DETERMINATION OF THE EQUleBRIUM CGNSTANT FOR AN ENZYME CATALYZED REACTION Thesis for the Degree of M. S. MICHiGAN STATE UNIVERSITY KAREN E. DeFAZlO 1968 WWW!fil’lfll’kfllWflflWllfl'flflflfilflfil we- 3 1293 00676 7838 JW’U’i 1332 11:1 ABSTRACT DETERMINATION OF THE EQUILIBRIUM CONSTANT FOR AN ENZYME CATALYZED REACTION by Karen E. DeFazio The study of energy transformationsixlliving organ- isms has become an integral part of understanding the nature of many enzymatically catalyzed reactions. In isolation, these reactions will reach a point of equilibrium at which no further net chemical change takes place. This equilib~ rium is eXpressed by a thermodynamic constant. The equilibrium constant for a reaction involving the crystalline enzyme, uridine diphOSphate glucose pyrophOSphory- lase, was determined. This enzyme catalyzed the biosynthesis of uridine diphOSphate glucose from uridine triphOSphate and glucose-l-phOSphate. A new Spectrophotometric method was available for the determination of inorganic pyrophOSphate. Thus, spectrophotometric determinations of all four compo- nents of the reaction were performed as a basis for the equilibrium calculations. Further, a new chromatographic method for the separation of all four components of the reac- tion was developed using polyethyleneimine-impregnated paper and a 2.0 M formic acid-0.4 M LiCl solvent. Employing radio- active substrates, the ccncentrations of the reactants were determined chromatographically. The equilibrium constant determined by both the spectrophotometric and the chromato- graphic procedures was found to be about 0.200 Thus, when Karen E. DeFazio the products were allowed to accumulate, the reaction pro- ceeded to about 30% uridine diphOSphate glucose formation from equivalent amounts of the substrates uridine triphos- phate and glucoseml-phoSphate. DETERMINATION OF THE EQUILIBRIUM CONSTANT FOR AN ENZYME CATALYZED REACTION By ‘N Karen E; DeFazio A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1968 ACKNOWLEDGMENTS The author wishes to eXpress her appreciation to Dr. R. G. Hansen for his guidance and interest throughout the course of this project. The author also wishes to thank the members of Dr. Hansen‘s laboratory for their helpful suggestions and assistance. The author is especially grateful to her parents, relatives, and friends for their encouragement and love throughout the course of this work. 11 VITA Karen E. DeFazio was born on July 29, 1943, at Elyria, Ohio. She graduated from Elyria High School in 1961. She attended Baldwin-Wallace College and received the B.S. degree in Chemistry in 1965 from the Department of Chemistry. Her graduate studies were pursued at Michigan State University in the Department of Biochemistry to complete the requirements for the degree of Master of Science. 111 TABLE OF CONTENTS INTRODUCTION . . . . o o . . . . . . LITERATURE BEVIE w 0 0 O 9 O O O O 0 General History of Uridine DiphOSphate PyrophOSphorylase . . . Development of Chromatographic Methods Separate All Reaction Components MATERIALS AND METHODS . . . . . . . Chemicals 0 O O O O O O 0 O O 0 Quantitative Measurements . . . Qualitative Measurements . . . . EXPERIMENTAL PROCEDURES AND RESULTS Glucose to Determination of Equilibrium Constant by Spectrophotometric Methods . DevelOpment of the Chromatographic System . . Determination of Equilibrium Constant by Chromatography o o o o o o 0 DISCUSSION 0 o o o o o o o o o o o o SUMFLABY o o o o o o o o o o o o o 0 LITERATURE CITED . . . . . . . . . . iv Page 10 10 10 12 13 13 l9 27 39 1+2 ML Table I. II. III. IV. VI. VII. VIII. IX. XI. XII. XIII. LIST OF TABLES Equilibrium study of UDP-glucose pyrophOSphory- lase reaction in E. Coli K12 . . . . . . . . Equilibrium study of UDP-glucose pyrophOSphory- laSe reaCtion in Calf liver 0 o o o o o o 0 Time course of the pyrophOSphorylase reaction . The effect of ammonium sulfate and enzyme con- centration on the equilibrium constant . . . The effect of the amount of magnesium on the equilibrium constant . . . . . . . . . . . . Determination of the equilibrium constant by the Spectrophotometric method . . . . . . . R values for separation of UMP from other nucleotides using a formic acid solvent . . R values for separation of nucleoside phos- phates and sugars with 2.0 M formic acid- 005 M Licl O O O O O O 0 O 0 0 0 O O G O O O R values for separation of nucleoside diphos- phate sugars with 2.0 M formic acid and lithiumCthrideoooooo 000 0000. Separation of nucleoside mono-, di-, and tri- phOSphates with 2.0 M formic acid and lithiumchloride.............. R values for separation of nucleoside mono-, f di-, and tri-phOSphates with 2.0 M formic aCid-003 PI L101 0 0 O O O 0 O O 0 O 0 0 O O Equilibrium study using luC-labelled sub» Strates o o o o o o o o o o o o o o o o o o Equilibrium studies using 14C- and 32P- labelled SUbStrateS o o o o o o o o o o o o Page 14 15 17 18 20 26 28 29 30 32 34 LIST OF FIGURES Figure Page 1. Separation of the four substrates: Glc-l-P, UDP-Glc, PPi, and UTP, using a formic acid- LiCl Solvent system . . . . . . . . . . . . . 22 2. Separation of the four substrates: Glc—l-P, UDP-Glc, PPi, and UTP, using a formic acid- NHLLCJ' SOlvent SyStem o o o o o o o o o o o o 2L!" 3. Equilibrium study using 14C- and 32P-labelled subStrateS o o o o o o o o o o o o o o o o o 35 4. Equilibrium study using 1”c.- and 32P-labelled substrates . . . . . . . . . . . . . . . . . 37 vi INTRODUCTION The study of energy-transformations in living organ- isms has become an integral part of understanding the nature of many enzymatically catalyzed reactions. We know that, in isolation, these reactions designated by the general equation A+B:C+D (1) will reach a point of equilibrium at which no further net chemical change takes place. The constant which eXpresses this chemical equilibrium and which, in turn, is related to the standard free energy change of the system is called the thermodynamic equilibrium constant, _ [c] [a] Keg " mm [B] ‘2’ where the brackets eXpress the concentration of the reactants and products at the point of equilibrium. The presence of an enzyme affects only the time and rate at which equilibrium is achieved, but should not affect the final Keq (l). The purpose of this thesis is to determine the equilib- rium constant of an enzymatically catalyzed reaction, Specifi- 1 _cally, the biosynthesis of UDPmGlc from UTP and Glcul-P UTP + GlCmI-P ;===2 UDPuGlC + PPi (3) 1The following abbreviations are used: UMP, UDP, and UTP, uridine moncw, dim, and triaphOSphate; UDPuGlc, uridine diphosphate glucose; UDP-Gal, uridine diphOSphate galactose; PPi, inorganic perphOSphate; Glc-iuP, glucose-l-phOSphate; AMP, ADP, and ATP, adenosine mono-, di-, and tri-phOSphate; ADP-G10, adenosine diphOSphate glucose; ADPmMan, adenosine 1 2 which involves the enzyme, uridine diphOSphate glucose pyro- phOSphorylase (UTch-D-glucose-l-P uridylyl transferase). The literature reports a range of 0.1 to 1.0 for the equilib- rium constant of the perphOSphorylase reaction. In view of the fact that the Keq should be independent of the enzyme source, attempts were made to more precisely measure its value using a new Spectrophotometric method for the determin- ation of PPi and a new chromatographic method for separation of all four components of the reaction. diphOSphate mannose; dmAMP, deoxyadenosine monophosphate; GMP, GDP, and GTP, guanosine mono-, di-, and tri-phOSphate; GDP-Glc, guanosine diphOSphate glucose; IMP, IDP, and ITP, inosine mono-, di-, and tri-phOSphate; IDP-Glc, inosine diphOSphate glucose; CMP, GDP, and CTP, cytidine mono-, di-, and tri-phOSphate; CDP—Glc, cytidine diphOSphate glucose; TMP, TDP, and TTP, thymidine mono-, dim, and tri-phOSphate; TDP-Glc, thymidine diphOSphate glucose; TPN, triphOSphopyri- dine nucleotide; LiCl, lithium chloride; PEI, polyethylm eneimine. LITERATURE REVIEW General History of Uridine DiphOSphate Glucose PyrophOSphorylase In 1950, Leloir and coworkers (2) isolated the nucleo- tide, uridine diphosphate glucose, from yeast. Shortly after- wards, Kalckar and Cutolo (3) discovered in a Zwischenferment preparation a pyrophOSphorylase enzyme which could cleave UDPuGlc to bring about the formation of UTP and Glc-l-P. They also found that this reaction was reversible with the enzyme acting as a uridyl transferase. Since then the enzyme has become known as the uridine diphosphoglucose pyrophOSphory- lase (UTPzd-D-glucose-l-P uridylyl transferase) which cata- lyzes the following reaction: UTP + Glc-i-P : UDP-Glc + PPi (3) In 1955, Munch-Petersen (4) purified the enzyme from yeast, established a Spectrophotometric assay for its activ- ity, and reported some of its properties. She did an equin librium study on the reaction by incubating 0.2 umole amounts of UDPmGlc and PPi with the enzyme and assaying the Glc-l-P formed in aliquots taken out at different time intervals. Glc-imP was analyzed by the addition of phoSphoglucomutase, cysteine, TPN, and Glc-6mP dehydrogenase. When readings at 340 mu were constant, indicating that all the Glc-l-P was used, excess UDP-glucose pyrophOSphorylase and PPi were added 4 to measure the residual UDP-Glc. It was concluded that the reaction stopped at 45% conversion, yielding an equilibrium constant near 1.0. Following this initial work of Munch-Petersen, a UDP- glucose perphOSphorylase was found in liver extracts (5), in mammary glands (6), in sugar beet leaves (7), in mung bean seedlings (8, 9), and in the plant Impatiens holstii (10). In 1957, Turner and Turner (11) isolated the enzyme from pea seed extracts and attempted to determine the equilibrium con- stant. With a concentration of 5.0 mM magnesium and at pH 7.9 and 30°, the Keq in the direction of synthesis of UDP-Glc and PPi was 0.14 which is significantly lower than 1.0 reported by Munch-Petersen (u). They observed that an increase in the magnesium ion concentration and a decrease in the pH of the reaction mixture each depressed the value of the con- stant. However, they did observe that precipitates were formed in the enzymic digests where the molar concentration of magnesium ions was equal to or greater than the concentra- tion of sodium pyrophOSphate. The presence of a complex for- mation with the sodium pyrophOSphate was suggested to have caused the depressing effect of the high magnesium ion con- centration on the pyrophOSphorylase reaction. In 1959, Palasi and Larner (12) purified the UDP- glucose pyrcphOSphorylase from skeletal muscle, and in 1961, Basu and Bachhawat (13) purified it from human brain. How- ever, no further equilibrium studies were reported until 1965 when Kamogawa and Kurahashi (14) preceded to purify the enzyme 5 from Escherichia coli K12. For studying the constant they prepared an incubation mixture which contained 0.30 umole of UDP-Glc, 0.28 umole of PPi, 3.8 ug of enzyme, and 3.0 umole of Mg012 in 1.0 ml of 0.5 M Tris-HCl buffer (pH 7.5). At various time intervals aliquots were withdrawn and the reac- tion stopped by boiling. They measured the amount of UDP- 010 by UDnglucose dehydrogenase, Glc-l-P by phOSphogluco- mutase and glucose-6uP dehydrogenase (4), UTP by nucleoside- diphOSphatekinase and hexokinase, and PPi by inorganic pyro- phOSphatase. Their results are shown in Table I. Averaging their values, Keq was calculated as 0.20 in the direction of synthesis of UDP-Glc and PPi, showing that the equilibrium was in favor of the degradation of UDP-Glc. In 1966, UDP-glucose pyrophOSphorylase was crystal- lized for the first time by Albrecht gt a; (15), and the source was calf liver. They did an equilibrium study by incubating the substrates with magnesium acetate, 2 mM, in 0.1 M Tris—acetate buffer (pH 7.8). After the addition of 0.35 ug of enzyme per ml, aliquots were removed at various time intervals and heated to boiling to stop the reaction. Equilibrium was attained in about 10 min at 30°. The con- centrations of the substrates and reaction products were determined as follows: GlculmP by phOSphoglucomutase and glucosen6mP dehydrogenase (4), UTP as described (16), UDP~ glucose after hydrolysis with venom pyrophosphatase and then measuring Glcmi-P as above, and PPi by difference. Their results are shown in Table II. The equilibrium constant in Table I. Equilibrium study of UDP-glucose pyrophOSphorylase reaction in E. Coli K12. Time ~ Minutes UDP-Glc PPi Glc-l-P UTP Keq 0 0.28 0.26 0.02 0.02 182.00 30 0.11 0.11 0.18 0.16 0.42 60 0.10 0.08 0.20 0.19 0.21 90 0.10 0.07 0.20 0.18 0.19 120 0.11 0.08 0.20 0.19 0.23 All concentrations are micromolar. Reference (14). .Amav mosmammmm .amaosadaas mam msoapmapsmozoo HH4 mm.o HH.o o Ha.o mH.o mm.o em.o mm.o m mm.o em.o o em.o :H.o we.o om.m mw.m N em.o mH.o o mfl.o wN.o m:.o wm.o om.o H ema Hesse HmapasH Hausa HmeHHH amuse HmapaqH Hausa HmeHQH psmafinmawm Ham oau:mao mnfisoao .Hmbfifi mamo SH Soapomma mmmamhosamosaoamm mmoosawlmmb wo mBSpm adaanaaaswm .HH canoe 8 the direction of synthesis of UDP-Glc was determined to be between 0.28 and 0.34. Development of Chromatographic Methods to Separate All Reaction Components The established importance of nucleoside diphOSphate sugar pyrophOSphorylases and their related substrates in car- bohydrate metabolism made necessary the development of chro- matographic methods capable of analyzing their incubation mixtures. The procedures involved ion-exchange column chro- matography, paper electrophoresis, paper chromatography, and thinulayer chromatography. The paper chromatography method was most useful for the analysis of enzymic reaction mixtures containing nucleo- side diphOSphate sugars and nucleoside mono-, di-, and tri- phOSphates. The solvent commonly used was ethanol and ammon- ium acetate (17, 18) and required a separation time of 24 to 64 hr. This being an extremely slow procedure brought about the investigations of more rapid and appropriate methods. It has been known for many years that polyethyleneimine and other basic polymers could be fixed on cellulose fibers, but it has not been realized that polyethyleneimine is an effective anionmexchanger which can be used in column, thin- layer, and paper chromatography. However, in 1963 Randerath published a paper indicating ribonucleotides could be separ- ated on PEI paper with a 1.0 M NaCl solvent in less than 1 hr (19). The same year it was reported that deoxyribonucleom tides could be separated from ribonucleotides by anionuexchange 9 thin layer chromatography (20). The plates were coated with the PEI-cellulose and developed with a solution of LiCl in aqueous boric acid. The borate added a net negative charge to the compound, thus increasing their distribution coeffi- cients on the anion-exchanger. In 1963, Dietrich (21) found that phOSphate deriva- tives of sugars could be separated from nucleotides using an ECTEOLA-cellulose powder on thin-layer plates. Randerath (22) found he could resolve complex nucleotide mixtures by two dimensional anion-exchange chromatography on PEI-cellu- lose thin layers. A LiCl solvent was used in one dimension and formic-Nanormate buffer (pH 3.4) in the other to develop the chromatogram. DPN, TPN, six nucleotide sugars, and four- teen common nucleoside-5'-mono-, di-, and tri~ph08phates were resolved in less than 3 hr. It was also possible to quanti- tatively elute small amounts of the nucleotides from the PEI plates (23). Finally, the effect of neutral and acid solvents on the development patterns of the nucleotides was compared (24). A formic aciduLiCl combination also showed some promise. In 1965, Verachtert §t_al_(25) published a method for characterizing nucleoside diphOSphate sugars in mixtures con- taining nucleoside mono-, di—, and tri—phOSphates. PEI paper with only LiCl as the solvent achieved a good separation in 3~4 hr. At the same time, Randerath (26) reported the separa_ tion of nucleotide sugars from nucleoside monophOSphates on PEI—cellulose thinulayer plates using either an acetic acid- LiCl solvent or a sodium borate-boric acid solvent. MATERIALS AND METHODS Chemicals All nucleotides and sugars were commercial products except IDP-Glc and TDP-Glc. These were synthesized by a pro- cedure which has been described (27). ADP-Man was synthe- sized enZymically using a calf liver extract (28). Polyethyleneimine was obtained as a 50% aqueous solu- tion from Chemirad Corp. (East Brunswick, New Jersey). The radioactive substrates U.L.-14C-Glc-1-P and 32PPi 1L’c—U.L. were obtained from New England Nuclear, and UDP-Glc- from ICN. UTP~B,y-32P was synthesized in the laboratory from 32PPi (S.T. Bass and R.G. Hansen, 1968, unpublished data). All enzymes were commercially prepared except for the UDP-glucose pyrophOSphorylase which was crystallized in the laboratory according to the method described by Albrecht gt 2.1 <15). Quantitative Measurements UDP-glucose pyrophOSQhorylase activity was determined by the method described by Albrecht gt_al (15). One unit of enzyme is defined as that amount required to liberate 1.0 umole of product per min at 25°. The chemical determination of the equilibrium constant was carried out by incubating 1.0 umole quantities of either GlcwluP and UTP or UDP-Glc and PPi with 1.0 umole of magnesium 10 11 acetate in 1.0 ml of 0.1 M Tris-acetate buffer (pH 7.8). After addition of the desired amount of enzyme, aliquots were removed at various time intervals and heated to boiling to stop the reaction. Equilibrium was attained in about 15 min at 30°. The concentration of the reaction products was deter- mined as follows: Glc-l-P by an end-point assay using phos— phoglucomutase and glucoseu6-P dehydrogenase (4, 15), UTP as described (16), PPi by further addition of UDP-Glc and UDP- glucose pyrophOSphorylase (29), and UDP-Glc by further addi- tion of PPi and UDP-glucose pyrophOSphorylase. A typical assay for determining Glc-l-P, PPi, and UDP-Glc follows: In quartz cuvettes with 1-cm light paths, 1.0 umole of magnesium acetate, 0.2 umole of TPN, an aliquot of the incubation mix- ture and enough phosphoglucomutase and g1ucose-6-P dehydro- genase to complete the reaction in 15 min or less at 25° were added successively to 0.1 M Tris-acetate buffer (pH 7.8) to make a final volume of 0.5 ml. When an end-point was reached, indicating that all the Glc-l-P was used, excess UDPaglucose pyrophOSphorylase and 1.0 umole of PPi were added to determine UDP—Glc, or excess UDP-glucose pyrophOSphorylase and 0.2 umole of UDP-Glc were added to determine PPi. All reactions were followed at 340 mu in a Beckman model DU Spectrophoto- meter equipped with a Gilford automatic sample changer and recorder (30). Radioactivity was measured in a Packard Instrument Company Tri-Carb liquid scintillation counter. The counting solution consisted of 770 ml of xylene, 770 ml of pudioxane, 12 462 ml absolute ethanol, 0.1 g of a-N-PO, 10.0 g of PPO, and 160.0 g of napthalene (31). Samples were placed in vials con- taining 15 ml of the counting fluid, shaken to achieve diSper- sion, and counted for 10 min each. Qualitative Measurements Polyethyleneimine-impregnated paper was employed for all chromatographic work. This was prepared by treating Whatman No. 1 paper with PEI (25). Between 0.05 and 0.10 umoles of nucleotides and sugars were applied as spots and 0.200 ml of each incubation mixture as streaks about 3 inches from the base of the paper, and development was achieved in a descending direction in 3 or 4 hr at room temperature, using the desired solvent. The ultraviolet-absorbing compounds were detected with a Mineralight ultraviolet lamp, and the sugar phOSphates with a molybdate Spray (32). EXPERIMENTAL PROCEDURES AND RESULTS Determination of Equilibrium Constant by SpectrOphotometric Methods The time course of the reaction was observed to determine the level of enzyme and length of time which was necessary to bring about equilibrium of the UDPuglucose pyrophOSphorylase. Two mixtures were incubated, one con- taining 1.0 umole quantities of the substrates Glc-lnP and UTP, the other containing 1.0 umole quantities of UDP-Glc and PPi. After the addition of 0.053 units of enzyme recrystallized three times, aliquots of each mixture were removed at various time intervals and were assayed for the substrates and reaction products. The results are shown in Table III. One can see that equilibrium in either direc- tion was achieved within 30 min at 30°, giving a Keq value of 0.23~0.25. The effect of ammonium sulfate in the reaction mix- ture on the equilibrium was investigated next. Four mixtures were prepared, each containing a different level of (NH4)2804 in the enzyme. 1.0 umcle quantities of the substrates GlcmleP and UTP were incubated for 1 hr at 30° with 1.0 umole of magnesium acetate in 1.0 ml of 0.1 M Trisoacetate buffer (pH 7.8). The results are shown in Table IV. No significant effect of the salt on the Keq value was observed. A test 13 Table III. 14 Time course of the pyrophOSphorylase reaction. Time Mixture Direction Incubation GIO-l-P UTP1 UDP-Glc PPi Keq 1 I 15 min 0.738 -—- 0.370 0.325 0.22 I 30 min 0.674 --- 0.313 0.361 0.25 I 1 hr 0.691 --- 0.325 0.370 0.25 I 2 hr 0.687 __- 0.333 0.358 0.25 I 3 hr 0.653 —-— 0.353 0.310 0.26 2 II 15 min 0.720 --- 0.275 0.411 0.22 II 30 min 0.708 --- 0.275 0.411 0.23 II 1 hr 0.682 --- 0.259 0.386 0.21 II 2 hr 0.727 --- 0.267 0.390 0.20 II 3 hr 0.725 --- 0.275 0.444 0.23 lUTP was not measured. Mixture 1 contained 1.0 umole of Glc-le, UTP, and magnesium acetate and mixture 2 contained 1.0 umole of UDP- Glc, PPi, and magnesium acetate in 1.0 ml of 0.1 M Trisa acetate buffer (pH 7.8). The reactions were each started with the addition of 0.053 units of enzyme. of synthesis is indicated: Keq is defined as micromolar. I The direction GlcmlmP + UTP -—-* UDP-Glc + PPi II Empecicfl Em] [Glen-P] Eur] ° All concentrations are 15 .amHoaoaoHa mam msoapmapsmosoo Had .HHH magma :H penance mm meow 63p mam Coapasammc wow one :oapomnao one . om pm a: a How Am.n may Hmwmsn mpmpoomamana : H.o no as o.a ad opmpmom adamms we go mHoaa o.H £pa3.pmpwnzosd who: mopdhpmflfim mo mmauapzmsv macs: o.H .omhsmmoa no: mm: mesa sm.o esm.o emm.o an- mmw.o eeuaaeao mo.H HH 6 em.o mem.o asm.o in- mae.o No.0 Hm.o HH m mm.o mmm.o emm.o an- mma.o o.m o.mm H e :m.o mmm.o smm.o in: mms.o m.o om.m H m mfl.o sam.o mam.o sun mes.o No.0 mm.o H m :H.o amm.o :mm.o In- tam.o Noo.o mmo.o H H see “mm eaoumo: map maauoao :ommfiemzv mean: godpoeeao eyepwaz H R mammsm .psmpmsoo Snag Inaaaswm map so noapmapsmosoo mfihusm cam mpdmaSm asfisosam mo pomhmm mQB .>H manma 16 involving the presence of (NH4)2804 in the enzyme was also performed. A portion of the enzyme crystals was dialyzed overnight against 0.01 M tricine buffer (pH 8.5). An equi- librium was then established with mixture 5 containing enzyme in (NH4)2804 and mixture 6 containing the dialyzed enzyme. The results are shown in Table IV. Again no sig- nificant effect was observed. Therefore, the presence of (NH4)2804 was ignored in Keq calculations. Since Turner and Turner (11) reported that the plant enzyme was affected by the magnesium concentration, incuba- tion mixtures were prepared with levels of magnesium acetate ranging from 0.01 mM to 5.0 mM. A magnesium level of 0.1 mM gave an equilibrium value in the same range as the 1.0 mM level (Table V). With the lower level of 0.01 mM magnesium equilibrium was not achieved in 1 hr of incubation. The enzyme turnover rate was probably inadequate due to the low level of magnesium. The high level of 5.0 mM caused a pre- cipitation of the PPi in the mixture and could not be assayed accurately. However, the mixture in the direction of syn- thesis of UDP-Glc and PPi was assayed and gave a Keq value near that observed when 1.0 mM magnesium was used. Further consideration of this issue should be given. Assuming now that the ammonium sulfate had little or no effect on the equilibrium constant and the 1.0 mM was a favorable level of magnesium concentration to use, a series of eXperiments were performed wherein the various substrates were incubated for 1 hr at 30°. The results are summarized in Table VI. The Keq value in direction I is 0.21 and in 17 .ommz cams mahnso Ho mpHss 3H.o .HmaoaOHoHs ohm msoapmesmosoo HHH .HHH canoe QH omsammo mm meow can the QOHHHQHHmo vmm one SoapomHHo one .ooms who: mmpmameSm Ho mmeszmsw macs: o.H .oom 96 H: H How pmpmosoQH who: moHSpNHa HH< .asHmmswms can SHHK ompmpdmaomhm Hmm 639* nun: *auunn smm.o mmm.o :mm.o oo.m HH 6 em.o mem.o smm.o Hme.o mem.o oo.m H m mm.o som.o em:.o mme.o mme.o Ho.o HH e No.0 NHH.o OOH.o mms.o mms.o Ho.o H m AH.o eHm.o sem.o 036.0 mme.o H.o HH m mH.o Hmm.o emm.o 3mm.o nee.o H.o H H eta HHH OHouHH: He: HuHuOHo wvamemm coHpoeHHH eespsz .HQMHmsoo ESHHDHHHSUm 65p 90 azawmswma Ho pagoam esp Ho pomwmm 639 .> magma 18 .HmHoEOHOHS who mSOHHMHmeosoo HHH .HHH maflme SH umsHmmp mm mawm map was mQOHpHsHHmp vex paw COHpomHHU 6:9 .oom as H: H Mom Am.m may Hmmmsn opdpmomlmHHB z H.o H0 H8 o.H :H mpdpmow asHmmswma Ho macs: o.H Ssz ompmpzosH ohms mopmemDSm Ho mmeHpsmsw macs: o.H .omHSmwma pom mms* mH.o mmm.o Hom.o mme.o mas.o mm.o HH mH.o mwm.o tom.o mme.o aoa.o mm.o H m mH.o mHm.o smm.o mme.o mms.o Hm.o HH mm.o mmm.o Hom.o aHe.o mee.o Hm.o H e eH.o mmm.o me.o mms.o mms.o HH.o HH om.o mmm.o OHm.o mme.o ase.o BH.o H m wH.o mmm.o eHm.o ame.o mHs.o AH.o HH om.o eon.o oom.o aae.o mme.o aH.o H m AH.o Hem.o omm.o *1--- mee.o am.o HH mm.o pmm.o mam.o *uunu mum.o sm.o H H eta HHH eHoamHe me: HiHseHo emwwmm :OHpeeHHH pzeaHHeme .eogpea OHHpmaoposaoppomam on» an asapmsoo ESHHQHHHsvm esp Ho QoHpmSHsHmpmm .H> manme 19 direction II is 0.18. Development of the Chromatographic System For separating the reactants and products, solutions containing 10.0 umoles each of the compounds UMP, UDP, UTP, UDPuGlc, UDP-Gal, PPi, and Glc-i-P were prepared. These were applied to PEI paper and their ion-exchange behavior as a function of decreasing formic acid concentration was studied. The results are shown in Table VII. Only the nucleotide, UMP and the sugar, Glc-i-P moved appreciably. This method can be applied to the separation of nucleoside monophOSphates from other nucleotides but will not separate the desired four substrates. A formic acid-LiCI solvent system was tested next. Concentrations of 0.5 M to 4.0 M formic acid containing 0.1 M, 0.5 M, 1.0 M, and 2.0 M LiCl were employed. Of these various mixtures the 2.0 M formic acid containing LiCl gave the best results. It was found that at salt concentrations below 0.3 M, the nucleoside di-, and tri—phosphates and PPi migrated very little. 0n the other hand, UMP and Glc-1-P moved appreciably. The two nucleoside diphOSphate sugars moved slightly and did not separate. With concentrations of LiCl above 0.3 M, the nucleoside dim, and tri-phosPhate moved appreciable with the diphOSphate preceding the tri- phOSphate. PPi moved between UDP and UTP, indicating for the first time the ability to separate PPi from UTP. The nucleoside diphOSphate sugars migrated still further and GlcnlnP moved the farthest followed by UMP. It was observed Table VII. R E solvent. 20 values for separation of UMP from her nucleotides using a formic acid Concentration of Formic Acid Compound 4.0 M 2.0 M 1.0 M 0.5 M UMP 0.47 0.34 0.25 0.21 UDP 0.01 0.01 0.01 0.01 UTP 0.01 0.00 0.00 0.01 UDP-G10 0.04 0.02 0.02 0.01 UDP-Gal 0.03 0.02 0.02 0.02 PPi 0.01 0.00 0.01 0.00 GlC-l-P 0.44 0.30 0.22 0.19 21 that Glc-l-P separated clearly from UDP-Glc, and the rate of movement of each compound increased regularly with increasing salt concentration. Most important, though, was the fact that the 2.0 M formic acid solvent containing 0.4-0.5 M LiCl separated clearly the four substrates Glc-l-P, UDP-Glc, PPi, and UTP. This separation is shown by the graph in Fig. 1. The formic acid solvent was then tried in combination with other salts instead of LiCl. NH4C1, NaCl, and KCl were all found to yield about the same results as LiCl. Only the separation of the substrates using a formic acid-NHuCI sol- vent is shown in Fig. 2. For some purposes NHucl may have an advantage since this salt is easily volatilized. NHACOOH and NaBorate were also tested in combination with formic acid, but did not separate the four substrates. However, the NaBorate salt did show some promise of separating UDP-Glc from UDP-Gal, but this was not pursued further. Boric acid with LiCl was also tried and gave very unsatisfactory results with general streaking of the Spots. In view of the resolving power of formic acid-LiCl for the compounds of principle interest, several preliminary eXperiments were conducted to see the effect of this solvent system on the separation of other nucleotides. Adenosine, guanosine, inosine, cytidine, uridine, and thymidine com- pounds were all tested using a 2.0 M formic acide0.5 M LiCl solvent system. The results are shown in Table VIII. A mixture of the nucleoside diphOSphate glucose derivatives were also separated with a 2.0 M formic acid—0.3 M LiCl 22 Figure 1. Separation of the four substrates Glc-l—P, UDP-Glc, PPi, and UTP, using a formic acid-LiCl solvent system. Between 0.05 and 0.1 umole of compound was Spotted and chromatography was performed from 3—4 hr at room temperature. 99.. 6.2.6.. 20a 2. as $.52 ON 0.. 0.. 0.0 . _ _ _ i\._\.HHV.\N . .1 10 0.0 .\ \A./\ i ll I... 0.0 so- ._\.o:\.ow loo 24 Figure 2. Separation of the four substrates Glc-l-P, UDP-G10, PPi, UTP, using a formic acid-NHACl solvent system. Between 0.05 and 0.1 umole of compound was spotted and chromatography was performed from 3-4 hr room temperature. 964 0.2%.. son 2. 6.12 8.52 n. . o. _ \_\.W.M.. 26 0m.0 HEHIB $0.0 :mEImmw Hw.0 cmSImQ< 30.0 OHUIHQU H0.0 OHUIHQH em.0 oHUImQU IIII oHOImmd no.0 mas 0H.0 HBO no.0 HRH 0H.0 mew mH.0 me¢ 0:.0 mma Hm.0 mmo 03.0 HQH 0m.0 mam m0.0 mag m m . o Has :0 . o .50 0x. . o HE E. . o .98 0 m . o 8.2 IIII msHoHahse 00.0 mQHUszo 00.0 msHmosH IIII msHmosmsu IIII msHmosmo¢ mm Undoaaoo hm undoaaoo Hm Undomaoo Hm ecsoaaoo Hm GQSoaaoo z 0.N QHHS memsm .HDHQ S m.OIUHom OHEHOM 0:0 mmmeHmOLQ mpHmooHos: Ho soHpmHmHmm How mosam> Hm .HHH> mHQMB 27 solvent system. These results are shown in Table IX. Sepa— rations of the mono-, di-, and tri—phOSphates are shown in Table X and their combination in Table XI. Determination of Equilibrium Constant by Chromatography A preliminary eXperiment was run to test if the sub- strates and reaction products of the incubation mixtures could, in fact, be separated on PEI paper with the 2.0 M formic acid—0.5 M LiCl solvent. Incubation mixtures were run as desoribed in the methods section. 0.200 ml of each reaction mixture was streaked on the paper. For visualiza- tion purposes 0.005 ml of 10.0 HM solutions of Glc-i-P, UTP, UDP-Glc, and PPi were also applied coincidentally. The chromatogram was deveIOped with a 2.0 M formic acid-0.5 M LiCl solvent for 3 or 4 hr at room temperature. The Spots were detected as described in the methods section. The com- ponents of the incubation mixtures separated and cochromato- graphed with the four authentic compounds. The purity of the radioactive substrates: U.L.-1uC- Glc-l—P, UTP—B,Y-32P, UDP-Glc-luc-U.L., and 32PPi was next tested. The radioactive compounds were cochromatographed with authentic unlabelled standards using a 2.0 M formic acid-0.4 M LiCl solvent for 3 or 4 hr at room temperature. The unlabelled compounds were detected as before, and any radioactive compounds by cutting 2.0 cm strips of the chro~ matogram and counting them in the scintillometer. The radioactive compounds were coincident with the authentic 28 Table IX. R values for separation of nucleoside diphOSphate sugars with 2.0 M formic acid-0.3 M LiCl. Compound Rf CDP-Glc 0.70 ADP-Glc _-__* GDP-Glc 0.54 TDP—Glc 0.46 UDP-Glc 0.40 IDP-Glc 0.37 *Could not detect. Table X. Separation of nucleoside mono-, di-, and tri-phos— phates with 2.0 M formic acid and lithium chloride. Concentration of LiCl __-- 0,5 M 1.0 M Nucleoside monophosphate diphOSphate triphOSphate Cytidine 0.88 0.67 0.50 Adenosine 0.85 0.65 0.47 Guanosine 0.58 0.55 0.38 Thymidine 0.40 0.51 0.38 Inosine 0.35 0.39 0.31 Uridine 0.33 0.45 0.33 30 Table XI. Rf values for separation of nucleoside mono-, di-, and tri-phOSphates with 2.0 formic acid-0.3 M LiCl. Compound Rf CMP 0.86 GDP 0.53 CTP 0.06 AMP 0.85 ADP 0.48 ATP 0.04 GMP 0.66 GDP 0.32 GTP 0.02 TMP 0.77 TDP 0.28 TTP 0.02 UMP 0.74 UDP 0.23 UTP 0.02 IMP 0.68 IDP 0.19 ITP 0.02 31 compounds on all chromatograms. The U.L.-14C-Glc-1-P was found to be pure, UDP-Glc-luc-U.L. contained a trace of UDP, UTP-B,Y-32P was pure except for a small amount of P1. The 32PPi appeared as if residual polyphOSphates may have been present. This seemed not to affect the UTP estimations. After validating the chromatographic system and radio- active chemicals, the first equilibrium eXperiment was per- formed using only the 14C radioactive compounds. Two mix- tures were prepared: the first containing 1.0 umole of Glc- 1-P and UTP with added U.L.—14 C-Glc-léP; the second contain- ing 1.0 umole of UDP-01c and PPi with added UDP-Glc-luC-U.L. Each was incubated with 1.0 umole of magnesium acetate and 0.17 units of crystallized enzyme in 1.0 ml of 0.1 M Tris- acetate buffer (pH 7.8) for 1 hr at 30°. The reaction mixtures were then streaked on PEI paper and developed with a 2.0 M formic acid-0.3 M LiCl solvent for 3 or 4 hr at room temperature. The chromatogram was cut into 2.0 cm strips and each strip was counted. The radio- activity coincident with the Glc-l-P and UDP-01c peaks was totaled and using a ratio of the counts with the preassayed amount of unlabelled substrates initially added to the mix- tures, the amount of products formed was calculated. The results are shown in Table XII. Experiments using both 11+0- and 32P-labelled substrates were next performed. Again reaction mixtures were prepared, those starting with 1.0 umole of Glc-l-P and UTP with added 14 U.L.- C-Glc-imP and UTP—B.Y-32P and those with 1.0 umole of 32 .HeHosOHoHa eHe msoHperseosoo HH4 .HHH wanes SH oesHHeo we eaem exp eHe msoHpHsHHeo vex use SOHpoeHHo ems .eememmeIeHa ohms mepermDSm oHoo Ho meSOEe HerHsH 039 .UepQSOo mes QHHpm zoee wee maHHpm ac 0.N opsH p50 macs maeHwopeaoaso 0:8 .eHSpeHemaep BOOM pe H: : Ho m Hog HUHH z H.0I0Hoe OHBHOH z 0.N :pHs oemoaebeo use Heaem Hmm so oexeeapm gasp eaez meHSpMHE e29 .oom pe as H Hem .w.m mm. HeHHSQ epepeoe ImHHB 2 H.0 00 H8 o.H SH eahwse oeNHHHeptho Ho mpHQS 0H.0 fine mpepeoe asHmesmea Ho macs: o.H 39H; depeQSOsH eH03 mepepmeSm epronHoeH eprermeH HHesp ans wsoae .mHmenpshm Ho SOHpoeHHo Cogs msHosemep .mepehmeSm oaoo 0eHHeeo 0:» Ho merHpsesw macs: o.H mm.o mmm.o mam.o mHm.o maa.o em0.o ooo.o emc.o ooo.o HH m AH.o Nmm.o ooo.o Nmm.o ooo.o wmm.o oem.o eec.o saw.o H H aea HeaHa HeHchH HecHa HeHaHeH HecHa HeHchH HeaHa HerHaH ccHaceaHm eaaawHa Hmm OHOImQD m9: mIHIOHU .neaeacnnam ecHHepeHIoeH echa ensue saHHnHHHaem .HHx ereH 33 UDP-Glc and PPi with added UDP-Glc-luC-U.L. and 32PP1. All mixtures were incubated with 1.0 umole of magnesium acetate and the appropriate amount of enzyme in 1.0 m1 of 0.1 M Tris—acetate buffer (pH 7.8) for 1 hr at 30°. The mixtures were each streaked on PEI paper and develOped with a 2.0 M formic acid-0.4 M LiCl solvent for 3 or 4 hr at room tempera- ture. The chromatograms were cut into 2.0 cm strips and each section counted. The counts under each peak were totaled with the results as shown in Table XIII. The Keq constant was calculated directly from the ratio of counts. Figures 3 and 4, representing eXperiments 5 and 6, respectively, show graphically how the peaks of each substrate separated on the chromatograms. .HHH eHQeB sH uesHHeu me esem esp eHe msOHpHsHHeu Gem use sOHpoeHHu ens .uepsSOO use mmHHpm So 0.N opsH p50 seSp eHeB waeHwopeSOHso esa .eHSpeHemaep soon He H: sum How HQHH 2 :.0IuHoe OHSHOH z 0.m SpHs uerHebeu use Hemem Hmm so uexeeapm sesp eHes meHSpNHa one .oom pe as H Hog .m.m mm. HeHHSQ epepeoeImHHB z H.0 no as o.H sH esthe Ho psSOEe epeHHQOHHQe eSH use epepeoe asHmeswes Ho macs: o.H 39H: uepen IzosH macs mepeameSm eeronHueH eproeHmeH HHeSp SpHs wsoae .mHmespshm Ho sOHp IoeHHu some msHusemeu .mepeameSm uHoo ueHHmeu m:p Ho merHpseSU macs: o.H 34 Hm.o seem smmH moon mNHH :H.o HH 0H.0 awn emeH owe mmem eH.o H mN.o sHom mHmH chm HHm: mH.o HH om.o ms: mamH mam down wH.o H mH.o Noam HNHH sHmm NHH: wH.o HH Hm.o mmu memH HOHH mean wH.o H eH.o :mmm OOHN Home meow 0H.0 HH mm.o mm: oomH emHH :mmm wH.o H AH.o moan omsH meme oeom mH.o HH «H.0 0mm omHH cmeH sHam mH.o H AH.o mmc meeH mseH swam Hm.o H aea Had chumo: was HIHIcHo emwwmm achecHHH pceaHHeaam epsst Hem epsSOU .mepenpmnsm ueHHeneHImmm use Io wsHms mmHuspm ssHHnHaHsum .HHHN eHQeE .H“__. ".0. 35 Figure 3. Equilibrium study using 14 c- and 32p- labelled substrates. starting with UDP-Glcluc + 32 Graph B represents equilibrium starting with 1 C-Glc-l-P + UTP-e.y—32P N 9 x E 0. 0 A B P e . w. m We a a a w m. ///////// Aw...“ m 37% VIII/Wll/llllllllllllly/gvM"fle. new” m avarfigflhwm a . ._. a 1.. ._. ._. No. x 2% 20 30 40 Cm l0 37 Figure 4. Equilibrium study using 1LIC- and 32P- labelled substrates. Details of eXperiment are described in the text. Graph A represents equilibrium starting with UDP-Glcluc + 32PPi ———-9.. Graph B repre- sents equilibrium starting with 14C-Glc-l-P + UTP- B.Y-32P ——-> . 40 _ e ‘ l.- . E en: \ We \\\\\ \\\... ... 0 .... .. m . a G mu H » I . , p . \\\\\\ M m\\\ H w 3 0. w. o m m .2 R W m - m . .- a w ...... p w wax/am u envy/3.... T . 5., . //////////’¢////////////’///////’///I////”’// .popo.o.o.o.o.0.l. U 7’III’IIIIIII //v.vn)u .0 ._. .m ..e m m A. No. x 2.6 Cm DISCUSSION The time course of the UDP-glucose pyrophOSphorylase reaction studied by spectrophotometric means showed that equilibrium, in either direction, could be achieved within 1 hr at 30°, provided that 1.0 umole quantities of each sub- strate and also magnesium were used. The value of Keq, which was defined as _ EDP-01c] Pi K... ‘ W ‘2’ ranged from 0.23 to 0.25 in the Spectrophotometric analysis. The presence of ammonium sulfate in the mixture showed no significant effect, yielding an equilibrium value of 0.14 to 0.27. Variations in the magnesium ion concentration appeared to have an effect on the constant. A favorable amount of magnesium was considered to be 1.0 mM. A level of 0.01 mM indicated that the turnover rate of the enzyme was inadequate for a 1 hr incubation. A 5.0 mM level of magnesium caused a precipitation of the PPi, effectively removing this product from the reaction mixture. Further consideration of the metal concentration should be interesting. Assuming that ammonium sulfate did not greatly alter the Keq and the 1.0 mM magnesium was a favorable level to use, a series of 1 hr incubation mixtures were equilibrated and found to give an average constant of 0.20. The equilibrium, 39 40 therefore, favors the degradation of UDP-Glc, leaving approx- imately 30% of UDP—Glc and PPi, and 70% of Glc-l-P and UTP. This value agrees with that observed in E. Coli K12 (14), falls within the 0.10 to 0.21 range reported by Turner and Turner (11), and is slightly lower than that reported by Albrecht (15). However, it is appreciably lower than the value of 1.0 originally obtained by Munch-Petersen (4). The fact that she did not have a crystalline enzyme may account for this difference. The presence of other enzymes and sub- strates in an unpurified preparation would be eXpected to alter the constant. A chromatographic method that was developed using a 2.0 M formic acid-0.4 M LiCl solvent on PEI paper was found to clearly separate the four components of the reaction mix- ture. This was the first time that Glc-1-P could be separated from UDP-01c, and UTP from PPi using one solvent system in a minimum of time. Applying this method to equilibrium studies gave very satisfactory results. Using only 14C—labelled sub- strates, the equilibrium constant was between 0.17 and 0.25. Using both 1°C- and 32P-labelled substrates, Keq ranged from 0.12 to 0.23 and was averaged to be 0.18. This agrees nicely with the average value of 0.20 obtained by the Spectrophoto- metric method. It is concluded that this new chromatographic method provides an excellent opportunity for further equilib- rium studies of enzymatic reactions and can be performed quite simply in a short amount of time. The application of the formic acid-LiCl solvent system 41 on the separation of other nucleotides yielded some inter- esting results. Under the acidic conditions the rate of migration decreased in the order: monophOSphate)>nucleo- tide sugars3>diphOSphate>-triphOSphates. Since polyethyl- eneimine is a resin that exchanges anions, the difference in negative charges of the compound affected their movement. The pattern is identical to that obtained by Randerath who used PEI-cellulose thin-layer plates instead of paper (24). Under neutral conditions using only LiCl as the solvent, Verachtert reported that the nucleotide sugars migrated faster than the monophOSphates (25). Randerath also reported this reversal on the thin-layer plates (24). Again under acidic conditions, the general rate of migration as affected by the base component was cytidine> adenosine) guanosine> thymidine:>uridine >inosine. There was some variation of this pattern when Spots migrated near the solvent front or moved only a short distance from the origin. The level of salt also appeared to have an effect on their pattern of separation. It is important to note the separation of adenosine and inosine compounds on the PEI paper. Until now, this was impossible using only a LiCl solvent (25). In summary, the separation of almost any two nucleo- tide substrates can be achieved using PEI paper and either an acidic or a neutral solvent. The rates of migration depend upon the salt concentration and the pH of the solvent, the net charge and the size and Spatial configuration of the molecule. SUMMARY The enzyme, uridine diphOSphate glucose pyrophOSphory- lase, catalyzing the biosynthesis of uridine diphOSphate glucose from uridine triphosphate and glucose-l-phOSphate, is available for the first time in crystalline form. The equilibrium constant for this enzymatic reaction was deter- mined by Spectrophotometric and chromatographic means. A new Spectrophotometric method for the determination of inorganic pyrophosphate was used. The Spectrophotometric determinations of all the components of the reaction were also performed as a basis for the equilibrium calculations. A new chromatographic method for the separation of all four components of the reaction was developed using polyethyleneimine-impregnated paper and a 2.0 M formic acid- 1[IO-- and 32P-labelled sub- 0.4 M LiCl solvent. Employing strates, the concentration of reactants at equilibrium was determined chromatographically. The separation of other nucleotides and sugars was also achieved using polyethyleneimine-impregnated paper and a formic acid-LiCl solvent system. The equilibrium constant determined by both the Spec- trophotometric and the chromatographic procedures was in good agreement at about 0.20. Accordingly, when the products were allowed to accumulate, the reaction proceeded to about 42 43 30% uridine diphOSphate glucose formation from equivalent amounts of the substrates uridine triphOSphate and glucose- 1-phOSphate. 9. 10. 11. 12. 13. 14. 15. 16. LITERATURE CITED Lehninger, A.L., "Bioenergetics," W.A. Benjamin, New York, 1965. p. 23. Caputto, R., Leloir, L.F., Cardini, C.E., and Paladini, A.C., J. Biol. Chem., 184, 333 (1950). Kalckar, H.M., Cutolo, E., and Munch-Petersen, A., Nature, 112, 1036 (1953). Munch—Petersen, A., Acta Chem. Scand., 2, 1523 (1955). Mills, G.T., 0ndarza, R., and Smith, E.E.B., Biochim. et Bigphys. Acta, 14, 159 (1954). Smith, E.E.B., and Mills, G.T., Biochim. et BiOphys. Acta. ig, 152 (1955). Burma, D.P., and Mortimer, D.C., Arch. Biochem. and Biophys., 6g, 16 (1956). Neufeld, E.F., Ginsburg, V., Putman, E.W., Fanshier, D., and Hassid, W.Z., Arch. Biochem. Biophys., 62, 602 (1957 . Ginsburg, V., J. Biol. Chem., 233, 55 (1958). Ganguli, N.C., J. Biol. Chem., 232, 337 (1958). Turner, D.H., and Turner, J.F., Biochem. J., 62, 448 (1958)- Villar-Palasi, C., and Larner, J., Arch. Biochem. Biophys., gg, 61 (1960). Basu, 2.K., and Bachhawat, B.K., J. Neurochem., Z, 174 (19 1). Kamogaga, A., and Kurahashi, K., J. Biochem., 51, 758 (19 5). Albrecht, G.J.. Bass, S.T., Seifert, L.L., and Hansen, 3.0.. J. Biol. Chem., 241, 2968 (1966). Verachtert, H., Bass, S.T., Seifert, L.L., and Hansen, R.G.. Anal. Biochem., 13, 259 (1965). 44 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29, 300 31. 32. 45 Paladini, A.C., and Leloir, L.F., Biochem. J., 51, 426 (1952). Pabst Laboratories, Circular O.R.—10, Milwaukee, 1956. Randerath, K., J. Chromatog., 12, 235 (1963). Randerath, K., Biochim. BiOphys. Acta, 6, 622 (1963). Dietrich, C. P., Dietrich, S.M.C., and Pontis, R.G., J. Chromatog., 15, 277 (1964). Randerath, D., and Randerath, K., J. Chromatog., 16. 126 (1964). Randerath, E., and Randerath, K., Anal. Biochem., 12, 83 (1965). Randerath, E., and Randerath, K., J. Chromatog., 16. 111 (1964). Verachtert, H., Bass, S.T., Wilder, J., and Hansen, R.G., Anal. Biochem., 11, 497 (1965). Randerath, E., and Randerath, K., Anal. Biochem.. 13. 575 (1965). Michelson, A.M., Biochim. Biophys. Acta, 91, 1 (1964). Verachtert, H.. Bass, S.T., and Hansen, R.G., Biochim. Biophys. Acta, 92, 482 (1964). Johnson, J.C., Shanoff, Mike, Bass, S.T., Boezi, J.A., and Hansen, R.G., submitted for publication in Anal. Biochem. Wood, 2.A., and Gilford, S.R.. Anal. Biochem., g, 601 (19 1 . Gordon, C.F., and Wolfe, A.L., Anal. Chem., 2g. 574 (1960). Block, R.J., Durrum, E.L., and Gunter, 2., "A manual of paper chromatography and paper electrophoresis." Academic Press, Inc., New York, 1958, p. 200. HICHIGRN STQTE UNIV. LIBRRRIES 31293006767838