the Solanaceae have high pectase contents. The activity of tobacco fibre from different batches of leaves variedbetween 0-047 and 0-09unit/g. fibre. The pectase content on adry weight basisof young tobacco leaves and old leaves which had turned yellow wasnot significantly different from that of thelarge green leavesusually used, when the plants were grownunder the sameconditions.
The plants listed in the table were of unknown manurial history and it was not known how the pectase content was influenced bymanurial
treat-ment. Advantage was taken to useanexperiment inwhich tobaccoplantsinfected with potato virus Y were grownwith different amounts and combina-tions of N, P and K. Infection with potato virus Y does not cause gross lesions and the plants in the well-manured groups werelarge andhealthylooking. The leaves of each group treated separately were
minced and washed as usual. Pectase determina-tions weremadeon samples of the sap plus wash-liquors and on thephosphate extracts of the fibres. Dry weight and N determinations were made on
both fractions. The total amountof pectase in the leaves of the group which had N, P and K was
fifteen times the amountin the control group. The activity per g. dry weight associated with the fibre
washighest in the groupsgivennitrogenand varied with the N content of thefibre. In the groupsgiven N and P theproportion of enzyme in the sapwas
about 30
%
compared with about 5° in all the Table 4. Pectase distribution in variousplantspeciesTotal
activity Activity units/g. Sap insap
Plant (pH) (%) Fibre Leaf
Nightshade (Solanum dulcamara) 5-6 9 0-060 0-024
Tomato(Lycopersicumesculentum) 6-0 33 0-063 0-023
Potato (Solanumtuberosum) 6-2 16 0-069 0-015
Elder
(Sambucus
nigra) 6-3 8 0-050 0-016*Peppermint (Mentha piperata) 6-3 17 0005 0-0016 Melilot (Melilotusaltissima) 8-6 43 0-016 0-014
*Clematis(Clematisjackmanni) 4-8 9 0-009 0-006
Whitebryony (Bryonia dioica) 6-3 16 0-022 0-011 Beakedparsley(Chaerophyllumsylvestre) 5-6 9 0-026 0-010 *Rhubarb(Rheumraponticum) 4-3 15 0-007 0-0034
Lemonpeel(flavedo+albedo) 0-062unit/g. peel Orangepeel (flavedo+albedo) 0-073unit/g. peel
*Representsasingle determination only. Forthe remainder the value istypicalofanumberof determinations.
108
M. HOLDENI946
other groups. The influence of manurial treatment on pectase content will be reported in more detail later.
DISCUSSION
Lineweaver et al. made their activity determinations at 300sothat all the values given here have tobe increased by 40
%
to make them comparable with theirs. Their preparations with a specificactivity of 2*5 units/mg. protein N have an activity about twice that of the most active preparations obtained in the present investigation. Adsorption on and elution from Celite did not result in suchalarge increase in specific activity as found by these workers, which may be due to the presence of a different type of impurity.The pH optimum for tobacco pectase given by Kertesz (1936) was at about 6-5, but no attempt was made to keep the pH constant so the value is probably in error. The values he obtained for
non-enzymicdemethylationwerehigher than those ob-tained here. Lineweaveretal. have shown that the relationship between activity, pH and salt
concen-tration is so complex that for anyoptimalpH value the experimental conditions must be defined. It would appear that tobacco pectase is less heat-sensitive thanorange-peelpectase,as atemperature of80° is needed to destroy all itsactivity,whereas orange-peelpectaseis destroyedat 45-50'.
The pectase content of tobacco leaves was in-fluenced by the manurial treatment of the plants with nitrogen and phosphorus but not appreciably by potassium. In allthe groups except thosegiven both N and P (these having the highest pectase contents), the percentage of the enzyme whichwas
sap-solublewaslow; nitrogenalone didnotincrease the proportion of sap-soluble enzyme but greatly
increased the amount associated with thefibre. The values given for the pectase content of the various species might have differed had the plants been grown under different conditions. However, it is clear that members of the Solanaceae have high pectase contents, and that tobacco leaf fibre is as good a source of the enzyme as citrus peel. Thehigh percentage of sap-soluble pectase and the abnor-mallyhigh pH of the sap of melilot are noteworthy.
SUMMARY
1. Pectase activity was determined by esti-mating the amount of methanol liberated from pectinunderstandard conditions.
2. Thesap oftobacco leaves contains only asmall proportion of the total pectase present, most being associated with the fibre.
3. Extraction of the enzyme isinfluenced by salt concentration and pH. The enzyme in the extract could be concentrated by precipitation with am-monium sulphate at 65
%
saturation from abuffer solution of pH 3 7, but not with ethanol. An increase inspecific activity of enzymepreparations was obtained byremoval of the proteinprecipitated atpH 4-6 in the presence of salt. The adsorption ofpectase onCelite from salt-freesolution was con-firmed, and elution with 0 2m-Na2HPO4 was moreeffective than with NaCl.
4. Theoptimum conditions for enzyme activity were found to be pH 8 and 55°. Acidification to
pH 3 and heating to 800destroyed the enzyme. 5. The pectase content of tobacco leaves was
influenced bymanurialtreatment. Thedistribution in the leaves often other species isgiven.
I wish to thank the Agricultural Research Council for agrant.
REFERENCES
Bawden, F. C. & Pirie, N. W. (1944). Brit. J. exp. Path. 25, 68.
Holden, M. (1945). Biochem. J. 39, 172.
Kertesz,Z.I. (1936). Ergebn. Enzymforsch. 5, 233. Lineweaver, H. & Ballou, G. A. (1945). Arch. Biochem.
6, 373.
MacDonnell,L. R., Jansen, E. F. & Lineweaver, H. (1945). Arch. Biochem. 6, 389.
Markham, R. (1942). Biochem. J.36, 790. Mehlitz, A. (1930). Biochem. Z. 221,217.
Neuberg, C. & Kobel, M. (1927). Biochem. Z. 190, 232.
The
Skin Protease
Inhibitory
Factor
of
Plasma
BY ANNE BELOFF, Department of Biochemistry, Oxford
(Received 25 October 1945)
This work arose from theobservations made (Beloff &Peters, 1944; 1945 a, b) on the influence of mode-rate-temperature burns upon a protease of skin. This paper describes the method used for estimating theinhibitory activity of plasma on the skin
proteo-lyticsystem, and the properties and nature ofthis inhibitory factor. The application of these obser-vations to the previous work on thermal burns will be described separately (Beloff & Peters,
