Paper-Chromatographic Separation of Chlorophylls and Carotenoids

336 C. A. BENASSI, G. COLOMBO AND G. ALLEGRI 1961Blackburn, S. & Lowther, A. S. (1951). Biochem. J. 48,

126.Buck, J. B. (1953). In Insect Physiology, p. 147.Ed. by Roeder, K. D. London: Chapman and HallLtd.

Clements, A. N. (1959). J. exp. Biol. 36, 656.Cromartie, R. I. T. (1959). Annu. Rev. Ent. 4, 59.Duchateau, G. H. & Florkin, M. (1958). Arch. int. Physiol.

66, 573.

Florkin, M. (1958). Proc. 4th int. Congr. Biochem., Vienna,12, 63.

Hackman, R. H. (1958). Proc. 4th int. Congr. Biochem.,Vienna, 12, 47.

Hunter-Jones, P. (1957). Nature, Lond., 180, 236.Kilby, B. A. & Neville, E. (1957). J. exp. Biol. 34, 276.Levy, A. L. (1954). Nature, Lond., 174, 126.Sanger, F. (1945). Biochem. J. 39, 507.Treherme, J. E. (1959). J. exp. Biol. 36, 533.

Biochem. J. (1961) 80, 336

Paper-Chromatographic Separation of Chlorophylls and Carotenoidsfrom Marine AlgaeBY S. W. JEFFREY

C.S.I.R.O. Marine Laboratory, Cronulla, Sydney, Au8tralia

(Received 23 January 1961)

Although many methods have been described forthe partial separation of chloroplast pigments bypaper chromatography (see review by Sestak, 1958)the species examined have been mainly higherplants and algae of the class Chlorophyceae, inwhich the main chloroplast pigments are chloro-phylls a and b, with p-carotene and lutein as themajor carotenoids. In the present work, a methodwas required for studying the pigment compositionof planktonic algae which occur in the oceanicwaters off Sydney. Representatives are found notonly of the Chlorophyceae but also of the Bacil-lariophyceae, Dinophyceae and Chrysophyceae,and one would expect to find chlorophylls a, b and ctogether with a wide range of carotenoids (Strain,1958). The method described below, which is amodification of the two-dimensional method ofLind, Lane & Gleason (1953), has enabled studies ofthe pigment composition of such diverse algalgroups to be made, since it provides a completeseparation of mixtures of chlorophylls a, b and c,carotenes and the xanthophylls lutein, violaxan-thin, neoxanthin, fucoxanthin, peridinin andastaxanthin, as well as a number of xanthophyllpigments which occur in relatively small quantities.

EXPERIMENTAL

Materials

Representatives from four classes of marine algae werestudied: green flagellates from the Chlorophyceae, diatomsfrom the Bacillariophyceae, naked dinoflagellates from theDinophyceae and golden-brown flagellates from theChrysophyceae (Fritsch, 1948). The material used wasobtained either from uni-algal, but not bacteria-free,cultures of marine algae, or from mixed natural plankton in

sea-water samples. The uni-algal cultures studied includedfour green flagellates [Dunaliella tertiolecta Butcher,Nannochloris atomus Butcher, Chlorella stigmatophoraButcher and Tetraselmis suecica (Kylin) Butcher], threediatoms [Phaeodactylum tricornutum Bohlen, Nitz8chiaclosterium (Ehr.) and Skeletonema costatum (Grev.)], onedinoflagellate (Gymnodinium sp.) and two golden-brownflagellates (Isochrysis galbana Parke and Sphaleromantissp.). Phaeodactylum, Dunaliella and Isochrysis were ob-tained from Dr Mary Parke of the Marine Laboratory,Plymouth. The other organisms were isolated from mixedplankton samples taken from the coastal waters offSydney.The organisms were grown in an Erdschreiber medium,

consisting of sea water enriched with nitrates, phosphatesand soil extract. The soil extract was prepared by auto-claving 1 kg. of soil with 1 1. of tap water at 15 lb./in.2 for2-3hr., andfiltering through an Eaton-Dikeman Paper 541.After diluting 1 vol. of the resulting extract with 2 vol. oftap water, concentrated stock nutrient mediawere preparedby adding 0-2 g. of NaNO3 and 0 03 g. of Na2HP04,12H20to every 50 ml. of soil extract, and autoclaving at5 lb./in.2 for 1 hr. The final culture medium was preparedwhen needed by adding 50 ml. of this enriched extract to1 1. of filtered sea water, and autoclaving at 5 lb./in.2 for1 hr. Stock cultures of algae were maintained in 200 ml. ofculture medium in 250 ml. Erlenmeyer flasks at 180, andillumination was from 40w fluorescent tubes giving a lightintensity of approx. 400 ft.-candles. Bulk cultures (4 1.)were continuously aerated in 51. Erlenmeyer or Haffkineflasks, and were used for analysis after 2-4 weeks' growth.The algae were harvested by continuous centrifuging at5000g and the cells were washed several times by centri-fuging at about 2000g for 10 min. This process removedmost of the bacteria, as judged by microscopic examinationunder phase contrast.In addition to these uni-algal cultures, mixed natural

plankton from sea-water samples was used for pigmentanalysis. At least 351. of sea water was needed to obtainsufficient pigments for chromatography. The sea water was

CHROMATOGRAPHY OF PIGMENTS IN ALGAEcollected in 3 gallon plastic jars and the algae were harvestedand extracted within 2 hr. of collection of the water sampleat sea.

Methods

Preparation ofpigment extracts. All reagents and solventsused for the extraction and chromatography were A.R.chemicals, and were used without further purification.Throughout the entire extraction period the tubes con-taining the pigments were kept in the dark as much aspossible.

Cells were harvested by centrifuging, and with bulkcultures or large volumes of sea water continuous centri-fuging at 5000g was used (Davis, 1957). The sedimentedcells (between 0-1 and 0 5 ml. of wet packed cells) wereextracted several times for 1-2 min. with small volumes(5-10 ml.) of 90% acetone until the residue was colourless.The combined acetone extracts were mixed with an equalvolume of diethyl ether in a separating funnel, and washedthoroughly with at least 5 vol. of 10% (w/v) NaCl. It isnecessary to use diethyl ether instead of light petroleum inthis purification step, since chlorophyll c is insoluble in thelatter solvent. The ether separated the pigments quanti-tatively from the acetone- and water-soluble impurities.After several washings with NaCl, the ether layer con-taining the pigments was collected, centrifuged briefly toremove suspended water droplets, transferred to a Petridish and evaporated to dryness by blowing a stream of coolair over the solution. After evaporation of the ether, thepigment residue was freed from the remaining water drop-lets by drying in a current of air. This procedure was foundmore satisfactory than drying under a vacuum, or by theuse of anhydrous sodium sulphate, which adsorbed thepigments. The pigment residue was then redissolved in theminimum quantity of diethyl ether and sometimes a traceof acetone was necessary to facilitate solution. Anyremaining water droplets were removed by centrifugingbefore chromatography, since for good resolution of thepigments on the chromatogram it was essential that theextract be completely dry.Pigments were not extracted with equal ease from all

species of algae. The diatoms and ,u-flagellates, which wereparticularly resistant, were first suspended in water (for1-3 min. for diatoms and 30 min. for the ,t-flagellates).This caused the cells to swell, and extraction then pro-ceeded with little difficulty.

Since, when plankton from sea-water samples was leftovernight in 90% acetone (Humphrey, 1960), there wasalways some breakdown (about 5-10%) of the chloro-phylls to pheophytins, the cells were suspended in water forabout 30 min. before acetone extraction, and 98% of thepigments were released by this treatment. If the residuewas then left overnight in acetone, only a trace (less than2% of the total) of material absorbing at 665 m,t was ob-tained. Chromatograms of extracts obtained by the watertreatment were free of pheophytin components, exceptwhen large amounts of decomposing cells were seen micro-scopically before extraction. In these cases the pheophytinswere presumably present in the original plankton sample.

Chromatography. Two-dimensional ascending chromato-graphy was carried out at room temperature (18-23°) in thedark. Whatman 3MM chromatography paper was cut into22 cm. squares, the extract, containing about 20-30kg. ofpigment, was applied at the origin, the paper was clipped

22

together to form a cylinder and placed in a jar previouslyequilibrated with fresh solvent mixture.

Solvents. Methanol, ethanol, propan-l-ol, propan-2-ol,butan-l-ol and isopentanol in concentrations up to 4%(v/v) in light petroleum, b.p. 60-80°, were tested, and 4%propan-l-ol in light petroleum was chosen as giving thebest resolution of carotenes, pheophytins and chlorophyllsa, b and c. Because the xanthophylls overlay chlorophyllsa and b, development of the chromatogram at right anglesto the first dimension was necessary for the separation ofthese pigments.

This was achieved by using, as the second solvent, 30%(v/v) chloroform in light petroleum, b.p. 60-80° (cf. Lindet al. 1953). The chromatograms were developed for about30 min. in each solvent, and allowed to dry in the dark fora few minutes after each run. Since the composition ofboth solvent mixtures was critical for good resolution ofthe pigments, the solvents were made up freshly each day.

If pigment extraction and chromatography were com-pleted in 2 hr., no breakdown of the labile pigmentsresulted, and chromatograms of young cultures of algaewere free of pheophytin spots.For quantitative measurements, portions of pigment

extracts were chromatographed and the spots correspond-ing to the various pigment fractions were eluted withethyl ether for the carotenoids and with acetone for thechlorophylls. The extinction of the eluate was read in aUnicam SP. 500 spectrophotometer, and the results areexpressed as mg. of pigment/g. dry wt. of cells.

Identification of pigments. Pigments were characterizedafter elution from the chromatogram by their absorptionspectra as described by Strain, Manning & Hardin (1944),Goodwin (1955), Smith & Benitez (1955), Strain (1958) andAnderson (1959). Good agreement with the published datawas obtained. Table 1 shows the absorption maxima ob-tained for the pigments studied. Examples from only afew organisms are given, but in all cases similar agreementwith the published data was obtained. It must be noted,however, that contamination of any one xanthophyllfraction with small quantities of similar pigments would notbe detected spectrophotometrically.That the astaxanthin found in Nannochioris atomus was

esterified was indicated by its chromatographic andspectrophotometric similarity with esterified astaxanthinprepared from eggs of Artemia salina (Gilchrist & Green,1960). In addition, esterified astaxanthin from bothsources was epiphasic when solutions of the pigment inlight petroleum were shaken with 90% methanol (Goodwin& Srisukh, 1949).The pigment fractions were further identified by co-chro-

matography with authentic samples in the two solventsystems (4% propan-l-ol in light petroleum and 30%chloroform in light petroleum). The pigments from twopreviously examined organisms were used as standards.Dunaliella tertiolecta, studied by Gilchrist & Green (1960),provided standard chlorophylls a and b, carotenes, lutein,violaxanthin and neoxanthin, and Nitzschia closterium,analysed by Strain et al. (1944), provided standard chloro-phylls a and c, carotenes, diatoxanthin, diadinoxanthin,fucoxanthin and neofucoxanthins. Since no previouslyexamined dinoflagellate cultures were available to us wehad no authentic source of dinoxanthin or peridinin, and inthese two cases provisional identification rested solely onabsorption data. In every other case co-chromatography of

Bioch. 10'61, 80

Vol. 80 337

Table 1. Absorption maxima of pigments in marine algae separated by paper chromatography

References: 1, Smith & Benitez (1955); 2, Anderson (1959); 3, Strain, Manning & Hardin (1944); 4, Goodwin &Srisukh (1949).

PigmentChlorophyll a

Chlorophyll bChlorophyll c

LuteinViolaxanthinNeoxanthinDiatoxanthinDiadinoxanthinFucoxanthinNeofucoxanthin ANeofucoxanthin BDinoxanthinDiadinoxanthinPeridininNeoperidininEsterified astaxanthin

a- and ,8-Carotene mixtures

Publishedmaxima(mP)

663, 615, 580,535, 430, 410

645, 595, 455626, 577-5,443*6

422, 445, 476421, 440, 470414, 436, 466453, 481448, 4784534471446i441*5, 471448, 478 J475464 [503488428, 452, 480

Reference1

11

Maximafound(mEL) Solvent

665, 618, 580, Acetone535, 430, 412

645, 598, 455 Acetone625, 580, 445 Ethyl ether

2 422, 445, 4742 421,441,4712 414, 437, 4663 453, 4813 448, 4783 4533 447*

Ethyl etherEthyl etherEthyl etherEthanolEthanolEthanolEthanol

3 446, 475* Ethanol

3 470* Ethanol4 501 Carbon disulphide

486 Pyridine2 429, 450, 478 Ethyl ether

* One spot obtained and assumed to be a mixture of these two components.

OrganismDunaliella tertiolecta

D. tertiolectaIsochrysis galbana

D. tertiolectaD. tertiolectaD. tertiolectaI. galbana1. galbana1. galbana1. galbana

Gymnodinium sp.

Gymnodinium sp.Nannochloris atomus

D. tertiolecta

a standard pigment with the appropriate pigment fractionfrom a previously unexamined organism resulted in asingle spot on the chromatogram in both solvent systems.This was taken as satisfactory evidence for the identifica-tion of the pigments.

RESULTS

Separation of pigmentsFig. 1 shows chromatograms obtained from

typical representatives of the Chlorophyceae,Bacillariophyceae, Dinophyceae and Chrysophy-ceae. The chlorophylls and carotenes showed goodseparation and in uni-algal cultures the xantho-phylls also gave good resolution. However, whenextracts from different organisms were mixed andchromatographed together some overlapping of thexanthophyll zones occurred. In mixtures ofdiatomsand dinoflagellates peridinin appeared betweenfucoxanthin and neofucoxanthins A and B, andoverlapped both components. In mixtures of greenflagellates and diatoms, violaxanthin, diatoxanthinand diadinoxanthin appeared as one spot on thechromatogram. a- and fl-Carotene did not separ-ate. Esterified astaxanthin (e.g. in Nannochlorisatomus or in sea-water samples containing a fewzooplankton) appeared as a pink zone which ranclosely behind the carotene fraction in bothsolvents. Good resolution of the pigments fromsea-water samples was obtained, and these corre-sponded to the pigments expected on the basis ofmicroscopic examination of algal types in thesample.

Mean R. values for the chloroplast pigments ofmarine algae are given in Table 2, and Table 3shows quantitative data on the pigment composi-tion of some algae. The extinction coefficients usedfor the carotenoids are those quoted by Goodwin(1955), but where these are unknown the extinctioncoefficient for carotene was used (Allen, Goodwin &Phagpolngarm, 1960). Extinction coefficients forchlorophylls a and b are given by Smith & Benitez(1955). No extinction values for pure chlorophyll care available, and the provisional value of 22-0given by Smith & Benitez (1955) was used. Thisextinction value was considered more accuratethan the 'specific pigment units' used for theestimation of chlorophyll c by Richards & Thomp-son (1952).A red cryptomonad, ahroomonas erroticon nom.

prov., which has recently been obtained in uni-algalculture, contained chlorophylls a and c, carotenes,three unidentified xanthophyll fractions, as well asa pink fluorescent water-soluble phycoerythrinfraction with an absorption maximum at 550 m,u,which is as yet unidentified.

Decomposition productsPheophytins were prepared as described by

Smith & Benitez (1955) and B. values for pheo-phytins a, b and c are shown in Table 2. Pheophytina was always present in extracts of old cultures andin plankton samples that contained much decom-posing material. Pheophytin b was not observed inany extracts of the algae examined. In extracts

338 S. W. JEFFREY 1961

Vol. 80 CHROMATOGRAPHY OF PIGMENTS IN ALGAE 339

1.0 _ (A) (B) 0

0-8 10

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Second dimensions-+Second dimnensions -

Fig. 1. Two-dimensional chromatograms of pigments in marine algae. (A) Dunaliella tertiokexta. (B) Isoclery8i8galbana. (C) Skelekmma coetatum. (D) Gymnodinium sp. (E) Mixed extract of Skeketonema coBtaum, Dunaliellatertiolecta and Gymnsodinium sp. (F) Sea-water extract conaning mixed diatoms and dinoflagellates. -Chromato-graphic solvent system: first dimension, 4% propan-1-ol in light petroleum; second dimension, 30% chloroformin light petroleum. 1, Carotenes (orange); 2, esterified astaxanthin (pink); 3, pheophytin a (grey-green);4, lutein (yellow); 5, diatoxanthin (yellow); 6, diadinoxanthin (yellow); 7,violaxanthin (yellow); 8, dinoxanthin+diadinoxanthin (yellow); 9, neodinoxanthin +neodiadinoxanthin (yellow); 10, chlorophyll a (blue-green);11, chlorophyll a' (blue-green); 12, chlorophyll b (olive green); 13, chlorophyll b'(olive green); 14, fucoxanthin(deep orange); 15, peridinin (reddish orange); 16, neofucoxanthin A and B (orange); 17, neoxanthin (yellow);18, chlorophyllide a (blue-green); 19, chlorophyll c (light green); 20, pheophytin c (light greenish-brown).

22-2

S. W. JEFFREY

derived from old cultures, or extracts that wereaged for several days, the chlorophyll a and b spotssometimes separated into two zones during de-velopment in the chloroform-light petroleumsolvent. These may be the isomerization productswhich Strain (1958) designated chlorophyll a' andchlorophyll b'.

Second chlorophyll a fraction

Several of the marine algae (Phaeodactylum tri-cormutum, Skeletonema costatum and the flagellateSphaleromantis sp.) and some of the sea-watersamples that were collected under conditions of a

diatom bloom showed a blue-green zone in addi-tion to the usual chlorophyll a spot. This new blue-green zone ran just ahead of chlorophyll c afterdevelopment with 4% propan-l-ol in light petrol-eum, but was never completely separated from it.This new chlorophyll did not move in chloroform-light petroleum. The absorption spectrum inacetone resembled closely that of chlorophyll a,

with maxima at 663, 615, 580, 535, 429 and 417 mp,but its chromatographic behaviour was quitedifferent. The unknown pigment could be separatedfrom chlorophyll c with ethyl ether as the chro-matographic solvent. Under these conditions, thecarotenes, xanthophylls and 'normal' chlorophylla travelled together just behind the solvent front,followed by a discrete blue-green spot (the second

Table 2. R vatlues of pigments from marine algaeseparated by two-dimen8ional chromatography

These RF values are mean values for pigments separatedfrom the ten marine algae studied. Because RF values inthese systems may vary according to the quantity of pig-ments put on the paper, with the time of development andthe organism used (Sestak, 1958), the values indicate therelative position of the pigments on the chromatogram,rather than absolute values.

PigmentsCarotenesChlorophyll a

Chlorophyllide aChlorophyll bChlorophyll cEsterified astaxanthinLuteinViolaxanthinNeoxanthinDiatoxanthinDiadinoxanthinDinoxanthinFucoxanthinNeofucoxanthin A and BPeridinin and neoperidininPheophytin aPheophytin bPheophytin c

RFFirst Second

dimension dimension0-96 0-960-84 0-290-38 00-65 0100-20 00-87 0-880*74 0730-65 0*480-32 0.050-57 0-60054 044054 0*440*49 0-280-49 0-080-51 0-230-87 0-960 70 0-890*00 0.00

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340 1961

CHROMATOGRAPHY OF PIGMENTS IN ALGAE

chlorophyll a derivative). Chlorophyll c moved alittle from the origin. The basicity test of Will-statter & Stoll (1913) showed this material to bea chlorophyllide derivative of chlorophyll a, and itwas subsequently found that the conversion ofchlorophyll a into the new form resulted from thepresence of a highly active chlorophyllase in someof the algae. The occurrence and properties of thisenzyme in marine algae will be discussed elsewhere.

DISCUSSION

Some workers have tacitly assumed that two-dimensional chromatography is too slow for theresolution of labile plant pigments (8estak, 1958),but the method which is described here is rapidand simple, and no apparent decomposition ofpigments occurs if the precautions of quick ex-traction and absence of light are observed.

Complete separation of all the minor xanthophyllcomponents was not achieved by the method,although for any one organism the xanthophyllresolution was considered satisfactory. In mixturesof organisms the xanthophyll spots showed acertain degree of overlapping, particularly peridininand fucoxanthin. These two pigments could beresolved by a third development in the direction ofthe second dimension with higher proportions ofchloroform to light petroleum (up to 60 %, v/v).

Astaxanthin, which is usually thought to be ananimal carotenoid, but which has been described inthe green alga Haematococcu8pluvialis (Kuhn, Stene& Sorensen, 1939; Goodwin & Jamikorn, 1954),was found in an esterified form in the green [,-flagellate Nannochloris atomus, which was isolatedfrom the coastal waters off Sydney. The concen-tration of this pigment increased appreciably withthe age of the culture.The xanthophylls in the two members of the

Chrysophyceae which were examined are of someinterest. Both organisms contained fucoxanthinand isomers as the major carotenoids and diato-xanthin as a minor pigment. Isochry8is galbanashowed in addition the presence of small quantitiesof diadinoxanthin, whereas Sphaleromantis con-tained a yellow pigment similar to dinoxanthin,with absorption maxima at 477 and 444 mu inethanol. Allen et al. (1960) identified fucoxanthinand diatoxanthin in members of the Chryso-phyceae, and Dales (1960) found fucoxanthin anddiadinoxanthin in cultures of Isochry8si galbana.The latter author claimed that chlorophyll c wasnot present in any of eight members of the Chryso-phyceae which he examined, whereas chlorophyll cwas found in all cultures of Isochry&i8 galbana andSphaleromantis studied in the present work. Thefailure to find chlorophyll c apparently resultedfrom the use of light petroleum, in which chloro-

phyll c is insoluble, instead of diethyl ether in theextraction procedure.The quantitative measurements showed that the

concentration of chlorophylls was approximately1-3 times the concentration of the carotenoids inthe organisms studied. The concentration of theaccessory chlorophylls b and c was of the sameorder of magnitude as that of chlorophyll a,although the chlorophyll c values are tentativefigures only, owing to uncertainty about the true ex-tinctionvalues for chlorophyll c. In the diatoms andgolden-brown flagellates fucoxanthin and isomersconstituted 80-90% of the total carotenoids, andperidinin accounted for 70% of the total carotenoidsin the dinoflagellate cultures. Each of the minor com-ponents in these organisms (carotene, diatoxanthin,diadinoxanthin etc.) represented about 5% of thetotal carotenoids. Lutein, in the green flagellates,accounted for 40 % of the total carotenoids, withcarotene, violaxanthin and neoxanthin present inquantities ranging from 10 to 20% of the total.The four green flagellates (Chlorophyceae)

studied contained chlorophylls a and b and caro-tenes and gave three prominent xanthophyll zonescorresponding to lutein, violaxanthin and neo-xanthin. The three diatoms (Bacillariophyceae) andthe two golden-brown flagellates (Chrysophyceae)showed a similarity in their pigment compositionwith chlorophylls a and c, carotenes and fucoxan-thin and isomers as the major xanthophylls. Thedinoflagellate (Dinophyceae) was distinguished bythe presence of peridinin in place of fucoxanthin asthe major xanthophyll, in addition to chlorophyllsa and c, carotenes and the minor xanthophyllsshown in Fig. 1. It is therefore apparent from thepresent work that chlorophylls a, b and c, thecarotenes and the xanthophylls lutein, fucoxanthinand peridinin are quantitatively the most im-portant photosynthetic pigments found in algaewhich comprise the phytoplankton organismsstudied.

SUMMARY

1. Two-dimensional paper chromatography withpropan-l-ol-light petroleum and chloroform-lightpetroleum mixtures separated the chloroplastpigments of marine algae belonging to the classesChlorophyceae, Bacillariophyceae, Dinophyceaeand Chrysophyceae.

2. The pigments separated included chloro-phylls a, b and c, their pheophytins, the carotenes,the xanthophylls lutein, violaxanthin, neoxanthin,fucoxanthin and isomers, peridinin, esterifiedastaxanthin and a number of minor xanthophyllcomponents.

3. The concentration of the accessory chloro-phylls b and c was of the same order of magnitudeas that of chlorophyll a. Fucoxanthin and peri-

Vol. 80 341

342 S. W. JEFFREY 1961

dinin, and their isomers, constituted 80-90% of thetotal carotenoids in the orga isms which containedthese pigments.

I wish to thank Dr R. W. Butcher, Fisheries Laboratory,Burnham-on-Crouch, Essex, for identifying some of theorganisms which were used in this investigation.

REFERENCESAllen, M. B., Goodwin, T. W. & Phagpolngarm, S. (1960).

J. gen. Microbiol. 23, 93.Anderson, J. M. (1959). Ph.D. Thesis: University of

California.Dales, R. P. (1960). J. Mar. biol. A88. U.K. 39,693.Davis, P. S. (1957). C.S.I.R.O. Audt. Div. Fi8h. Oceanogr.Report no. 7.

Fritsch, F. E. (1948). The Structure and Reproduction of theAlgae, vol. 1. Cambridge University Press.

Gilchrist,B.M. &Green,J. (1960). Proc.Roy.Soc. B,152, 118.Goodwin, T. W. (1955). In Modern Method8 of Plant

Analysi8, vol. 3, p. 272. Ed. by Paech, K. & Tracey, M.Heidelberg: Springer Verlag.

Goodwin, T. W. & Jamikorn, M. (1954). Biochem. J. 57,376.

Goodwin, T. W. & Srisukh, S. (1949). Biochem. J. 45,268.

Humphrey, G. F. (1960). C.S.I.R.O. Aust. Div. Fieh.Oceanogr. Tech. Pap. no. 9.

Kuhn, R., Stene, J. & Sorensen, N. A. (1939). Ber. chem.dt8ch. Ge8. 72, 1688.

Lind, E. F., Lane, H. C. & Gleason, L. S. (1953). PlantPhyeiol. 28, 325.

Richards, F. A. & Thompson, T. G. (1952). J. Mar. Ree.11, 156.

SestAk, Z. (1958). J. Chromat. 1, 293.Smith, J. H. C. & Benitez, A. (1955). In Modern Methods of

Plant Analyese, vol. 4, p. 142. Ed. by Paech, K. &Tracey, M. Heidelberg: Springer Verlag.

Strain, H. H. (1958). Chloroplaet Pigmente and Chromato-graphic Analy8ie, 32nd Priestley Lecture, UniversityPark, Pennsylvania.

Strain, H. H., Manning, W. M. & Hardin, G. (1944). Biol.Bull., Woods Hole, 86, 169.

Willstatter, R. & Stoll, A. (1913). Inveetigatione onChlorophyll. Lancaster: Science Press Printing Co.

Biochem. J (1961) 80, 342

Intrinsic Factor: Active and Inhibitory Components fromthe Mitochondria of Human Gastric Mucosal Cells

BY W. H. TAYLOR, BARBARA J. MALLETT AND K. B. TAYLORDepartment of Chemical Pathology, A8hton Street, Liverpool 3, and Nuffield Department of Medicine,

Radcliffe Infirmary, Oxford

(Received 13 February 1961)

Cell particles, separated by high-speed ultra-centrifuging from preparations of human and ratgastric mucosa, have been shown to contain asmuch as half of the intrinsic-factor activity of thewhole preparation (Taylor, 1955). The activity ofthe human particles may be removed by disruptionwith water, in which the active material subse-quently dissolves, but in the rat the activityremains bound to the disrupted particles (Taylor,Mallett, Witts & Taylor, 1958 b). The activity fromthe human particles may be further concentratedinto a 'substance E' (O'Brien, Taylor, Turnbull &Witts, 1955), which on paper electrophoresis,electrophoresis in the Tiselius apparatus andSvedberg ultracentrifuging cannot be resolved intomore than one component. Substance E promotesthe absorption of 6°Co-labelled vitamin B12 inpatients with pernicious anaemia in doses of about10 mg., but this represents a low order of activityand it has been provisionally suggested that sub-stance E may be a precursor of intrinsic factor(O'Brien et al. 1955). Further investigations into

the activity of substance E and related studies arenow reported.

METHODS

Substance E was prepared as described by O'Brien et al.(1955) from human gastric mucosa.Paper electrophoresis was carried out at room temper-

ature for 16 hr. in a horizontal bath with a current of 1 mA/4 cm. width of paper strip and diethylbarbituric acid-sodium diethylbarbiturate buffer (pH 8-6, I 0.06). Thepaper strips were stained with 1% bromophenol blue inethanol saturated with mercuric chloride.

Ultracentrifugal studies were carried out in the OxfordSvedberg ultracentrifuge under the guidance of ProfessorA. G. Ogston, F.R.S.

Intrinsic-factor activity was assayed in patients withpernicious anaemia in remission by giving 0*5 pg. of 60Co-labelled vitamin B1,2 in aqueous solution by mouth, aloneand then with preparations of intrinsic factor, and com-paring the amounts ofradioactivity appearing in the stools.Measurements of activity were made in a ring of Geigercounters, similar to that described by Bradley (1957). Inorder to compare quantitatively results obtained withdifferent fractions and patients, the increase of absorption