Linear gradient elution of nucleotides from Dowex-1-formate: Application to the erythrocyte

:ANALYTICAL

Linear

BIOCHEMISTRY

Gradient Formate: SAMUEL

3, 285-297 (1962)

Elution of Application A. MORELL, TIBOR

Nucleotides from to the Erythrocyte VALBORG

E. AYERS,

Dowex-l-

AND

J. GREENWALT

From the Department of Biochemistry, Milwaukee Blood Center, Inc., and Department of Biochemistry, Marquette University School of Medicine, Milwaukee, Wisconsin

Received December

the

28, 1960

INTRODUCTION

Ion-exchange chromatography of nucleotides was introduced by Cohn and Carter (1) and has been applied extensively to analytical and preparative biochemistry. Modifications employing “extended gradient” elution from Dowex-1-formate were developed by Hurlbert, Schmitz, Brumm, and Potter (2). The separation of complex mixtures of nucleotides from tissue extracts was greatly facilitated by the use of their ‘Lammonium formate” and “formic acid” systems (3). The latter syst,em has been used frequently in studies on the acid-soluble phosphates of the erythrocyte (46). In this system relatively high concentrations of formic acidammonium format,e mixtures are required for elution; AMP, ADP, and ATP, for example, are eluted at approximately 1.0, 2.6, and 3.7 M formic acid, respectively. A nongradient elution pattern of ribonucleotides from Dowex-1-formate at considerably lower acid-salt molarities has been described by Bergkvist and Deutsch (7). In their system, formic acid is maintained at 0.1 M and AMP, ADP, and ATPI are eluted at 0, 0.3, and 0.5 M ammonium formate, respectively. Since formic acid contributes considerably more background absorption than ammonium formate, application of the latter elution technique to a gradient system combines the advantages of both methods. In studying phosphate distribution and P3?exchange in the erythrocyte, ‘The abbreviations used are AMP, ADP, and ATP for adenosine-5’-phosphate, -diphosphate, and -triphosphate, respectively; C, U, G, and IMP, IDP, and ITP for Ihe corresponding 5’-phosphates of cytidine, uridine, guanosine, and inosine, respecttively ; 2,3-DPG, 2,3-diphosphoglyceric acid; Pi, orthophosphate ; F-1,6-P for fructose 1,6-diphosphate; RBC, red blood cells; PCA, perchloric acid; TCA, trichloroacetic acid ; F.A., formic acid ; Am.F., ammonium formate; ACD, acid-citrate-dextrose, NIH formula “A.” 285

286

MORELL,

AYERS,

AND

GREENWALT

the acid-soluble phosphates identified by paper chromatography were ATP, ADP, Pi and 2,3-DPG (8, 9). The objective of the present investigation was to apply ion-exchange techniques to studies on phosphate partition in the erythrocyte. The work of Bergkvist and Deutsch (7) served as the basis for developing an efficient single-column linear gradient elution pattern for the acid-soluble ribonucleotides and sugar phosphates of the erythrocyte. The procedure herein described should be applicable to other tissue extracts and offers several analytical and preparative advantages: (a) low background absorption due to formic acid, especially at the shorter wavelengths, 250-260 rnp; (b) low salt contamination when isolating products from “peak” tubes; and (c) improved resolution of similarly charged compounds. The latter is accomplished by interrupting the gradient at an eluant concentration that most effectively separates the mixture and that may often be calculated from available linear-gradient data. As described below, Bartlett’s elution patterns (10) were applied in this manner to the separation of ATP and 2,3-DPG, the two principal acid-soluble phosphates of the erythrocyte. PROCEDURES

Apparatus A Gilson Model V-15 fraction collector equipped with a recording ultraviolet absorption meter, 254 rnp sensitivity, was used.2 One-liter aspirator bottles (Corning No. 1220), connected through a capillary stopcock, served as “reservoir” and “mixer” to obtain a linear gradient by simple gravity flow (11). The bottles were supported on rings clamped to extension rods resting on the housing of t,he absorption meter. An electric stirrer, independently supported from the ceiling to avoid vibration, was found to be more satisfactory than the magnetic type. The resin bed was supported on a coarse grade of fritted disk (Corning No. 36060-2C), which was sealed to a 12 X 1 cm Pyrex tube. The latter was sealed to the bottom of a 125-ml TS 24/40 Erlenmeyer flask (12) which accommodated either (a) a separatory funnel for adsorption and washing, or (b) the tube leading from the mixer bottle for elution. All connections were “glass to glass.” Flow rates were controlled by means of a Teflon needle valve (Corning No. NV-99800). A Beckman model DU spectrophotometer was used for nucleotide analysis and a Beckman model B spectrophotometer (with 150 X 19 mm cuvettes, Coleman No. 6-304) was used for phosphate and sugar analysis. A Vortex test tube mixer3 was used in the sugar determinations. A Sorvall ’ Gilson 3 Kraft

Medical Electronics, Mfg. Co., New York,

Middleton, N. Y.

Wisconsin.

LINEAR

GRADIENT

ELUTION

OF

NUCLEOTIDES

model SS-1 and an International model CL were used for centrifuging the higher (7200 rpm) and lower (3000 rpm) speeds, respectively. Preparation

287 at

of Resin a,ld Columns

A IO-lb batch of Dowex l-xl0 chloride, 200400 mesh, capacity 1.2 meq/ml, was washed free of ‘lfines.” The resin was converted to the formate form by washing on sintered fumrels with 3 M sodium formate until the effluents were free of chloride. Excess sodium formate was removed by copious washing with water. Aliquots of the stock resin slurry were used for all experiments to avoid variations in different commercial lots. A fresh resin bed was prepared for each adsorption-elution experiment. A loosely packed layer of Pyrex wool (0.5 cm) was placed over the sintered bottom of the column and the resin added to a height of 12 cm, after allowing time for settling. A disk of Whatman No. 1 filter paper and a thin layer of glass wool were placed on top. The column was then washed as follows by gravity flow: (a) 250 ml 4 M F.A. in 1 hf An1.F.; (b) 20 ml concentrated F.A. (approx. 90%), followed by 100 ml water; (c) 250 ml 4 M Am.F. in 1 M F.A., followed by 100 ml water; (d) 250 ml 1 M Am.F. followed by 500 ml water. The total capacity of the column (packed resin volume = 9.4 ml) yas 11 meq. Preparation

of dcid Extracts

of Ery throcy tes

Freshly drawn ACD blood was centrifuged for 5 min at 3000 rpm and 4OC. After removing the plasma and buffy coat, the cells were suspended in cold 0.85% saline (10 vol), again centrifuged, and resuspended in cold saline to a hematocrit of either 25% for perchloric acid (PCA) or 50% for trichloroacctic acid (TCA) extraction. For direct comparison with the results of paper chromatography (8, 9), most of the samples studied by ion-exchange were PCA extracts. PCA Extract. The 25% washed cell suspensionwas mixed in an ice bath with an equal volume of 10% PCA and centrifuged for 15 min at 7200 rpm and 4%. The supernatant solution was filtered and neutralized (ice bath) with 40% KOH (Hydrion paper, pH 7-S). After centrifuging for 15 min at 7200 rpm and 4OC t,o remove insoluble KClO,, the clear solution was stored at -20°C until used. On thawing, additional insoluble KClO, which formed was removed by centrifuging. Each milliliter of the PCA extract represented 0.10 to 0.14 ml of packed RBC. A volume equivalent to 8 ml RBC was diluted to 250 ml for adsorption on the column. TCA Extract. The 50% washed cell suspension was cooled in ice and shaken vigorously for 15 set with 2 vol of cold 15% TCA. The clear filtrate was extracted 3 times with cold ethyl ether. After removing the

288

MORELL,

AYERS,

AND

GREENWALT

dissolved ether with a stream of nitrogen, 0.25 ml of 1 M sodium acetate was added per 10 ml of filtrate, which resulted in a pH of 5.0 2 0.2. The “RBC equivalent” of the extracts varied from 0.15 to 0.19 ml/ml filtrate. A volume equivalent to 8 ml RBC was diluted to 250 ml and adjusted to pH 7-8 for adsorption on the column. Adsorption

and El&ion

Adsorption was conducted at a flow rate of 0.7 ml/min, followed by washing with 100 ml of water at 0.9 ml/min. The 250-ml aliquots of diluted extract contained 150 + 20 pmoles of total P. Four principal components, ATP, ADP, Pi, and 2,3-DPG comprised over 75% of the total P. Preliminary tests had shown t,hat the column capacity was adequate for quantitative adsorption of the phosphates from both the PCA and the TCA extracts. The phosphates present comprised only 2 to 3% of this capacity. Resolution on subsequent elution, however, appeared to be influenced by the presence of perchlorate, which reduced the capacity of the column. A calculation of the solubility of KClO, at O°C showed that approximately 3 meq perchlorate, or about one third of the total capacity of the column, was present. Exchange of formate by perchlorate apparently occurred during adsorption, with a corresponding loss in resolving capacity. Elution rates were 0.4 to 0.6 ml/min, depending upon the gradient. Fractions were collected in lo-ml portions per tube. Preliminary studies resulted in the adoption of the schedule shown in Table 1. TABLE LINEAR Tube No. (IO ml/tube)

O-50

50-150 150-165” 165-265b

GRADIENT TAnar

-

Mixing

bottle

Water, 250 ml 0.1 M F.A., 500 ml

M F.A., 150 ml 0.1 M F.A., 500 ml 0.1

0.5

1 ELUTION

SCHEDULE

gradient

Reservoir

0.1 AT F.A., 0.1 M F.A., 500 ml

bottle

Rate (n&/tube)

250 ml 0.5 M Am.F.,

M Am.%‘.,

0.5 3-l Am.F.,

22 17 14

0.5 M F.A., 500 ml

2.0 M Am.F.,

6 Linear gradient interrupted at tube 150; nongradient to tube 165 (Figs. 3,5, or to tube 170 (Figs. 4 and 6). b For Figs. 3, 4, and 7; 170-270 for Figs. 5 and 6. Experiments were discontinued elution of GTP, which occurred at tube No. 215 for Figs. 3, 5, and 7 (0.3 M F.A., Am.F.); and at t,ube No. 240 for Figs. 4 and 6 (0.38 M F.A., 1.56 M Am.F.).

17

and 7), after 1.25 M

Analysis of Eluant Fractions

Optical densities at 250, 254, 260, and 280 rnp served for identification and quantitative analysis of the nucleotides in each lo-ml fraction. The

LINEAR

GRADIENT

ELUTION

OF

NUCLEOTIDES

289

spectral constants employed for the 5’-mono, -di, and -triphosphates of the nucleosides are shown in Table 2 (13, 14). Phosphate was determined TABLE SPECTRAL S'-Phosphates of

range

.idenosine Cytidine Uridiw Guanosine Inosine

2-7 2-4 2-7 4-i 3-i

CONSTANTS

USED

2

FOR ANALYSIS

(13,14)

OF ~TUCLEOTIDIB

PH

14.9 13.0 9 .9 11.7 10.5

260 280 260 260 25-l

0.81 0.45 0.73 1.16 1.69

0.95 0.63 0.88 1.16

0.20 2.11 0.40 0.67

1.47

0.24

by the method of Schaffer, Fong, and Kirk (15) either directly for Pi or after wet-ashing for total P (16). Ketose and aldose sugar phosphates were obtained by first determining ketoses with the specific carbazole reagent of Dische and Borenfreund (17) and then subtracting ketose from the total reducing values obtained in the anthrone test of Mokrasch (18). .40

8

.35

7

.30

6

c.25 0 -t 2 X.20 cn

5

PH 4

IO

20

Ml. FIG. 1. Decrease in molar to ammonium formate.

absorbancy

30

40

50

N H,OH at 250 rn+ during

titration

of formic acid

290

MORELL,

AYERS,

AND

GREENWALT

The method is similar to that employed by Bartlett (IO), who utilized rate differences in the anthrone test for assaying mixtures of aldoses and ketoses. 2,3-DPG was determined by analysis for tot.al P. Background

Ultraviolet

Absorption

As pointed out by Hurlbert et al. (2, 3), the background absorption of formic acid-ammonium formate solutions is due principally to the free acid. The decrease in absorption which occurs at 250 m,u during the titration of formic acid to ammonium formate is shown in Fig. 1. The molar absorbancy of the acid decreased from 0.35 to 0.04 on neutralization with NH,OH. Since crystalline ammonium formate exhibited only half this value (E,,, = OX?), background absorption was minimized by using the salt to prepare all of the eluant solutions. It is interesting to note that the absorbancy of the free acid drops rapidly with increasing wavelength: E 260 = 0.021, E,,, = 0.007. Background absorption during elution was determined at 250, 254, 260, and 280 mp., the spectrophotometer being adjusted to 100% transmission with distilled water. Table 3 shows the eluant absorption gradient and

AfP

.4 FIG.

gradient (x-x)

2.

.a LITERS

1.2 THROUGH

Background absorption for elution of AMP, system described (0-O) and comparison (2).

1.6 2.0 COLUMN ADP, and ATP by the linear with that of Hurlbert et al.

LINEAR

GRADIENT

ELUTION

TABLE BACRGR~UND

Tube No.

10 20 30 40 5oa 60 70 80 90 100

0.029

110 120 130 140 150"

0.041

160 165”

0 041

170 180

190 200 210 215d

0.127

Effluent

260

Ernuent

0.019

O.OOT

0.025 0.030 0.034 0.037 0.0-K:! 0.044 0.047 0.049 0.051 0.051 0.052 0.053 0 ,053 0.051 0.052 0.055 0.057 0.075 0.086 0.105

0.012

0.122 0.129

3

254 Inp

Eluant

Eluarlt

O.Oli

0.016 0.016

0.0‘46

0.018 0.020 0.019 0.024 0.024 0.025 0.035 0.02‘l 0.025 0.025 0.024 0.025 0.025 0.026 0 ,033 0.035 0.044 0 ,049 0.051

EFFLUEXT

III,, Effluent

280 m&d

Eluant

0.004

0.007 0.007

0.009

0.010 0.012 0.013 0.014 0.014 0.014 0.014 0.015 0.015 0.014 0.013 0.013 0.014 0.016

0.004

0.007 0.007

0.015 0.016 0.023

0.018 0.019

Effluent

0.006 0.007

0.009 0.010 0.008 0.008

0.014 0.010

291

NUCLEOTIDES

ABSORPTION FOR ELUANT AND (OPTICAL DENSITIES vs. WATER)

250 mp Elusnt

OF

0.013

0.008 0.008 0.008 0.008 0.007

0.010 0.012 0.009 0.009 0.009 0.010 0.010 0.010 0.010 0.009 0.010 0.010 0.010 0.010 0.014 0.011

a Linear gradient eluant to tube 50, water to 0.1 M HCOOH. b Linear gradient eluant for tubes 50-150,O.l M HCOOH to 0.1 M HCOOH in 0.5 M HCOONH,. c Nongradient eluant for tubes 150-165, 0.1 M HCOOH in 0.5 M HCOONH,. d Linear gradient eluant for tubes 165-215, 0.1 M HCOOH in 0.5 ih’ HCOONHa to 0.3 M HCOOH in 1.25 M HCOONHa.

the effluent values obtained at every tenth tube during a blank run through a 12 X 1 cm column prepared as described above. Even with the extensive washing procedure used in preparing the resin for ion-exchange, significant amounts of absorbing substances were removed during elution. Appropriate corrections for background absorption, based on Table 3, were routinely applied. In Fig. 2 the background eluant absorption for AMP, ADP, and ATP is compared with that of Hurlbert et nl. (2). RESULTS

The linear gradient elution of a synthetic mixture of nine purine nucleotides, the 5’-mono-, -di-, and -triphosphates of adenosine, inosine,

292

MORELL,

AYERS,

AND

GREENWALT

and guanosine, is shown in Fig. 3. An aqueous solution (250 ml) containing 3 pmoles of each sodium salt was adsorbed on the column. Seven peaks were obtained. The diphosphates and the triphosphates of adenosine and

i AMP

2

PH ELUAN

.4

W4P

.6

.a

GMP

1.0 LITERS

ADP+IDp

1.2 THROUGH

2.4 II

-.IM

FIG. 3. Linear Dowex-l-form&e.

.IM .5U

EA.

gradient

separation

I. 4 1.6 COLUMN

4.3 F.A. AMF.

Ii-7 --l.25M

IlP+ATP

GDP

I.0

GTP

2.0

4.3 .3 M F.A. AM.F.

of a mixture of nine purine-5’-nucleotides

2.2 +I +I

from

inosine were not separated. Ratio analysis, 250/260 rnp, established the sequences as ADP-IDP and ITP-ATP, respectively. Recoveries were practically quantitative, -t5a/, of theory for the individual nucleotides. Analysis of a TCA extract of normal adult erythrocytes is shown in Fig. 4. The elution positions for AMP, ADP, ATP, and GTP were identical to those obtained in Fig. 3. Orthophosphate, UMP, and IMP were eluted as a mixture. Two unidentified nucleotides were found, one each preceding AMP and ADP. Separation of 2,3-DPG and ATP was accomplished by interrupting the gradient at 0.1 M F.A.-0.5 M Am.F. a$ shown. Since 2,3-DPG is nonabsorbing, its elution position is indicated by an arrow. The results are summarized in Table 4.

1.4 -

I.2 ; g 1.0 z g

.0-

2 .6 -

.4-

.2 1

.2

.4

A

.s

.fJ

I

I .o LITERS

I.2

1.4

THROUGH

I.6

l.S

2.0

2.2

COLUMN

PH ELUAN

FIG.

5MAM.F.

4. Linear

gradient

elution

of TCA TABLE

ACID-SOLUBLE

PHOSPHATES PCA

Compound

/moles P/ 100 ml RBCc

AMP ADP ATP 2,3-DPG GTP UMP + IMP Unidentified nucleotides Pi

1.8 10.5 422.5 1020.2 20.5 6.7 11.7 96.0

Total

1619 .gd

eluted

extract

-

of normal

adult

erythrocytes

4 OF THE

ERYTHROCYTE

extract” ‘7, total &ted

P

0.1 2.5 26.1 63.0 1.3 0.4 0.7 5.9

4.5 55.4 393.3 1103.3 47.0 11.5 9.7 92.2

0.3 3.2 22.9 64.3 2.7 0.7 0.6 5.3

100.0

1716.9’

100.0

Q Pooled extracts of erythrocytes of six normal adults; duplicate columns agreed within f 5 ‘%. b Extract of erythrocytes of one normal adult. c 2,3-DPG, Pi, and total P by orthophosphate analysis: pmoles culated from optica densities of Figs. 4 and 6. d Recovery of total P in the extract (1617 pmoles/lOO ml RBC) e Recovery of total P in the extract (1898 pmoles/lOO ml RBC) 293

analysis

on separate

P for nucleotides was 100.2$&. was 90.5%.

cal-

294

MORELL,

AYERS,

AND

GREENWALT

Separation of another synthetic mixture, a PCA filtrate of ten reference compounds, is shown in Fig. 5. The ATP and 2,3-DPG content simulated

1.6 1

CUP

AMP

&lMPGMP

F-1.6-P

ADP

2.3 DPG

ATP GTP

1.4 -

.6 -

.4 -

.2 A

J

I

I

I

1

.2

.4

.6

.6

1.0 LITERS

2.4

PH

ELUAN

II

FIG.

mixture extracts.

-.I

M F.A.

I I.2 THROUGH

I

1.4 COLUMN 4.3 .IM F.A. .5M AM.F.

5. Linear gradient separation of a mixture was treated with PCA and neutralized

I

I

1.6

I.6

-.JM 1.25U

of ten reference as described for

I 20

22

4.3 F.A. AU.E

compounds. The the erythrocyte

that of an acid extract of erythrocytes. The mixture contained 3 pmoles each of AMP, CMP, GMP, IMP, UMP, and GTP: 4 each of ADP and F-1,6-P; 10 of ATP; and 40 of 2,3-DPG. After treatment with PCA and neutralization with KOH, the solution was chromatographed as described for the erythrocyte extracts. Recoveries were practically quantitative, &5% for the individual compounds. The elution positions of F-1,6-P and 2,3-DPG, both nonabsorbing, are indicated by arrows. The separation of the purine monophosphates, IMP and GMP, was less complete than found in Fig. 3, where PCA was not used. Since 2.2% of the total P was converted to orthophosphate, the nucleotides were stable during the entire analytical procedure.

LINEAR

GRADIENT

ELUTION

OF

KUCLEOTIDES

295

Analysis of a PCA extract of normal adult erythrocytes is shown in Fig. 6. The elution positions of AMP, Pi, UMP, IMP, ADP, 2,3-DPG,

x

Pi WP+IMP

MAP

x

w-c%

ADP

GTP

1

\1 1.6 -

1.4 -

3 E

I.2 -

z N l.Os c iii 5 .60 .6 -

2

.4

.6

.6

I.0 LITERS

I.2 THROUGH

2.4

PH

ELUAN

FIG.

-

.I M EA.+

6. Linear

1.4 COLUMN 4.3

.IM F.A. .5MAM.F.

1

gradient

elution

of PCA

extract

I.6

1.6

LO

b 3 -

of normal

1

adult

.38M I. 56M

2.2 4.3 +/ F.A. AM. F. -I

erythrocytes.

ATP, and GTP coincided with the reference compounds of Fig. 5. In comparison with the TCA extract (Fig. 4), an unidentified nucleotide mixture was found where DPN and CMP had appeared. Unidentified nucleotides preceding AMP and ADP were again found. As shown in Table 4, good agreement was obtained for the principal components of the PCA and TCA extracts of the erythrocytes. A schematic summary of the linear gradient elution pattern obtained for t.he acid-soluble phosphates of the erythrocyte is shown in Fig. 7. DISCUSSION

One of the principal advantages of linear gradient elution, as compared with extended gradient procedures, is the relative ease with which pub-

296

; .I

MORELL,

L/”

t

I

I

I

.2

4

AYERS,

AND

0.5;

--/A-’

HCOOH

6

GREENWALT

.6

1

I

I

I

I

I

I

I.0

1.2

1.4

1.6

1.8

LO

linear

gradient

.

I

FIG. 7. Schematic summary of Dowex-1-formate for acid-soluble phosphates of the erythrocyte.

elution

positions

lished data can be extrapolated to solve a particular problem. Bartlett (lo), for example, presented a schematic summary of the effect of pH and formate concentration (1 to 4 M) on the linear gradient elution positions of various glycolytic intermediates, including 2,3-DPG and ATP. Application of his data to the low formate concentrations of Bergkvist and Deutsch (7) indicated that, at pH 4.3, interruption of the gradient would probably be required to facilitate a difficult separation. When the gradient was interrupted (150 ml at 0.1 M F.A.-O.5 M Am.F.) separation of 2,3DPG and ATP was readily achieved. In chromatographing acid-soluble phosphates on Dowex-1-formate, consideration should be given to the type of acid being employed for deproteinization and the relative ease with which it can be removed prior to adsorption. For example, removal of trichloroacetic acid by extraction with ether was found to be more effective than separation of perchlorate by neutralization with KOH. Exchange of perchlorate for formate during adsorption had apparently resulted in a sufficiently reduced column capacity to account for the more complete separations of nucleotides by the TCA extraction procedure. It is of interest to note that, in the linear gradient procedure described here, where the pH varies from 2.4 to 4.3, the relative positions within groups of phosphates change as follows during elution: C-A-U-I-G, C-U-A-I-G, and C-U-I-A-G for the ?-mono-, -di-, and -triphosphates,

LINEAR

GRADIENT

ELUTION

OF

297

NUCLEOTIDES

respectively. In the systems described by Hurlbert et al. (2), the elution positions remain constant: C-A-G-U for LLformic acid,” pH 1.5-3.0; and C-U-A-G for “ammonium formate,” pH 3.0-5.0. SUMMARY

A single-column linear gradient elution procedure for separating ribonucleotides and sugar phosphates on Dowex-1-formate is presented. In the pH range 2.44.3, effective separations of mono-, di-, and triphosphates were obtained at relatively low concentrations of formate. Application of the method to the erythrocyte is described. The advantages of linear gradient elution, as compared with extended gradient procedures, are illustrated. ACKNOWLEDGMENTS This investigation was supported by Grant RG-5243 from the National Institutes of Health, U. S. P. H. S. The authors thank Dr. V. R. Potter, McArdle Memorial Laboratory, University of Wisconsin, and Mr. David Reitz, Pabst Laboratories, for many helpful suggestions regarding the separation of nucleotides from Dowex-lformate. REFERENCES 1. COHN, W. E., AND CARTER, C. E., J. Am. 2. HURLBERT, R. B., SCHMITZ, H., BRUMM,

Chem. Sot. 72.4273 (1950). A. F., AND POTTER, V. R., J.

Biol. Chem.

209, 23 (1954). 3. HURLBERT,

R. B., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 793. Academic Press, New York, 1957. 4. WILLOUGHBY, H. W., AND WAISMAN, H. A., Cancer Research 17. 942 (1957). 5. VANDERHEIDEN, B. S., HUENNERENS, F. M., AND GABRIO, B. W., Federation Proc.

17, 327 (1958). 6. MILLS, G. C., AND SUMMERS, 7. BERGKVIST, R., AND DEUTSCH, 8. GREENWALT,

9. GREENWALT,

L. B., Arch. Biochem. Biophys. 84,7 (1959). A., Acta Chem. Stand. 8.1877 (1954). T. J., AND AYERS, V. E., Blood 15,698 (1960). T. J., AYERS, V. E., AND MORELL, S. A., J. Lab. Clin. Med.

(1959). 10. BARTLETT, G. R., J. Biol. Chem. 234, 459 (1959). Il. BOCK, R. M., .&ND LING, N. S., Anal. Chem. 26, 1543 (1954). 12. HERBERT, E., AND POTTER, V. R., J. i?ioZ. Chem. 222,455 (1955). 13. BOCK, R. M., LING, N. S., MORELL, S. A., AND LIPTON, S. H.,

54, 820

Arch. Biochem.

Biophys. 62, 253 (1956). 14. Circulars No. OR-IO, -15, Pabst Laboratories, Milwaukee, Wisconsin, 1956, 1959. 15. SCHAFFER, F. L., FONG, J., AND KIRK, P. L., Anal. Chem. 25, 343 (1953). 16. LEPAGE, G. A., in “Manometric Techniques” (W. W. Umbreit, R. H. Burris, and J. F. Stauffer, eds.), 3rd ed., p. 273. Burgess Publishing Co., Minneapolis, 1959. 17. DISCHE, Z., AND BORENFREUND, E., J. Biol. Chem. 192. 583 (1951). 18. MOKRASCH, L. C., J. BioZ. Chem. 208. 55 (1954).