E“ I'll!!!" HIllllflllfllllllllflllllfllflllfllll 3 1293 253 LIBRARY Michigan State University This is to certify that the thesis entitled THE PURIFICATION AND SOME PROPERTIES OF PURPLE ACID PHOSPHATASE FROM BEEF SPLEEN presented by Sandy Shieufun Lin has been accepted towards fulfillment of the requirements for Wdegree in —Science—— Ma. grew Major professor Date May 15 o 1979 0-7639 OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. THE PURIFICATION AND SOME PROPERTIES OF PURPLE ACID PHOSPHATASE FROM BEEF SPLEEN BY Sandy Shieufun Lin A THESIS Submitted. to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Chemistry 1979 ABSTRACT THE PURIFICATION AND SOME PROPERTIES OF PURPLE ACID PHOSPHATASE FROM BEEF SPLEEN by Sandy Shieufun Lin The existing purification procedure for purple acid phosphatase from beef spleen was relatively inefficient and has been extended using cellu- lose phosphate chronatography. The recovery is much higher than that obtained.with other techniques. The purity is increased.by two and a half fold. ' The Michaelis - Henten constant, K‘ . for p-nitrophenyl phosphate . has been found to be 2.0 an. A plot of 1/Vo versus 1/(3) according to the Lineweaver - Burke equation was used to calculate the Km . The relationship between the structural features of inhibitors and the binding specificity of the enzyme is investigated through the inhi- bition studies of the structural analogues of the substrate, PNPP. The results suggest that the binding specificity is not related to the charge distribution on the aronatic moieties of the structual analogues. but related to the nature of phosphorus linkage. A catalytic reaction pathway is postulated from the products inhi- bition studies. ACKNOWLEDGEMENT The author is indebted to Dr. B. A. Averill for his direction, assistance. and encouragement in making this investigation possible. The author would also like to express thanks to Dr. Babcock for lending the Haring blendor used in the preparation of crude beef spleen honogenate. II Acknowledgement . . . Table of Contents . . List of Tables . . . . List of Figures . . . List of Abbreviations TABLE OF CONTENTS I O Intrducti m 0 0 O O O O O O O O O O O O O O O O O O O O 0 II. Experimental . . . A. Materials and Math“: 0 O O O O O O O O O 0 O O O O O B I Pratein Micatim O O O O O O O O O O O O O O O O O I III C Discu881on O O O O O O O O O O O O O O O O O O O O O 0 O 0 IV. SW 0 O O O O O O O O O O O O O O O O O O O O O O O 0 References 0 O 0 O O O O O O O O O O O O O O O O O O O O O O 0 III Page ii iii iv vi sages?“ Table I 'II III VI LIST OF TABLES Purification of Beef Spleen Acid Phosphatase Acid Extraction Saturated Ammonium Sulfate Fractionation Inhibition Study with PABP a: Pin?“ Inhibition Study with Hydrolysis Products. p—nitrophenol - andP1 Activation of- Beef Spleen Acid Phosphatase by Ferrous Ion and Inactivation by Ascorbic Acid in The Absence of PNPP Page 15 16 i7 LIST OF FIGURES Figure Page 1 Elution Profile of Beef Spleen Acid Phosphatase from Cit-52 Cellulose Chromatography Column . 11 2 Elution Profile of Beef Spleen Acid Phosphatase from Sephadex G75 Chromatography Column 13 3 Elution Profile of Beef Spleen Acid Phosphatase from Cellulose Phosphate Column 18 it Determination of Km for p-nitrophenylphosphate 19 The Chemical Structure of PNPP. PABP and PABP‘. 22 Enzymatic Reaction Pathway 25 PNP PABP LIST OF ABBREVIATIONS p—Nitrophenylphosphate p-Nitrophenol p—Aminobenzylphosphonic acid Ascorbic acid (2-(N-Korpholino) ethanesulfonic acid) . Carboxymethyl 52 Z-Aminoethylphosphonic acid p-acetamidobensylphosphonic acid VI I . INTRODUCTION The enzymes that catalyze the hydrolysis of phosphate esters may be divided into three classes, namely, alkaline phosphatases, non-purple acid phosphatases and purple phosphatases. Host of the violet phospha- tases are acid phosphatases, except the alkaline phosphatase isolated from Micrococcus sodonensis (1). These enzymes play a vital role in the regulation of the physiological level of inorganic phosphate and phosphorylated metabolites. A class of alkaline phosphatases cmtaining zinc has been studied in great detail, as has the class of acid phospha- tasee. that do not contain any known prosthetic group and are not violet in color. Although a few isolated reports have appeared on a series of violet acid phosphatases, this class of enzymes remains poorly charac- terized as to the nature of the violet chromophore and the hydrolytic catalytic preperties associated with an iron containing enzyme. This class of enzymes has been isolated from diverse sourses such as bovine spleen (2,3). porcine intrauterine fluids (1+, 5), sweet potato (6,7), kidney been (8), the mold Neurospora crassa (9). All are reported to be basic glycoproteins. The violet acid phosphatase from beef spleen was first purified by Glomset in 1959 (3) . The electronic spectrum of this enzyme is reported by them to have an intense absorption band at 550 nm and a shoulder at 310-320 nm similar to that reported by other workers. The enzyme has a pH optimum of about 5N6. The enzyme fran beef spleen showed activity toward a variety of 1 substrates including aromatic phosphate esters, phosphoproteins, neu- cleotides and inorganic pyrophosphate. Aliphatic phosphate esters such as glycerol phosphate were not hydrolyzed to any appreciable extent by the enzyme. In marked contrast, aromatic phosphate esters.and,phOSpha- mides were hydrolyzed by the enzyme at a rapid rate. The enzyme was inhibited by various chelating reagents at low concentration with ATP and casein as substrates (10). Among these inhibitors molybdate was the most effective one. The enzyme was reported to be activated by reducing agents (11). Of these ascorbic acid and thioglycolic acid were the best activators. Furthermore the enzyme was inactivated by short exposure to reducing agents in the absence of substrates. casein and.ATP, upon preincubation at room temperature. (Ferrous ion protected against this inactivation (12). Although the existence of enzyme activity in diverse sources as described above has been known for over 20 years. only a few of these enzymes have been obtained in the purified preparatian and demonstrated to contain tightly bound iron. They are those isolated from beef spleen (2). porcine uterine fluids (h). and kidney bean (8). The number of'ferric ions per molecule of beef spleen.acid.phosphatase is reported by Revel and Backer (12) to be one and the molecule weight was h0.000 daltons. In 1978 the Australian group reported the presence of two iron atoms per molecule of enzyme from beef spleen (13). The equivalent weight per iron atom was 20,000 daltons. However the Swedish group had.discounted the possibility of metal ion involvement on the basis of arc spectrography, EPR measurements and chemical analysis of certain metals (3). There are two objectives of the research in our laboratory: (1) to determine the relationship of the structure of the prosthetic group to 3 the enzymatic activity; 2. to establish the nature of the unusual chro- mophore. including identification of the metal. its ligands and their geometrical arrangement in the violet phosphatases. No other iron con- taining enzymes have been demonstrated to have phosphatase activity. It is unique among iron containing enzymes in that it is hydrolytic whereas most iron-containing enzymes participate in electron transfer reactions or oxidation/reduction catalysis. In line with our laboratory's interest. the development of a separation technique in an attempt to improve the purity and recovery is described. In addition to this. the Hichaelis- Kenton constant.‘K. . was»determined_for p-nitrophenyl phosphate as substrate. A catalytic reaction pathway is postulated through products inhibition studies.‘The activation of this enzyme by ferrous ion in the presence or absence of ascorbic acid (reducing agent) and substrate. PNPP . is describedt The inactivation of this enzyme by ascorbic acid in the absence of substrate and ferrous ion is also described in this thesis. II . EXPERIMENTAL A. Materials and Methods 1. Reagents and materials Bovine spleen was obtained from the local slaughter house at Okemos. Michigan. (2-(N-horpholino) ethanesulfonic acid) was the Ultrol brand of Calbiochem. La J olla. Ca; ascorbic acid was from Hallinckrodt Company. Paris Kentucky . Para-nitrophenylphosphate disodium salt hexahydrate was. the product of Aldrich Chemical Company, Inc . Milwaukee Wis. Ammonium sulfate, enzyme grade, was the product of ICN pharmaceutical Inc: Life Science Group, Cleveland, Ohio. 2. Instrumentation Conductivity and pH were measured with a digital electromark analyzer. Harkson Scientific. The absorbance of UV a: Visible light was measured by a Beckman nu spectrometer adapted with a Gilford photometer 252 and a Linear recorder. Protein solutions were concentrated through an Amicon micro-ultrafiltration system. 8 MC. and high performance thin- channel ultrafiltration system, T0? 10, under N with Amicon diaflo PM 10 2 membranes. Dialysis tubing was the product of Fisher Scientific Company. Pittsburgh, Pa. 15219. 3. Preparation of column for chromatography All the glass columns used were purchased from Fisher a Porter Inc. The column buffer was 0.1 M NaOAc buffer. pH 1L9 unless otherwise speci- fied . 5 a. Ion exchange chromatography ( CMSZ cellulose ) CM-SZ cellulose was purchased from Hhatman LTD. Springfield.flill. Maidstone, Kent. 600 g of the above gel was added to 3 l of 0.1 N NaOAc buffer; pH 459 and gently stirredt The pH was adjusted to 4.9 and the fines were decanted. The above process was repeated until the wash so- lution was found to have a.pH of 4.9 and the volume of the slurry was 3 la The slurry was then added to a glass column 2.5 cm in diameter and gravity packed to a height of 62 cm, The column was then washed with one column volume of 0.1M NaOAc buffer; pH 4.9. b. Cel.permeation chromatography ( Sephadex:C75 ) Sephadex G75 was purchased from Pharmacia Fine Chemicals, Division of Pharmacia Inc. Piscatawa. lew'Jersey. 20 g of medium mesh gel was added to 500 ml of 0.1 H NaOAc buffer; pH 4t9 and gently stirred» The pH was adjusted to 4.9 and the gel was treated in the same manner as described.above. The volume of the resulting slurry was 300 ml. The slurry was then added to a glass column 1.25 cm in diameter and gravity packed to a height of 65 cm, The column was then washed with one column volume of 0.1 M NaOAc buffer. 0.1 mu in.EDTA, pH 4.9, and kept at 490. c. Cellulose phosphate chromatography Monosodium cellulose phosphate was purchased from Hhatman LTD Springfield.nill, Haidstone. Kent. 2.2 g of cellulose phosphate was added.to 60 ml of 0.1 h-NaOAc buffer. pH 4.9 and the gel was treated in the same manner as described above. The Slurry was poured into a glass column 1 cm in diameter and gravity packed to a height of 7 cm. The column was then washed with one or two column volumes of column buffer and kept at 4°. 4. Enzymatic activity and protein concentration determination 6 a. Assay for acid phosphatase The assay was performed either by the continuous method (2) or the fixed time interval method (5). The assay temperature was 25°. A Beckman DU spectrometer was used to record the absorbance at 410 mm, The apparent extinction coefficient, 5*, of p-nitrophenol was determined to be 1266 l/mole at 25°, pH 6.0 with pKa = 7.15. The assay mixture (final volume 1.25 ml) contained 100 an has buffer ( pH 4.9 ), 10 ml! ASC, 20 M ferrous ammonium sulfate, an aliquot of enzyme and 10 mh p-nitrophenyl phosphate ( PNPP ). The mixture, excluding PNPP, was equilibrated at 25°. PNPP was then added to»the mixture and the reaction was started.by addition of an aliquot of enzyme. In the routine continuous assay procedure the in- creasing absorbance of phenolate at 410 nm was followed at pH 6.0 with a recorder attached to the Cilford spectrophotometer; The rate of hydrolysis was then calculated.from.Beer's law. In the non-routine procedure ( fixed time interval ), the enzymatic reaction was terminated at fixed.times by the addition of 6rN NaOH solution, and the absorbance at 410 nm was measured on the Cilford spectrometer. A Linear plot was obtained from the plot of the change in absorbance per minute versus time ( minute ). The extinction coefficient,£,,(i7.000 1/mole) was used to calculate the rate of hydrolysis. b. Definition of a unit of activity and specific activity. One unit of acid phosphatase activity is defined as the amount of enzyme required to catalyze the hydrolysis of 1)(mole of p-nitroPhenyl phosphate per minute under the above specified conditions. Specific activity is defined as units/mg, i.e. units of enzymatic activity per milligram of protein. 7 c. Determination of protein concentration The protein concentration was determined according to the Lowry method (14) or estimated from A280 by assuming a solution having 1280 of 1 contains 1 mg/ml of protein. During the earlier stage of preparation, the fraction having A280 contains 4 mg/ml protein according to the Lowry method. B. Protein purification All chemicals used were reagent grade and all solutions were made with distilled, deionized water. All operations were performed at 4° C, unless otherwise specified. Fresh bovine spleen were obtained from a local slaughter-house, stored at -20°C , and thawed at 4° immediately prior to acid extraction. The acid extraction was carried out at 25°, the acid extract was stored for no more than 24 hours, and the ammonium sulfate fractionation was performed essentially according to Singer and Fructon (is) . The fraction after salting out was dialyzed, then subjected to a series of chromatography steps, including Cit-52 cellulose, C75 Sephadex and cellulose phosphate. 1. Acid extraction 1.65 Kg of frozen beef spleen was thawed at 4°C and ground with 3310 ml of cold water in a 1 gallon Haring blendor (2 mine at medium speed, 1 min at high speed). To the "homogenate” ( 5800 ml ), 2 N 8230“ ( 185 ml ) was added with stirring until the pH was 3.0. After one hour the pH had risen to 3.5, and 40 ml 2 N H250“ was added to readjust the 11-1 to 3.0. Toluene ( 26 ml ) was then added, and the mixture was stirred at room temperature for 22 hours. It was then centrifuged at 2000 g for 30 minutes at 4°, and the supernatant fluid was filtered through glass wool to give 4000 ml of a brown-yellow solution (" acid extract ") . The enzymatic activity and protein concentration were measured. 2. 60. - 90 % saturated ammonium sulfate fractionation To 4000 ml of the acid extract, 1287 g of solid ammonium sulfate (60 % saturation) was added in small portions gently with stirring. The mixture was then centrifuged at 2500 xg for 30 minutes. The pellet, con- taining no activity, was discarded. To the supernatant fluid 9 ( 3600 ml ), 817.0 g of (“192301.1 was added ( 90 S saturation ) as des- cribed above. The mixture was centrifuged at 2500 x g at u°. The preci- pitate collected was then suspended in a minimum amount of 0.1 H NaOAc buffer, pl 4.9, resulting in 300 ml of solution, which was then dialyzed for 48 hours at 4° versus 2 changes of 3 l of 0.1 h NaOAc buffer, pH 4.9. The conductivity of the resultant dialysate was near that of the acetate buffer alone. The total activity and concentration of the dialyzed protein were measured. The dialysis tubing was prepared by boiling it in a 0.001 M EDTA solution, and then boiling in distilled water. The tubing was washed with cold, deionized water and then followed by the addition of the aliquot enzyme. 3. 014-52 cellulose column The dialyzed fraction of total volume 920 ml and activity of 552 units was loaded to a 014-52 cellulose column described previously on page 5.The bound protein was then washed by several column volumes of 0.1 h NaOAc buffer, In n.9, until the 1280 was less than 0.05 and then eluted with a linear gradient consisting of 2.0 l of column buffer and 2.0 l of 0.6 14 NaCl in column buffer, followed by 300 ml solution of 0.6 14 NaCl in column buffer. The fractions were collected in an auto- matic fractionator, the A280 profile and the enzymatic activity profiles are shown in fig. 1. Fractions with a specific activity greater than 1.0 were pooled and the combined fractions were concentrated down to 5 ml by Amicon microultrafiltration ( 8 NC ) under N through Amicon 1114-2 2 membrane . 4. Sephadex G75 The fraction with a volume of 5 ml from Cit-52 cellulose column was then loaded on to a Sephadex C75 column as described on page 5. The 10 protein was then eluted with 0.1 M NaOAc buffer, pH 4.9. The protein concentration and the enzymatic activity profile were plotted ( fig. 2 ). Fractions with a specific activity greater than 11+.0 were pooled and con- centrated to 1 ml by ultrafiltration as above. The concentrated enzyme was then frozen in liquid nitrogen and kept at 1+0. A summary of the purification scheme is presented in Table I with calculated yields. 5. Further purification by cellulose phosphate column A sample containing 11+9 units of activity was loaded on to ‘the cellu- lose phosphate column.as described on page 5. and then eluted with a linear gadient containing 100 ml of 0.5 M NaCl in 0.1 H NaOAc buffer. pi 15.9 and 100 ml of 0.0 M NaCl in 0.1 H NaOAc buffer. 1!! 0.9. Fractions with a specific activity ‘g‘eater than 19.0 were pooled and concentrated as described above. The average specific activity was 35.00 units/mg. The elution profile is shown in fig. 3. A summary of the purification scheme is presented in Table I. 11 .HI Na as: sodoomum some mo osdae> one .haa>a¢o¢ oaasshnsm . moaouao some .oo~< . moaowao unmade .ssoaoo hemenmouesouno omoHsHHou «also 8am Surgeons 3% seamen seem as £08m 5.33m « unawah é‘lW/SUNH) 12 Ail/\IJDV ELLVWAZNB O. 0. ‘3 a) o- 8' I l I l o o N \l '0 .9 o: - LLJ . CD 3% Z ' Z . 9 5' '5 40 me +—' , U— u . <1 0 . ' as ' < 0.1 "o 2 v I g e 1 l 1 1 1 1 o N O. Q 0. <3? N. 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S 8 25 n2. 3“ e8 - n... lemma 3% a 3 Beware . 338% . asses . H33 assesses a coupon—caveman sang 5.235 wggvdm HHH manna. ABSORBANCE AT 280nm 18 .0 o) 0.8 l T é é a m ENZYMATIC ACTIVITY UNITS/ ML 1 p m FRACTION NUMBER FIG. 3 l5 Elution Profile of Beef Spleen Acid Phosphatase from Cellulose Phosphate Column A linear gradient of 0.5 M - 1.0 M NaCl in NaOAc buffer ( pH 4.9 ) was used for elution. The volume of each fraction was 5 n1. Closed circle : AZBO' Open circles : Enzymatic activity. 19 Figure 1! Determination of Kll for p-nitrophenylphosphate Va is the initial velocity of the catalytic reaction. (8) is the concentration of ‘p-nitr0phenylphosphate . The standard method was perfomed at 25°, 11-! 6.0 with various concentration of substrate, (3), a 0.5 m. 1.0 m, 2.0 m. 4.0 m. .5.0 1:14. (smurf/mm) 0“ 20 —T Vm Ill! itLllLllLllllJllllllllLLl /|_._'__9_4 5 l0 '5 20 25 KM {-g-y M"'>dccc caccasuca ccccc cocoa occac can: a: m .35 z... e mmzm A3 3 3 cans. c cam 5d. 33m 83325 3 £33. 22 Figure 5 The Chemical Structures of PNPP, PABP and map" 23 PNPP‘ PABP RABP FIG. 5 2# Table V Inhibition Studies with Hydrolysis Products, p-Nitrophenol and?i Enzymatic activity Sample I. 10 mM P1 0.002 Control I 3' 0.195 Sample II. 0.118 1.5 mM p-nitrophenol Control 11" 0.120 %’inhibition 99.00 0.00 Containing the same assay components as that in sample I except 10 mM P1. Containing the same assay components as that in sample II except 1.5 mM p-nitrophenol. 1.0 mM PNPP was»used in all cases. 25 Figure 6 Enzymatic Reaction Pathway $.03. aza N am. 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III. DISCUSSION The enzyme prepared by the published procedure (2) , which involves acid extraction. ammonium sulfate fractionation. CM-52 cellulose chro- matography ( Table I ), was obtained in poor yield and in.a relatively impure form. As seen in Table I. the specific activity ( 0.07 units/mg ) of the crude acid extract of beef spleen was relatively low compared to that obtained by Campbell and Zerner (which was 0.37 u/mg) (2). Pre- sumably this could be due to the nutritional difference between the beef spleen used or could be due to the effect of pH value and toluene concen- tration in the acid extraction step.‘The recovery obtained in the ammonium sulfate fractionation step ('Table I ) was only half that of Campbell and Zerner (2). Singer and.Fructon's method was used in both cases. The 1+ fold purification obtained from 60 - 90 % ammonium sulfate fractionation ( Table I ) is relatively low. In general the average puri- fication in this step was on the order of 25. The 40 fold purification obtained by Campbell and Zerner seems to be remarkably high. The effect of the pH of acid extract on the purity and the effect of the per cent ammonium sulfate fractionation on the recovery were therefore examined in an attempt to obtain a highly purified enzyme. Table II lists the specific activity of the acid extract obtained at different pH value and with different toluene concentrations. We conclude that the pH value of acid extract and the concentration of toluene does not make much differ- ence in the enzymatic purity of acid extract. The results in Table III 28 29 show that the ammonium sulfate fraction giving the maximum recovery is the same as that used'by Campbell and Zerner (2). i. e. 60 - 90 %. Slight change in the per cent ammonium sulfate used over the range of 60 - 90 i does not make much difference in the recovery and purity. The recoveries in both the CM-SZ step and the Sephadex G75 step were quite low. both in our preparation and in that of Campbell and Zerner. The cellulose phos- phate chromatography was thought to be potentially an efficient tech- nique in increasing the yield and degree of purification. As seen in Fig. 3, a fraction containing 1’49 units per‘ milligram was eluted as a single peak corresponding to a position equivalent to about 0.6 :4 NaCl in NaOAc. plilt.9andtowardsthe trailingedgeofthemajorproteinpeak(Fig. 3). A 2’ 1/2 fold purification ( Table I ) is obtained. and a high recovery ( 70 x ) is obtained in this technique. Purification by cellulose phos- phate column is simple and can be used to advantage for the production of large quantities of enzyle . As seen in Fig. 5. PABP and PABP' are both structural analogues of substrate, PNPP, in that they both have an aromatic moiety and a phospho- rus containing group similar to that of PNPP. Consequently they were thought to be' potential inhibitors of PNPP. In line with this, an inhi- bition study was then carried out in an attempt to establish the res laticnship between the structural feature and binding specificity of the beef spleen acid phosphatase toward these two compounds. First of all the inhibition study was done on PABP. the results indicate that PABP is not an inhibitor of Pm ( Table IV ). Two possible factors are proposed to account for the difference of binding specificity between PB? and PNPP . One is the charge distribution , the other is the nature of the phosphorus linkage. The second pKa of the phosphonic acid group on PABP was estimated to be 8.5 (16), the 11b of amino group on PABP was esti- 30 mated to be 4.0. The fact that PABP is not very water soluble at the assay pH 6.0 suggests that some of it may exist as zwitter ion with a positive charge being localized on amino group and a negative charge being localized on the phosphorus containing group, whereas PNPP has mononegative charge on the phosphate group (pK a2 of phosphoric acid is close to 7.0 ). and no charge on the aromatic moiety. Should the enzyme be able to differentiate between the difference in charge distribution on the aromatic moieties of PABP and PNPP, then the enzyme should be inhibited by the derivative of PABP formed after the charge on aromatic moiety is removed. mar". a derivative of PAH? formed by acylation of the amino group, was then subjected to an inhibition study ( Table IV ). The result indicates that PABP* is not an inhibitor either. This implies that the binding specificity has nothing to do with the charge distrie bution factor. but has to do with the nature of phosphorus linkage. It seems that phenolic linkage is crucial for binding. The enzyme. however, also shows activity toward a class of phosphoenidates (1?). hence the phenolic linkage can not be the only factor toward the binding specifi- city of beef spleen acid phosphatase. Perhaps more weight has to be given to the nature of C-P bond in phosphonates as analogues of natural phosphates as regard to the binding specificity. During the past 15 years there has been developed significant interest in the preparation and investigation of phosphonic acids and their derivatives which appear as the analogues of naturally occurring phosphates. Crofts and Kosolapoff have measured the second pKa' s for a series of phosphonic acids and found them to be in the range 7.7 - 8.2 when a primary alkyl youp is attached to phosphorus (16). This‘is to be compared with the corresponding monoalkyl phosphates (18). A typical decrease in acidity 31 of phosphonic acids was found upon substitution of an alkyl group for the phenolic oxygen in the phosphorus containing group (19). Dittredge and Roberts demonstrated the existence of a glycerol ester of 2-aminoethyl- phophonic acid in the lipid extract of anemones, which yields AEP on hydrolysis (20). The resistance of AB? to the action of hydrolytic enzymes which are present in sea anemones suggests that substances with C-P bonds provide groups which are similar to phosphate group in the dissociation constants. but which are more likely to be stable to the action of phosphatases or other hydrolytic enzymes. Only a few reports have appeared on the nature of C-P bond in phosphonic acid and its derivatives. Zeleznick (21) and hastalerz (22) have demonstrated the biological cleavage of the C-P bond by several species of bacteria. No attempt has been made here to cover the literatures dealing with the use of many synthetic phosphonic acid analogues of naturally occurring subs- tances as potential inhibitors. which may be used as immobolizing agents coupled to the solid matrix support of the column used in affinity chro- matography. a potentially powerful separation technique. Little has been done on the investigation of catalytic reaction mechanism tn the violet acid phosphatases over last 20 years. From the inhibition studies on the catalytic reaction products, P and p-nitro- i phenol. we were able to propose a catalytic reaction pathway for the beef spleen acid phosphatase . Two reaction pathways were considered primarily ( Fig. 6 ). According to microscopic reversibility, if route 1 is the actual pathway then inhibition by p-nitrophenol should occur. If route 2 is the actual one, then inhibition by inorganic phosphate should occur. Table V indicates that the enzyme is inhibited by phosphate but not by " p-nitrophenol to any extent. This implies that route 2 is the major I 32 catalytic reaction pathway in which the p-nitrOphenol diffuses away first, followed by the loss of P1 once the intermediate E P1 PNP was formed. The Lineweaver’Burke equation is very useful in enzyme kinetic studies. A straight line is obtained from the plot of i/Vo versus 1/(S) from this equationt The Michaelis.Menten constant, K. . can thus be determined from the slope of the plot; it is an approximate value for the intracellular level of the substrate. It is a constant for a given enzyme. at fixed pH, ionic strength and temperature. It provides a mean to tell whether two enzymes from different source are the same or not. The K. of beef sp1een acid phosphatase for p-nitrophenyl phosphate has not ever been determined before. It is determined to be 2.0 an approximately (Fig. 4), a relatively high value compared to the normal level ( less than 1 an ) of intracellular substrates. Perhaps it is because that PNPP is not a physiological substrate. . In agreement with the observation of previous workers (12), it is found that enzyme activity is considerably enhanced by ascorbic acid in the presence of substrate, PNPP. The enzyme is inactivated by short ex- posure to ascorbic acid in the absence of substrate. PNPP ( column.E in Table VI ). A hypothesis to explain these observations is that the enzyme is isolated from beef spleen as a ferric - enzyme complex ( resting foam ). which is‘not very active and is of considerable stability to the heat. For activity, it is visualized that the ferric - enzyle has to be reduced to form a ferrous - enzyme complex which is more readily disso- ciable. Ascorbic acid is found to be the most effective reducing agent. The ferrous - enzyme complex is stablized in the presence of substrate. Hithout substrate the ferrous ion dissociates from the enzyme, resulting in the loss of activity. Addition of ferrous ion in the presence or 33 absence of substrate and / or ascorvic acid simply favors the equilibrium condition for the ferrous - enzyme complex. The result from column E a F in Table VI supports this hypothesis. The addition of ferric ion in the absence of ferrous ion and ascorbic acid does not activate the enzyme to any extent ( column C of Table VI ). Preincubation of the enzyme in buffer and ferrous ion in the absence of ascorbic acid for variable time intervals before starting the reaction by the addition of substrate results in variable activity. The results from column A a B in Table VI shows that the activity decreases as the preincubation period is in- creased. This is due to the oxidation of ferrous ion upon exposure to air. In addition to that, the degree of activation by ferrous ion is also de- pendent on the concentration. As the concentration of ferrous ion in- creases. the degree of activation increases. 1. IV. mm The inefficient purification of beef spleen acid phosphatase by the published procedure (2) has been improved by further purification on a column of cellulose phosphate to advantage of increasing the re- covery by 6 times that of Cit-52 cellulose column and Sephadex G75 gel permeation column. A two and half fol'd purification is- obtained on this column. 2. The K]. of beef spleen acid phosphatase toward substrate p-nitrophenyl- 1%. phosphate was determined to be approximately 2.0 mi! from the plot of the Lineweaver Burk equation. - Inhibition studies with two analogues .of substrate. PAH? and Pm'. revealed that the binding specificity of enzyme was not related to the type of phosphorus linkage in the compound. Further work has to be done in order to establish a clear relationship between the structural features of the substrate or inhibitor and the binding specificity of beef spleen acid phosphatase. The enzyme was activated by reducing agent ( ascorbic acid ) in the presence of substrate. PNPP. However. reducing agents such as ' ascorbic acid inactivated the enzyme in the absence of!!!” and ferrous ion. Ferrous ion prevents this inactivation. As the concen- tration of ferrous ion increases. the degree of activation increases. In contrast to that.‘ ferric ion was found to have no effect of acti- vation toward this enzyme. 5. 35 An enzymatic reaction pathway is postulated from the result of the inhibition studies with the catalytic reaction products. p-nitro- phenol and inorganic phosphate. The pathway involves the formation of an intermediate. E-PifPNP. followed by the sequential release of p-nitrophenol and inorganic phosphate. BIBLICBRAPHY (1) R. H. Glew and E. C. Heath. J. Biol. 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