THE JOURN.

~ OF BIOLOGICAL CHEMISTRY
Vol. 241, No. 8, Issue of April 25, 1966
Printed in U.S.A.

Chloroperoxidase
I. ISOLATION AND PROPERTIES OF THE CRYSTALLINE GLYCOPROTEIN*

(Received for publication, August 25, 1965)

DAVID R. MORRIS$ AND LOWELL P. HAGER~

From the Biochemistry Division, Department oj Chemistry and Chemical Engineering, University of Illinois,
Urbana, Illinois 61803

SUMMARY purified chloroperoxidase preparation was shown to catalyze the

Chloroperoxidase from Caldariomyces fumago has been reaction shown in Equation 1 (3).
isolated in crystalline form. The prosthetic group of chloro-
peroxidase is ferriprotoporphyrin IX. On the basis of the ::
OOCCH&H&CHzCOO- + Cl- + Hz02 + 2H+ +
heme content, the minimal molecular weight of chloroperoxi- (1)
dase is 40,200. On the basis of hydrodynamic measure- 0
ments, the molecular weight of chloroperoxidase is approxi- COn + -OOCCH&H&H&l + 2HzO
mately 42,000. Therefore, in solution at neutral pH,
chloroperoxidase behaves as a monomeric species. In order to obtain suitable material for further investigation
The spectral properties of the oxidized and reduced forms of the mechanism of this reaction, chloroperoxidase has been
of the enzyme and also of the azide and cyanide complexes extensively purified and is now available in the crystalline state.
are reported. In this communication we report on the purification and partial
The amino acid composition of chloroperoxidase is reported. physical and chemical characterization of chloroperoxidase.
Chloroperoxidase is rich iu aspartic acid, glutamic acid, and MFTHODS AND MATERIALS
serine and proline residues. These four amino acids con-
stitute 45 % of the total amino acid content. Enzymatic Assay-The standard purification assay for chloro-
Chloroperoxidase is a glycoprotein. Approximately 25 peroxidase is based on the loss of absorbance at 278 rnp
to 30% of the molecule is carbohydrate. Chromatography accompanying the conversion of 1, I-dimethyl-4-chloro-3,5-
of acid hydrolysates reveals that glucosamine and arabinose cyclohexanedione (monochlorodimedon) to l , l-dimethyl-4,4-di-
are major constituents. chloro-3,5-cyclohexanedione (dichlorodimedon) (4). The stand-
ard assay mixture contains 306 pmoles of potassium phosphate
buffer, pH 2.75, 60 pmoles of potassium chloride, 6 pmoles of
hydrogen peroxide, 0.3 pmole of monochlorodimedon, and a
suitable aliquot of enzyme in a total volume of 3 ml. The re-
action was started by the addition of enzyme. Incubation was
Studies on the biosynthesis of the chlorine-containing fungal at room temperature. The change in absorbance at 278 mp as
metabolite caldariomycin, produced by the mold Caldariomyces a function of time was continuously recorded on a Gilford-modi-
fumago, have led to the question of the exact nature of the en- fied Beckman DU spectrophotometer. The rate of reaction was
zymatic halogenation reaction involved. Reports from this linear for 3 to 4 min. One unit of chloroperoxidase activity has
laboratory demonstrated the synthesis of &chlorolevulinic acid been defined as the formation of 1 kmole of dichlorodimedon
from P-ketoadipic acid and inorganic chloride catalyzed by ace- per min under the standard assay conditions.
tone-dried mycelial powders of this organism (1). This crude Protein Determinations-Protein concentrations at intermedi-
enzyme system was fractionated, and the peroxidatic nature of ate purification stages were estimated by the method of Lowry
the chlorination reaction was established (2). This partially et al. (5) with bovine serum albumin as a standard. Protein
* This investigation was supported by Grant GB-2786 from the determination by dry weight analysis on crystalline chloro-
Na.tional Science Foundation. Portions of this work were taken peroxidase gave a value 35% higher than did the Lowry method.
from a thesis submitted by D. R. Morris in partial satisfaction of Amino Acid Analysis-Hydrolysis of the crystalline enzyme
requirements for the degree of Doctor of Philosophy, University was carried out on approximately 2-mg aliquots of the lyophi-
of Illinois, 1964. A preliminary report of this work was presented
at the 148th Meeting of the American Chemical Society, Septem- lized, dialyzed protein. A weighed sample was dissolved in
ber, 1964, Chicago, Illinois. freshly distilled constant boiling hydrochloric acid, sealed in an
$ This author held a predoctoral fellowship from the National evacuated tube, and held at 110” for 24 or 48 hours. Amino
Institutes of Health at the time of the investigation. Present acid analysis was carried out with the Beckman model MS amino
address, Princeton University, P.O. Box 704, Princeton, New acid analyzer according to the method of Moore, Spackman, and
Jersey.
$ Reprint requests should be addressed to this author. Stein (6).

1763

This is an Open Access article under the CC BY license.

1764 Chloroperoxidase. I Vol. 241, No. 8

Performic acid oxidation was performed by the method de- concentrated material from two 15-liter cultures was dialyzed
scribed by Him (7). against three 40-liter changes of deionized water. This dialysis
Materials-Hydrogen peroxide was purchased from Barium step and all subsequent steps in the purification were carried
and Chemicals, Inc., and was standardized periodically by the out at 4” unless otherwise indicated. The dialyzed material
oxidation of iodide to iodine catalyzed by molybdate and sub- was then freeze-dried, and the resulting powder was stored at
sequent titration of the iodine formed with sodium arsenite. -20”.
DEAE-Sephadex was supplied by Pharmacia. The dialysis Ethanol Fructionation-The freeze-dried powder obtained
tubing supplied by LKB for use with their ultrafilter was used from two 15-liter cultures (approximately 40 g) was dissolved in
directly. Dialysis tubing obtained from Visking Division, approximately 400 ml of distilled water and dialyzed overnight
Union Carbide, was heated to 60” for 3 hours in 1 rnhf EDTA against 4 liters of distilled water. Ethanol (precooled to -10”)
and 1% sodium bicarbonate and washed twice at 60” for 3 hours was then added to this preparation with vigorous stirring in a
with glass-distilled water before use. Monochlorodimedon was -10” ice-salt bath until an ethanol concentration of 45% was
prepared from dimedon by chlorination with sodium hypo- reached (82 ml of absolute ethanol per 100 ml of the preparation).
chlorite (4). After stirring for 15 min, the preparation was centrifuged at
-10” for 15 min at 15,000 x g, and the black precipitate was
EXPERIMENTAL PROCEDURE discarded. Ethanol was then added to the supernatant until a
Growth of Mold and Preparation of Crude Enzyme-The strain concentration of 65% (v/v) was reached (104 ml of absolute
of C. fumago used in these experiments was that previously used ethanol per 100 ml of the original preparation). This prepara-
by Shaw, Beckwith, and Hager (8). Stock cultures of C. fumqo tion was then allowed to stir slowly in the salt bath until the
were maintained at 4” on oatmeal agar slants (8). Inoculum precipitate coagulated on the bottom of the beaker. The super-
was grown for 7 to 9 days as a surface mat on Difco malt ex- natant was then completely drained off and discarded. The
tract agar slants prepared in 32-0~ prescription bottles. The resulting gelatinous brown precipitate was redissolved in approxi-
surface mat from three bottles of inoculum was used to inoculate mately 30 ml of 0.1 M phosphate buffer, pH 6.0, and stored at
30 liters of a modified Czapek-Dox medium. The surface mats -20”. Normally, two batches of ethanol-fractionated material
were aseptically removed from the inoculum bottles, transferred would be pooled at this point for further purification steps.
to a sterile Waring Blendor jar containing approximately 500 Column Chromatography on DEAE-Sephadex-Approximately
ml of modified Czapek-Dox medium, and homogenized for 30 25 g of DEAE-Sephadex A-50 were first swollen in water and
set on the Blendor. The homogenate was equally divided and washed with 500 ml of 0.5 N hydrochloric acid, then with 500 ml
used to inoculate two 5-gallon Pyrex bottles. Each 5-gallon of 0.5 N sodium hydroxide, and finally was thoroughly washed
bottle contained 15 liters of growth medium. The growth with water. The Sephadex was then neutralized with phosphoric
medium was the modified Czapek-Dox medium described by acid to pH 6.0 and equilibrated with 0.1 M phosphate buffer,
Clutterbuck et al. (9) for the growth of C. fumago further modi- pH 6.0, by stirring in a 2-liter beaker with several changes of the
fied by a supplement of 3.3 g of Difco yeast extract per liter. buffer. The equilibrated resin was then packed in a 3.7-cm
The mold was grown under submerged conditions at 20-23” diameter column to a height of 13 cm. Two pooled batches
with stirring and vigorous aeration until the enzyme level in the obtained from the 45 to 65% ethanol fractionation step (appros-
medium had reached at least 5 units per ml (approximately 6 imately 60 ml) were adsorbed to the column, and the column
to 7 days). The mold mycelium was then filtered off on a 33-cm was washed with 1 column volume of 0.1 M phosphate buffer,
stainless steel Buchner funnel. pH 6.0. Chloroperoxidase was then eluted from the column
The filtrate containing the enzyme was then concentrated with a linear ionic strength gradient prepared by placing 350
approximately lo-fold with a vacuum laboratory evaporator ml of 0.1 M phosphate buffer, pH 6.0, in the mixing chamber and
supplied by Precision Scientific Company. Care was taken 350 ml of 0.2 M phosphate buffer, pH 6.0, in the reservoir. Frac-
during the evaporation to keep the temperature below 45”. The tions of 9 ml each were collected. A typical elution diagram is
shown in Fig. 1. The first peaks of chloroperoxidase activity
were artifacts which were probably introduced by the elevated
1’ temperature during concentration of the medium since they were
seen in preparations which had not been exposed to this treat-
ment. Those fractions in the last peak with specific activities
of 860 and higher were combined and dialyzed against 0.01 RI
phosphate buffer, pH 5.0.
Column Chromatography on Calcium Phosphate Gel-Cellulose-
Calcium phosphate gel columns were prepared by mixing 100 ml
of a 30 mg per ml suspension of calcium phosphate gel, prepared
as described by Swingle and Tiselius (lo), with 250 ml of a 10%
(w/v) suspension of coarse Whatman cellulose powder in 0.01 M
phosphate buffer, pH 5.0. This suspension was packed in a column
3.8 cm in diameter. The dialyzed DEAE-cellulose column frac-
tions were adsorbed on the calcium phosphate gel column and
TUBE NUMBER
eluted with a gradient consisting of 350 ml of 0.01 M phosphate
FIG. 1. Chromatography of chloroperoxidase on DEAE-Sepha- buffer, pH 5.0, in the mixing chamber and 350 ml of 0.2 M phos-
dex. The chloroperoxidase activity (---) and the protein c( n- phate buffer, pH 5.0, in the reservoir. All fractions having a
centration (- - -) are plotted as functions of the eluate volur e.
Each tube contained 9 ml. specific activity of 1200 or higher were combined.
Issue of April 25, 1966 D. R. Morris and L. P. Hager 1765

Crystallization of Chloroperoxitkse-The combined fractions TABLE I

from the calciumphosphategel column were concentratedby 6- Purijka~ion of chloroperoxidase
hour dialysis against saturated ammoniumsulfate and centri-
Fraction Volume Total Total Specific
fuged at 34,000 X g for 30 min. The precipitate wasredissolved activity protein activity
in 3.5 ml of 0.01 M phosphatebuffer, pH 6.0, to give a protein
pWlCS/
concentration of approximately 8 mg per ml (5 to 10 mg per ml ntl nin x 10-4
hasproved to be satisfactory). The concentratedcolumn frac- Crude extract (dialyzed)“. 890.0 7.97 8600.0 9.27
tions were then dialyzed overnight against 60% saturated am- Ethanol (4565%). 65.1 7.08 571 .o 124.00
monium sulfate. The enzyme preparation was then removed DEAE-Sephadex eluate. 186.0 4.77 48.5 984.00
from the dialysis bag, and saturated ammonium sulfate was Calcium phosphate-cellu-
addeddropwiseto a final concentrationof 70y0 saturation. The lose eluate. . 128.0 3.79 30.2 1250.00
samplewasthen allowedto standat 4”, and crystallization began 1st crystallization. . 2.0 2.49 16.9 1480.00
2nd crystallization.. . 2.0 1.36 8.2 1660.00
6 to 24 hours later. Crystallization was usually complete 24
3rd crystallization. .. . 1 .O 0.64 4.0 1600.00
hours after it started. A photograph of the brown needlesof
crystalline chloroperoxidaseis shownin Fig. 2. The resultsof a LIPrepared from 80 g of lyophilized powder.
typical purification are shownin Table I.
ma1molecularweight of 40,200was calculated for the enzyme,
RESULTS which had been crystallized three times. For this calculation,
ProstheticGroup of Chloroperoxidase-Crystallinechloroperoxi- the protein concentration of the enzyme preparation was cor-
dasewasshownto contain ferriprotoporphyrin IX by the forma- rected to a dry weight basis.
tion of the formic acid hemochromogen,with the use of the Spectral Propertiesof Chloroperoxidase-fig. 3 showsthe ab-
method describedby Deeb and Hager (11). With the reported sorption spectra for the native and dithionite-reducedforms of
extinction coefficientof 2.56 X lo5 M-’ cm-l at 408 rnp, a mini- chloroperoxidaseand for thoseforms of chloroperoxidasein com-

FIG. 2. Photograph of chloroperoxidase crystals X 1000

1766 Chloroperoxidase. I Vol. 241, No. 8

coefficients reported in this section are based on heme concentra-

tion.
The dithionite-reduced form of chloroperoxidase has a complex
Soret region with a maximum at 409 rnE.1and shoulders at approxi-
mately 420 and 450 mp. The visible band in the reduced form
is at 550 rnh. The millimolar extinction coefficients of the re-
duced form at 409, 420, 450, and 550 rnp are, respectively, 68.7,
66.5, 38.5, and 13.7. The complexity of the reduced spectrum
of chloroperoxidase is probably due to denaturation of the enzyme
by dithionite or the side products of dithionite oxidation, or both.
This contention is supported by the fact that reoxidation of re-
duced chloroperoxidase yields an inactive product. The denatur-
ation of myoglobin (12) and hemoglobin (13) by dithionite has
been reported.
1 I I I I I The azide derivative of chloroperoxidase shows absorption
400 400 560 640 maxima at 432, 549, 583, and 665 rnF and millimolar extinction
WAVELENGTH - m,u coefficients of 88.5, 10.9, 7.8, and 3.0, respectively. The cyanide
complex has absorption maxima at 437 and 555 mp. The milli-
FIG. 3. Absorption spectra of chloroperoxidase and its deriva-
molar extinction coefficients of the cyanide complex are, respec-
tives. The spectra of the native (--) and dithionite-reduced
(- - -) forms of chloroperoxidase were measured in 0.1 M citrate- tively, 77.8 and 11.5.
phosphate buffer, pH 5.4. The spectrum of the azide derivative Chemical Composition of Chloroparoxidase-Table II gives the
(-.-.-) was measured in 0.1 M citrate-phosphate buffer, pH 4.2, amino acid composition of chloroperoxidase. The most striking
and 0.56 M sodium azide. The spectrum of the cyanide complex characteristic is the great predominance of the acidic amino acids
(. . . .) was scanned in 0.1 M citrate-phosphate buffer, pH 5.4, and
0.06 M potassium cyanide. over the basic residues. Also interesting is the high proline con-
tent of chloroperoxidase.
TABLE II The molecular weight of chloroperoxidase, on the basis of its
Amino acid composition of chloroperoxidase amino acid composition (without tryptophan, plus hemin), can
be calculated to be 28,640 (Table II). The minimal molecular
The amino acid analysis was carried out as described under
“Methods and Materials.” weight calculated on the basis of heme content is approximately
40% higher than this value. This fact, together with the fact
No. of residues per mole of enzyme that protein concentration by dry weight analysis invariably
Percentage gives values 35% higher than those obtained by the Lowry
Amino acid Average or by weight
24-hr 48-hr extrapolated
hydrolysate hydrolysate V&e

Met 2.00 2 0.94

Cysa 2.00 2 0.66
1.3
I
LYS 4.00 4.00 4 1.83
Arg 6.42 6.46 6 3.34
His 7.06 6.84 7 3.42
Ile 9.42 8.84 9 3.63
.2
Tyrb 8.28 6.92 10 5.82
Val 11.8 11.8 12 4.24
Phe 13.6 12.6 13 6.82
GUY 14.8 13.8 14 2.85
Thr* 13.7 11.1 17 6.13
1. I
Leu 20.4 18.8 20 8.07
Prob 21.8 20.8 23 7.96
Ala 25.0 22.2 24 6.08
Glub 22.4 19.2 26 12.0
Serb 20.6 12.7 33 10.2 I
I I I I I I RIGIN
Aspb 37.2 35.2 39 16.0 I 2 3 4 5 6 7

99.99 FIG. 4. Chromatography of the sugar obtained from a hy-

Total 261
drolysate of chloroperoxidase. A 1.77.mg sample of twice crystal-
lized chloroperoxidase was lyophilized to dryness and hydrolyzed
(1 Determined as cysteic acid in a performic acid-oxidized for 3 hours under reduced pressure at 110” in 0.1 ml of 2 N hydro-
sample. chloric acid. This sample was then spotted on Whatman No. 1
b Extrapolated to zero time, assuming first order decay. paper together with 106 pg each of the indicated standards. The
origin spots are numbered as follows: 1, glucose; 2, xylose; S,
glucosamine; 4, hydrolyzed enzyme sample; 5, mannose; 6, arab-
plex with cyanide and azide. The spectrum of native chloro-
inose; 7, ribose. The chromatogram was developed for 18 hours
peroxidase has absorption maxima at 403, 515, 542, and 650 rnl.c. with butanol-acetic acid-water (40: 10 : 50) as a solvent (14). The
The millimolar extinction coefficients of these peaks are, respec- spots were visualized with the triphenyltetrazolium chloride
tively, 75.3, 11.5, 10.8, and 4.2. These and the other extinction method of Trevelyan, Procter, and Harrison (15).
Issue of April 25, 1966 D. R. Morris and L. P. Hager 1767

the plot of In G with respect to r2 for the overspeeded run. The

linearity of this plot indicates that the preparation is homogene-
4.0
ous and that the system is not an associating one.
An equilibrium experiment with the short column method of
Van Holde and Baldwin (20) was also performed on crystalline
chloroperoxidase with the use of interference optics. Weight aver-
s 20,w 3.5 age and z-average molecular weights of 42,000 and 41,900, re-
spectively, were calculated.1 The close agreement of these two
values is further indication of the homogeneity of this crystalline
enzyme.

3.0 DISCUSSION

Chloroperoxidase is very similar to the plant peroxidases iso-

I I I I I lated from horseradish and Japanese radish in terms of amino
3 6 9 12
acid and carbohydrate content (21-23), molecular weight (24,
mg/ml (DRY WEIGHT)
25), and spectral properties (25, 26). Horseradish peroxidase
FIG. 5. Dependence of sedimentation constant on concentration and Japanese radish perxodiase have been assigned molecular
of chloroperoxidase. The indicated concentration of chloro- weights of 39,800 (24) and 55,700 (25), respectively. Chloro-
peroxidase was dissolved in 0.1 M potassium phosphate buffer, pH peroxidase, with an approximate molecular weight of 42,000,
6.0, and the sedimentation constant (~20,~) was measured in the
Beckman analytical ultracentrifuge, model E. more closely resembles horseradish peroxidase with respect to
molecular weight. Horseradish peroxidase, Japanese radish
peroxidase, and chloroperoxidase are all glycoproteins. Horse-
method, leads one to the conclusion that chloroperoxidase con-
radish peroxidase contains approximately 18% (21), and Japa-
tains approximately 25 to 35% by weight of a nonprotein sub- nese radish peroxidase contains 28% (22), compared to the 25
stance. Fig. 4 shows a paper chromatogram in which an acid to 30% carbohydrate content of chloroperoxidase. On the basis
hydrolysate of crystalline chloroperoxidase was examined for
of carbohydrate content, chloroperoxidase more closely resembles
reducing sugars. As can be seen from this chromatogram, Japanese radish peroxidase. However, Japanese radish peroxi-
chloroperoxidase contains glucosamine and arabinose. When dase contains mannose and xylose as principal monomeric com-
the Somogyi modification of Nelson’s test for reducing sugars ponents in addition to arabinose and hexosamine whereas only
(16) was performed on twice crystallized chloroperoxidase which glucosamine and arabinose were detected in chloroperoxidase
had been hydrolyzed as in Fig. 4, it was calculated that the en- hydrolysates. In addition, chloroperoxidase and Japanese radish
zyme contained 12,300 g of reducing sugars per 40,000 g, dry peroxidase share a distinct pattern with respect to amino acid
weight (based on a molecular weight of 180 per reducing group).
composition. Both of these glycoproteins contain a great excess
Calculation from these data yields a carbohydrate content of of aspartic acid and glutamic acid residues over the sum of the
30.7%. Since the enzyme contains arabinose, a pentose, in basic amino acid residues, and both are rich in serine and proline
addition to glucosamine, the value of 30.7 y0 carbohydrate would residues (23).
be a slight overestimate. Thus, it would appear that chloro- Despite these apparent similarities, the halogenation activity
peroxidase contains a carbohydrate component at a minimum
level of 25% of the total weight of the enzyme (estimated from
amino acid analysis) and at a maximum level of 30% (reducing
sugar analysis).
With the knowledge that chloroperoxidase contains 25 to 30%
carbohydrate and since the amino acid composition is known,
it is possible to calculate the partial specific volume of the enzyme.
The partial specific volume for the protein part of chloroperoxi-
dase, calculated by the method of Cohn and Edsel (17), is 0.75.
Assuming a partial specific volume of 0.60 for the carbohydrate
portion of the enzyme (18) and a carbohydrate content of 25%,
the calculated 0 for chloroperoxidase is 0.71.
Ultracelztrifuge Studies on Chloroperoxidase-Fig. 5 shows a
plot of the dependence of SZO,~ on the concentration of chloro-
peroxidase. As can be seen from this graph, the sedimentation
coefficient of chloroperoxidase is inversely proportional to the
protein concentration. Extrapolation of this plot to infinite I I I
dilution gives a sedimentation coefficient of 4.1 S. 45.50 45.75 46.00 46.25
An overspeeded equilibrium experiment was carried out with R2
crystalline chloroperoxidase by the method of Yphantis (19). FIG. 6. Overspeeded sedimentation equilibrium of chloroper-
With the use of the partial specific volume of 0.71 calculated oxidase. The concentration (fringe displacement) is expressed as
above, a molecular weight of 42,000 was found.’ Fig. 6 shows inches on the microcomparator. The distance from the axis of
rotation (R) is expressed in centimeters. The centrifuge speed
1 These calculations were carried out on the IBM 7094 computer was 29,500 rpm. The protein concentration was 1.3 mg per ml in
using programs loaned to us by Mr. Robert Dyson. 0.1 M phosphate buffer, pH 6.0.
1768 Chloroperoxidase. I Vol. 241, No. 8

of chloroperoxide places it apart from horseradish and Japanese 9. CLUTTERBUCK, P. W., MUKHOPADHYAY, S. L., OXFORD, A. E.,
radish peroxide in terms of catalytic activity. We have tested AND RAISTRICK, H.; Biochem. J., 34. 664 (1940).
10. SWINGLE. S. M.. AND TISELIUS. A.. Biochem. J.. 43.171 (1951).
horseradish peroxidase under a variety of conditions and cannot 11. DEEB, S.‘S., AND HAGER, L. P.,‘J. biol. Chem., i39,‘1024 (1964j.
detect the chlorination or bromination activity which is char- 12. URNES, P., Doctoral dissertation, Harvard University, 1963.
acteristic of chloroperoxidase (4). Therefore, there must be a 13. HAMADA, K., OKAZAKI, T., SHUKUYA, R., AND KAZIRO, K.,
basic difference in the constitution of the active sites of these J. Biochem., 52,374 (1962).
14. PARTRIDGE, S: M., Biochem. J., 42, 238 (1948).
peroxidases. 15. TREVELYAN. W. E.. PROCTER. D. P.. AND HARRISON. J. S..
Nature, 166, 444 (1950). ’ ’
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