TRANSAMINASEACTIVITY IN HUMANBLOOD

By ARTHURKARMEN,FELIX WROBLEWSKI,AND JOHN S. LADUE

(From the Sloan-Kettering Institute, Department of Medicine, Memorial Center,New York City, N. Y.)

(Submitted for publication April 3, 1954; accepted July 15, 1954)

Enzymatic transamination consists of the en-zyme catalyzed reversible transfer of the alphaamino nitrogen of an amino acid to an alpha-ketoacid with the synthesis of a second amino acid anda second alpha-keto acid. Enzymes catalyzingdifferent transamination reactions are found widelydistributed in animal tissues and have been shownto change in activity in some tissues during dis-ease (1-3). These observations prompted thepresent study to determine if transaminase ac-tivity could be demonstrated in human serum andblood cellular elements and, if so, to study anyvariations in activity of this enzyme in the bloodof normal and diseased man.

METHODSAND MATERIALS

The two transaminases found most active in animaltissues are:

1. "Glutamic-oxalacetic transaminase"Aspartate + alpha-keto glutarate = glutamate +

oxalacetate2. "Glutamic-pyruvic transaminase"

Alanine + alpha-keto glutarate = glutamate + py-ruvate

When aspartate or alanine are incubated with alpha-keto glutarate and a source of enzyme, the rate of pro-duction of glutamate may be taken as a measure oftransaminase activity. The amount of glutamate pro-duced after a given incubation period under standardizedconditions was measured by quantitative paper chromato-graphic analysis (4).

One-tenth molar solutions of I-aspartate, I-alanine, andalpha-keto glutarate were prepared in 0.06 M phosphatebuffer and the pH of the solutions adjusted to pH 7.6.For serum transaminase determinations, 0.5 ml. of clear,non-hemolyzed serum, 1.5 ml. of 0.06 M phosphate buf-fer, pH 7.6, and 0.5 ml. of either the alanine or aspartatesolutions were incubated for ten minutes at 37°C. Atthis time, 0.5 ml. of the alpha-keto glutarate solution wasadded and the incubation continued for 18 hours. Forwhole blood hemolysate transaminase determinations,equal volumes of blood and distilled water were shakentogether for ten minutes, 1.0 ml. of the hemolysate wasadded to 1.0 ml. of the phosphate buffer, and the sub-strates added as above. The time of incubation of thehemolysate substrate mixture was three hours.

At the end of the incubation period, proteins were sepa-rated by adding 7.0 ml. of absolute ethyl alcohol, centri-fuging for ten minutes, and washing the precipitate with5 ml. of 70 per cent ethanol. The supernatant was evap-orated to dryness over a water bath and the residue dis-solved in 1.0 ml. of 0.06 Mphosphate buffer. Aliquots of0.05 ml. were then applied to Whatman No. 1 filter paperand chromatographed by the descending method for 18hours, using phenol saturated with water as solvent andwater saturated with phenol to saturate the atmosphere ofthe tank. The papers were then removed and dried in airat room temperature (5). The position of the aminoacids was located by spraying the paper with a 0.1per cent solution of ninhydrin in butanol and heatinggently with an infra-red lamp.

The areas of paper corresponding to glutamate werecut out, rolled, and placed in test tubes. Elution of theamino acid from the paper and quantitative color de-velopment with ninhydrin were performed in one opera-tion by adding the reagents and treating the paper asdescribed in the procedure of Troll and Cannan (6).Areas of paper containing standard amounts of gluta-mate were analyzed concomitantly. Papers correspond-ing to incubation mixtures containing 0.5 ml. of serum,or 1.0 ml. of hemolysate were used as blanks.

RESULTS

Reliability of the method

No loss of glutamate was encountered in theincubation period with serum or in the process ofpaper chromatography. Satisfactory recovery ofknown quantities of glutamate added to serum andincubated for 18 hours, or applied to paper di-rectly, was obtained (Table I).

Presence and properties of the enzyme in serum

Incubation of aspartate and alpha-keto glutarateor alanine and alpha-keto glutarate, without addi-tion of serum, failed to form detectable quanti-ties of glutamate after 18 hours of incubation. In-cubation of 0.5 ml. of serum and any one of thesubstrates singly, similarly showed no formationof measurable glutamate. Thus, non-enzymatictransamination was inferred not to occur underthe conditions of these experiments, and concen-

126

TRANSAMINASEACTIVITY IN HUMANBLOOD

TABLE I

Recopery of added glutamate

Micromoles Micromoles %Aliquot added recovered Recovery

A. From paper after phenol chromatography

1. 0.405 0.410 101.12. 0.405 0.405 100.03. 0.405 0.415 102.34. 0.405 0.424 104.7

B. From serum after eighteen-hour incubation

1. 8.00 8.10 101.12. 8.00 8.57 107.03. 8.00 7.60 95.0

tration of alpha-keto glutarate and amino groupdonors in serum and hemolysates was inferred tobe negligible. Incubation of 0.5 ml. of serum withaspartate and alpha-keto glutarate resulted in theformation of from 4.4 to 15.0 micromoles of glu-tamate in 18 hours, depending on the serum sam-ple tested.

The amount of glutamate produced was foundto be directly proportional to the time of. incubationwhen identical mixtures of serum, buffer, and sub-strates were incubated for varied, intervals of time(Figures 1 and 2). The rate of production of

9

8

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"0 3 6 9 12 15 18 21HOURSOF INCUBATION

FIG. 2. RATE OF GLUTAMATzPRODUCTONVia TRANs-AMINATION (WHoLz BLOD H SAMOLYATE MNM SnuUCOMPAxD)

glutamate was seen to be directly proportional tothe quantity of serum, when 0.5 ml. and 1.0 ml.samples of the same sera were incubated withidentical substrate mixtures (Table II).

Pyridoxal phosphate has been shown to act ascoenzyme in transamination reactions and O'Kaneand Gunsalus have determined the coenzyme satu-rated curve (7). Addition of a buffered solutionof pyridoxal phosphate in a concentration of tenmicrograms per ml. was found to have no meas-urable effect on the transaminase activity of theserum. Addition of a boiled and filtered extractof rat liver, used as source of possible activators,

TABLE I}

The effect of enzyme concentration on rate of transaminationincubation period-twenty hours

Micromolesglutamateproduced

Sample by 0.3 ml.

1. 5.62. 4.93. 6.64. 6.45. 5.96. 5.2

Ratemicromokcs/

m1.11w.0.560.490.660.640.590.52

Micromolesglutamateproducedby1.0 ml.

10.49.7

, 11.011.712.3

9.8

Ratemia'ois/

mnl./fr.0.520.480.550.590.610.49

HOURSOF INCUBATION

FIG. 1. TIME RATE OF GLUTAMATEPRODUCTION ViaTRANSAMINATION (Two 0.5 ML. SERUMSAMPLES)

127

ARTHURKARMEN, FELIX WR6BLSWSKI, AND JOHN S. LADUE

was similarly found not to affect the observedtransaminase activity of serum.

Increased concentration of aspartate in a givenserum incubation mixture was seen to cause agreater increase in the observed rate of glutamateproduction than an increase in the concentrationof alpha-keto glutarate, demonstrating that com-plete saturation of the enzyme with substrate hadnot been achieved at these concentrations. Theseresults are in essential agreement with those re-ported for transaminase preparations from pigheart muscle (7, 8).

The effect of pH on serum transaminase ac-tivity was studied by altering the composition ofthe buffer used. Phosphate buffer, 0.2 M, ofseveral pH values was substituted for the 0.06 Mbuffer. The pH of each incubation mixture wasdetermined before and after incubation. An in-crease in pH from 0.1 to 0.2 pH units was ob-served in each sample after the incubation periodand the average of the pre- and post-incubationvalues was taken as the pH of the mixture. Thefinding of maximal activity between pH 7.0 and8.0 (Figure 3) is in essential agreement with theresults of Cohen (9) and others using pig heartmuscle as source of transaminase.

No change in transaminase activity with timewas noted in serum samples stored from ten min-utes to 96 hours at room temperature, or for pe-riods of from one hour to two weeks in the re-frigerator (O to 5°C.). The transaminase activitywas not changed by freezing or lyophilization of

0.70

0-60

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0.0

2 0.20

O _- 5.0 6.0 7.0 8.0 9.0 10.0

pH UNITS

FIG. 3. EFFECT OF VARYING HYDROGENION CONCENTRA-TIONS ON SERUMTRANSAMINASEAcTnTY

TABLE III

Distribution of transaminase acivity in serum ofhealthy adults

Aspartate- Alanine-Trans- alpha-keto alpha-keto

aminase glutarate glutarateactivity

micromolesl No. of No. ofml./hr. samples % samples %

0.20-0.39 0 0 2 5.10.40-0.49 7 7.8 11 28.20.50-0.59 22 25.0 16 41.10.60-0.69 24 27.2 5 12.50.70-0.79 15 17.1 2 5.10.80-0.89 5 5.7 1 2.50.90-0.99 9 10.2 1 2.51.00-1.09 5 5.7 1 2.51.36 1 1.1

Total numbertested 88 100 39 100

Mean transaminase Aspartate- 0.622i0.191 Std.activity alpha-keto deviation

micromoles/ml./hr. glutarate

Alanine- 0.525 d0. 146 Std.alpha-keto deviationglutarate

the serum. No change in activity was noted insera subjected to 560 C. for 25 minutes. Seraheated to 1000C. for ten minutes were found tohave a decrease in activity to 10 per cent of theoriginal transaminase activity.

No difference could be detected between trans-aminase activity in serum and in plasma from thesame donors by using oxalate, citrate, or heparinas anticoagulants.

Transaminase activity in the blood of normalhumans

The serum glutamic oxalacetic transaminaseactivity in 88 normal humans varied from 0.41 to1.36 micromoles per ml. per hour with a meanactivity of 0.622 + 0.191 standard deviation. Se-rum glutamic pyruvic transaminase activity in 39samples was found to be between 0.21 and 1.01micromoles per ml. per hr. with a mean value of0.525 + 0.146 (Table III). The glutamic oxal-acetic transaminase activity found in hemolysatesranged from 5.0 to 8.7 micromoles per ml. per hr.with a mean value of 6.86 ± 0.78 while the glu-tamic pyruvic transaminase in hemolysates variedfrom 1.6 to 3.3 micromoles per ml. per hr. with amean value of 2.48 ± 0.36 (Table IV).

In each of 29 hemolysate samples, the ratio of

128

TRANSAMINASEACTIVITY IN HUMANBLOOD

TABLE IV

Distribution of transaminase activity in whole bloodhemolysates of healthy adults

Aspartate-alpha-keto Alanine-alpha-ketoglutarate glutarate

Trans- Trans-aminase aminaseactivity activity

micromokesl No. of miromoksl No. ofml./hr. samples % ml./hr. samples %

5.0-5.9 3 10.3 1.5-1.9 3 10.36.0-6.9 12 41.4 2.0-2.5 12 41.47.0-7.9 12 41.4 2.5-3.0 1 1 38.08.0-8.9 2 6.9 3.0-3.3 3 10.3

Total No. 29 100.0 Total No. 29 100.0

Mean transaminase Aspartate- 6.8640.78 Std.activity alpha-keto deviation

micromoles/ml./hr. glutarate

Alanine- 2.4840.36 Std.alpha-keto deviationglutarate

glutamic oxalacetic transaminase activity to glu-tamic pyruvic transaminase activity varied be-tween 2.04 and 3.60 micromoles per ml. per hr.with a mean value of 2.70 0.40. In 39 deter-minations of serum transaminase activity, thisratio was found to be 0.725 to 1.67 micromolesper ml. per hr. with a mean of 1.15 + 0.23. Noconsistent relationship was noted between indi-vidual serum and corresponding hemolysate trans-aminase activities.

In no instance was transaminase activity absentin the sera of the normal humans tested or in any

of the sera of hospitalized patients with variousdisease states tested. Increased activity was foundin the sera of one patient with lymphomatous dis-ease, one with extensive rhabdomyosarcoma, twowith acute leukemia, one with acute hepatitis, andtwo patients with arteriosclerotic heart diseaseand recent myocardial infarction. Serum glu-

TABLE V

Transaminase activity in blood of hospitalized patients in micromoles per ml. per hr.

Clinical diagnosis

Carcinoma of lungCarcinoma of lungCarcinoma of palateCarcinoma of eyelidCarcinoma of breastFibromyoma of uterusLymphomaLymphomaLymphomaLymphomaLymphomaLymphomaLymphomaLymphomaRhabdomyosarcomaRhabdomyosarcomaChronic pulmonary tuberculosisChronic pulmonary tuberculosisAcute leukemiaAcute leukemiaAcute leukemiaAcute hepatitisDiabetes mellitusPortal cirrhosisPortal cirrhosisGeneralized arteriosclerosisCerebral hemorrhageArteriosclerotic heart disease with CHFASHDwith anginal syndromeASHDwith myocardial infarction-

see Figure 5

Serum tranamnsglutamic

Oxalacetic Pyruvic

0.54 0.410.51 0.380.89 0.700.75 0.470.80 0.730.81 0.590.46 0.510.71 0.470.52 0.390.74 0.531.06 0.700.36 0.320.47 0.370.71 0.481.70 1.600.40 0.350.92 1.240.58 0.441.28 1.510.36 0.551.72 1.251.97 1.750.46 0.350.99 1.280.49 0.380.50 0.630.85 0.411.16 0.810.95 1.25

Hemolysate tranmaminsglutamic

Oxalacetic Pyruvic

8.86 2.071.73 2.546.25 2.608.05 1.80

8.75 2.7311.3 2.70

7.85 1.566.45 2.536.80 1.666.35 2.007.00 1.60

9.137.267.208.955.265.207.957.208.468.703.106.139.405.008.65

7.462.003.601.603.401.603.73

10.42.733.501.40

3.263.402.203.27

Average normal value 0.62 0.52 6.86 2.48Normal range* 0.24-1.04 0.23-0.82 5.30-8.42 1.76-3.20

* Normal range represents the normal mean value plus or minus two standard deviations.

129

ARTHURKARMEN, FEELIX WR6BLEWSKI, AND JOHN S. LADUE

rtote-AlphoketoglutoroteSystem

Q., p

4. ~b\;:~ Mean -

cud Value ofNormals

5 10 15 20DAYS AFTER ADMISSION

Alanine-AI phoketoglutorateSystem

-~~~p0T dCyi

IQI(/)

MeanValue ofNormals

5 10 15 20DAYS AFTER ADMISSION

FIG. 4. SERuMTRANSAMINASEACTIVITY IN PATIENTS WITH ADMISSIONDIAGNOSIS OF MYOCARDIALINFARCTION

tamic oxalacetic transaminase activity and serum

glutamic pyruvic transaminase activity was seen

to vary together in most cases of marked depar-ture from the normal range. Somewhat greatervariation was found in the transaminase activityof the hemolysates from the same patients (TableV).

Figure 4 represents serial determinations ofserum transaminase activity in five patients ad-mitted to the hospital with the admitting diagno-sis of acute myocardial infarction at various timesafter the onset of acute infarction. Of the five pa-

tients shown, the clinical picture subsequent to ad-mission substantiated the diagnosis of transmuralmyocardial infarction in Nos. 1, 2, 4, and 5. Ele-vated values were found in patients Nos. 1 and 5,the only patients with acute infarctions studiedduring the first week after infarction. The find-ing of 2.02 units in patient No. 1 represents thehighest value found in any of the samples testedand almost twice the value taken as the upper

limit of normal.

DISCUSSION

The method of transaminase assay by quanti-tative paper chromatography of the glutamateproduced was chosen because of its sensitivity andthe simplicity of the equipment required. Othermethods of measuring transamination reactionsmay be divided into two main types: Those utiliz-ing either specific chemical or enzymatic decar-boxylation of one of the products and subsequentmanometric measurement of carbon dioxideevolved and those utilizing the high ultraviolet ab-sorption of oxalacetate at wavelength 280 milli-micra to follow the glutamic oxalacetic transami-nase reaction by measuring a change in opticaldensity as oxalacetate is produced or consumed.The high bicarbonate content of blood togetherwith low transaminase activity makes a methoddepending on carbon dioxide evolution difficultto ap,ply to seru. Low transaminase activity andhigh protein content together with the known in-stability of oxalacetate make the usual spectro-photometric assay difficult to apply to serum.

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130

TRANSAMINASEACTIVITY IN HUMANBLOOD

The results demonstrated the enzymatic natureof the observed transamination and suggest thatserum glutamic oxalacetic transaminase has simi-lar chemical characteristics to transaminase de-rived from other sources. The failure of pyridoxalphosphate or boiled liver extract to increase themeasured transaminase activity may be taken asevidence that the enzyme was fully activated inthe serum samples tested. The difference in com-parative amounts of the two transaminases in se-rum and hemolysates could represent a differencein the rates of diffusion of the two enzymes acrossthe cellular membranes of the blood cells, or, asseems more likely, this difference could be takenas suggestive evidence for a source of the serumenzymes apart from the blood cells.

The sera of patients with acute myocardial in-farction were thought worthy of particular atten-tion because of the possibility that destruction ofcardiac muscle, reported rich in transaminase ac-tivity, might result in a release of this enzyme intothe blood stream and might thus increase the se-rum transaminase activity. The finding of in-creased serum transaminase activity in the twopatients studied during the first week after infarc-tion is compatible with such a possibility.

SUMMARY

1. The presence of glutamic oxalacetic and glu-tamic pyruvic transaminase activity in human se-rum, plasma, and whole blood hemolysates has

been demonstrated. A method is presented fortheir measurement by estimating the rate of gluta-mate formation employing quantitative paperchromatography.

2. The chemical properties of the enzyme in se-rum, including the variation in activity withchanges in concentration of enzyme, substrate, andhydrogen ion were found similar to those re-ported for transaminases in animal tissues.Freezing or lyophilizing the serum, or storing itat 0 to 5°C. for periods up to two weeks resultedin no significant change in transaminase activity.Boiling destroyed the activity.

3. Glutamic oxalacetic and glutamic pyruvictransaminase activities were found to be approxi-mately equal in normal human serum. Wholeblood hemolysates were found to have ten timesas much glutamic oxalacetic transaminase activityas serum. Glutamic oxalacetic transaminase wasfound to be approximately 2.7 times as active asglutamic pyruvic transaminase in hemolysates.

4. A survey of transaminase activity in theblood of hospitalized patients revealed departurefrom the normal range of activity in several dis-ease states.

ACKNOWLEDGMENT

Wewish to acknowledge gratefully the interest, adviceand assistance of Drs. Aaron Bendich, Oscar Bodansky,Severo Ochoa, and Rulon Rawson.

APPENDIX

A NOTEONTHE SPECTROPHOTOMETRICASSAYOF GLUTAMIC-OXALACETICTRANSAMINASEIN HUMANBLOOD

SERUM

By ARTHURKARMEN

(Department of Pharmacology, New York University College of Medicine)

A spectrophotometric method was devised inwhich the transamination reaction (Reaction 1)is coupled to the reduction of oxalacetate to mal-ate by reduced diphosphopyridine nucleotide(DPNH), in the presence of an excess of puri-fied malic dehydrogenase (Reaction 2). The oxi-dation of DPNH, and therefore the transamina-tion reaction, is followed by measuring the- de-

crease in light absorption at wave length 340 mu,at which the reduced pyridine nucleotides have anabsorption peak.

(1) a-Keto glutarate + aspartate t L-gluta-mate + oxalacetate

(2) Oxalacetate + DPNH+ H+ ;± L-malate+ DPN+

131

ARTHURKARMEN, FELIX WR6BLEWSKI, AND JOHN S. LADUE

METHODS

Materials. Aspartic acid, alpha-keto glutaric acid andreduced diphosphopyridinenucleotide were obtained com-

mercially. Purified malic dehydrogenase, prepared frompig heart muscle by the method of Straub (10), was

tested and found free of detectable transaminase or glu-tamic dehydrogenase activity.'

Expermental procedure. From 0.1 to 1.0 ml. of serum,

1.0 ml. of 0.1 Mphosphate buffer, pH 7.4, 0.5 ml. of 0.2 Maspartate in buffer, pH 7.4, 0.2 ml. of DPNH(1 mngm.per ml.) and 0.1 ml. of a solution of purified malic de-hydrogenase (50 micrograms of enzyme protein per ml.)were mixed and brought to a final volume of 2.8 ml. in a

cuvette having a 1.0 cm. light path. The blank containedall reactants listed except DPNH. After 10 minutes,0.2 ml. of 0.1 M alpha-keto glutarate in buffer, pH 7.4,was added. The optical density at wavelength 340 mAwas followed for five minutes, and the rate of decrease of

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SERUMMIXTURE

OKETOGWTARATEADDED

DIALYZED

SERUMMIXTURE

+P"

.KETOGLUTARATEADDED

STANDARDASSAY

EFFECT OF DIALYSISOF SERUM

5 10 15 20TIME IN MINUTES

FIG. 5. SPECTROPHOTOMETRICASSAY OF SERUMTRANSAMINASE

Top and bottom curves, all components except a-ketoglutarate present initially; a-keto glutarate added attime indicated by arrows. Middle curve: all compo-nents present initially, additional DPNHat time indicatedby arrow. DPNHadded at zero time in all cases. Theincrease in optical density when a-keto glutarate is addedis due to the absorption of light by a-keto glutarate at thiswavelength.

1 I am indebted to Dr. Martin Schwartz for thispreparation.

27r

24~

21

1 3

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01

F 25

2 21

z

z 18

E m 12

5 1 I5 2 A 30 35 40O45 50 - J10o0MICROMOLESOF ASPARTATE IN 3.0 ML.

50 MICROMOLESASPARTATE PRESENT

MICROMOLESOF o(KETOGLUTARATE IN 3.0 ML.

FIG. 6. SERUMTRANSAMINASEACrIVITY AS A FUNCTIONOF SUBSTRATECONCENTRATION

optical density taken as the measure of the transaminaseactivity of the serum. The reaction was followed in a

Beckman model DU spectrophotometer at room temper-ature. The activity is expressed as units per ml. of se-

rum per minute. One unit equals a ecrease in opticaldensity of 0.001 under the conditions desb4,bed.

Comments on procedure. When DPNHis added toserum without addition of substrates the optical densityof the mixture decreases for six to seven minutes, indi-cating the oxidation of a finite quantity of DPNH. Afterthis reaction has stopped, completion of the transamina-tion system results in a steady decrease in optical densitywhich is taken as a measure of the transaminase activityof the serum (Figure 5, top curve).

The rate or amount of DPNHoxidation by serum

alone is not changed by the addition of malic dehydro-genase and either aspartate or alpha-keto glutarate. Theamount oxidized is not appreciably affected by addingmore DPNH (Figure 5, middle curve). Addition of0.2 ml. of a solution of potassium pyruvate (4 mgm. perml.), after this reaction has stopped, results in completeoxidation of the DPNHpresent at a measurable rate,demonstrating the presence of lactic dehydrogenase inserum. Dialysis of the serum against 0.1 M phosphatebuffer reduces the amount of DPNHoxidized withoutchanging the measurable transaminase activity of theserum (cf. Figure 5, top and bottom curves).

132

40 MICROMOLESOCKETOGLUTARATE

PRESENT

I a I I I I I I I a e It11

TRANSAMINASEACTIVITY IN HUMANBLOOD

It is, therefore, suggested that the oxidation of DPNHby serum alone depends on the presence of pyruvate andlactic dehydrogenase in serum. The reaction stops whenall the pyruvate is enzymatically reduced to lactate byDPNH.

Omitting malic dehydrogenase from otherwise com-plete transamination reaction mixtures resulted in a vari-able decrease in the observed rate of DPNHoxidation.That the reaction was still measurable was taken as sug-gestive evidence either for the presence of malic dehydro-genase in serum or for the decarboxylation of oxalacetateto pyruvate and subsequent DPNHoxidation by lacticdehydrogenase. Addition of more malic dehydrogenaseto any serum transaminase assay resulted in no furtherincrease in the measured rate of reaction, indicating thatthe enzyme was present in excess in the assay mixture.

RESULTS

Study of the variation of serum transaminaseactivity with substrate concentration indicated thatmaximal activity was obtained when the three ml.reaction mixture contained more than 50 micro-moles of aspartate and 5 micromoles of alpha-ketoglutarate (Figure 6). One hundred micromolesof aspartate and 20 micromoles of alpha-keto gluta-rate were used in subsequent determinations.

Proportionality of the observed rate of reactionto the amount of serum present was observed overa wide range of serum transaminase activities(Figure 7).

Transaminase activity was measured in the seraof 50 normal humans in addition to those deter-

MILLILITERS OF SERUM

FIG. 7. TRANSAMINASEAcTIvrY AS A FUNCTION OF

SERum CONCENTRATiONThree different serum samples.

mined by the chromatographic assay method.The values found ranged from a low of nine to ahigh of thirty-two units per ml. per minute witha mean value of 19.6. Conversion of these unitsto micromoles per ml. per hour, using the extinc-tion coefficient for DPNHdetermined by Horeckerand Kornberg (11) gives a mean value of 0.57,which falls within the range of 0.24 to 1.04 micro-moles per ml. per hour found by the quantitativepaper chromatographic assay.

ACKNOWLEDGMENT

The author wishes to express his appreciation to Dr.Severo Ochoa for invaluable help in performing thiswork.

REFERENCES

1. Awapara, J., Effect of protein depletion on the trans-aminating activities of some rat organs. J. Biol.Chem., 1953, 200, 537.

2. Cohen, P. P., Hekhuis, G. L., and Sober, E. K,Transamination in liver from rats fed butter yel-low. Cancer Research, 1942, 2, 405.

3. Braunstein, A. E., Transamination and the integra-tive functions of the dicarboxylic acids in nitrogenmetabolism. Adv. Protein Chem., 1947, 3, 11.

4. Awapara, J., and Seale, B., Distribution of transami-nases in rat organs. J. Biol. Chem., 1952, 194,497.

5. Brush, M. K., Boutwell, R. K., Barton, A. D., andHeidelberger, C., Destruction of amino acids dur-ing filter paper chromatography. Science, 1951,113, 4.

6. Troll, W., and Cannan, R. K., A modified photometricninhydrin method for the analysis of amino andimino acids. J. Biol. Chem., 1953, 200, 803.

7. O'Kane, D., and Gunsalus, I. C., The resolution andpurification of glutamic-aspartic transaminase. J.Biol. Chem., 1947, 170, 425.

8. Nisonoff, A., Henry, S. S., and Barnes, F. W., Jr.,Mechanisms in enzymatic transamination: Vari-ables in the spectrophotometric estimation of glu-tamic-aspartate kinetics. J. Biol. Chem., 1952, 199,699.

9. Cohen, P. P., Kinetics of transaminase activity. J.Biol. Chem., 1940, 136, 585.

10. Straub, F. B., Reinigung der Apfelsiuredehydrase unddie Bedeutung der Zellstruktur in der Apfels&ure-dehydrierung. Ztschr. f. physiol. Chem., 1942, 275,63.

11. Horecker, B. L., and Kornberg, A., The extinctioncoefficients of the reduced band of pyridine nucleo-tides. J. Biol. Chem., 1948, 175, 385.

133