immacwas mu mmamou or A g-Amvmss ”flush {or Hm beam cf Ph. D. MCI-{EGAN STATE UNIVERSETY John P’hiliép Riehm 19161 ‘lJE‘5l '2 This is to certify that the thesis entitled Proteolysis and Inhibition of a P-Amylase presented by John Phillip Riehm has been accepted towards fulfillment of the requirements for Ph.D. degree in Chemistry Major profess r Date November 222 1261 0—169 LIBRARY Michigan State University 1 PLACE IN RETURN BOX to remove this checkout from your record. [ To AVOID FINE return on or before date due. ! MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE slot c:/CIRCIDaleDue.p65-p.15 w; . .-... - '1 PROTEOLYSIS AND INHIBITION OF A [B-AMYLASE By John Phillip Riehm AN ABSTRACT OF.A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1961 ABSTRACT PROTEOLYSIS AND INHIBITION OF A B—AMYLASE By John Phillip Riehm The inhibitory effect of various reagents which are known to react with sulfhydryl groups has been examined on the enzyme sweet potato @— amylase with the hope of Specifically labeling the "active site" of the molecule. Both N-ethyl maleimide and p—chloromercuribenzoate are effective inhibitors. N—ethyl maleimide reacts with four sulfhydryl groups in aqueous solution and 15 to 16 in BM urea. The inactivation of B—amylase by p—chloromercuribenzoate which is reversible, involves the reaction of six sulfhydryl groups. Fifty percent inactivation of B-amylase activity by both reagents corresponds to reaction of one sulfhydryl group, suggesting that only one sulfhydryl group is required for catalysis. The reaction of sweet potato B-amylase with the exopeptidases has been examined with the hope of degrading the molecule to a smaller active fragment. fl—Amylase appears to be resistant to proteolysis catalyzed by carboxypeptidase A and leucine aminopeptidase. Nevertheless, carboxy— peptidase B does catalyze a small degradation of the molecule, thus indicating the presence of either a lysine or arginine residue at the carboxyl-terminus of the molecule. The combined action of carboxypepti— dases A and B results in much more degradation; nevertheless, this treatment has no effect on the amylase activity of the B—amylase molecule. John Phillip Riehm A new method has been devised for the quantitative determination of reaction between N-ethyl maleimide and sulfhydryl groups of proteins This method depends on the determination of S—cysteinosuccinic acid produced from acid hydrolysates of the N-ethyl maleimide-treated proteins. PROTEOLYSIS.AND INHIBITION OF A B—AMYLASE By John Phillip Riehm A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistny 1961 ACKNOWLEDGMENT The author wishes to express his sincere appreciation to Dr. John C. Speck, Jr., whose interest, co-operation and guidance have greatly facil- itated the progress of the present study. Acknowledgment is also due to the Department of Chemistry and the National Institutes of Health for funds provided in support of this work. ii I. II. INTRODUCTION . . TABLE OF CONTENTS Mechanism of B-Amylase Action The Exopeptidase Inhibitors . . EXPERIMENTAL . . . Apparatus . Reagents . Correlation Absorbance at 280 mu of Protein Concentration to Assay of B—Amylase Activity . . . . . Purification of B-Amylase on Sephadex G—75 Quantitative Amino Acid Analysis The Exopeptidases . . . . . . . . . . Inhibitors Quantitative Determination of S—Cysteinosuccinic Acid on the SteinrMoore Column AND DISCUSSION Correlation of Protein Concentration to Absorbance at 280 mu Purification of B-Amylase on Sephadex 6-75 Results of Amino Acid.Analysis' Action of the Exopeptidases on Sweet Potato D-Amylase . . Inhibitors PAGE 12 12 13 111 111 15 16 18 21 23 30 30 3O 31 31 39 TABLE OF CONTENTS (Cont.) PAGE 6. Quantitative Determination of S-Cysteino- succinic Acid from N-Ethyl Maleimide- treated Proteins . . . . . . . . . . . . . A6 IV. SWY o o o o o o o o o o o o o o o o o o o o o o o o 53 V. REFWCES O D O O O O O O O O O O O O O O C O O O O O 55 iv TABLE II. III. IV. VIII. IX. LIST OF TABLES PAGE Correlation of Protein Concentration to Absorbance at 280mu . . . . . . . . . . . . . . . . . . . . . 30 Amino Acid Content of Sweet Potato B-Amylase . . . . . 33 Amino Acid Content of Sweet Potato B—Amylase . . . . 311 Action of Carboxypeptidase A on Unpurified B—Amylase . 35 Action of Carboxypeptidase A on Purified fi-Amylase . . 36 Action of Carboxypeptidase B on B—Amylase . . . . . . 38 Action of Leucine Aminopeptidase on Sweet Potato B- Amylase . . . . . . . . . . 39 Spectrophotometric Determination of p—Chloromercuribenzoate with B—Amylase . . . . . . . . . . . . . . . . . . bl Reactivation of B—Amylase Inhibited by p—Chloro— mercuribenzoate and Mercuric Chloride . . . . . . h2 Spectrophotometric Determination of N— —Ethyl Maleimide with B— AmylaSe . . . . . . . . . h3 Action of Other Inhibitors on B—Amylase . . . . . . . b3 Color Yield from the Reaction between Ninhydrin and S— Cysteinosuccinic Acid . . . . . . . h8 Conversion of S— —Cysteino— (N— ethy1)- succinimide to S— —Cysteinosuccinic Acid . . . . . . . h8 Comparison of the Spectrophotometric Method with Yields of S—Cysteinosuccinic Acid . . . . . . . . 50 m LIST OF FIGURES FIGURE PAGE 1. Purification of B—Amylase on Sephadex G-75 . . . . . . 32 2. Correlation of Sulfhydryl Group Reaction with Inhibition of B—Amylase by p—Chloromercuribenzoate. to 3. Inhibition of Sweet Potato B-Amylase by N-Ethyl Maleimide hh h. Correlation of Sulfhydryl Group Reaction with Inhibition of B—Amylase by N—Ethyl Maleimide at pH 8.0 . . . . . . . . . . . . . . . . . . . . . . . AS 5. Elution Curve of S-Cysteinosuccinic Acid from a 150—cm Stein—Moore Column. . . . . . . . . . . . . b7 6. Elution Curve of a 72—Hour Acid Hydrolysate of S—Cysteino—(N—ethyl)-succinimide from a 30—cm Stein—Moore Column . . . . . . . . . . . . . . . . 51 vi - g . . n . . - . . . . . . . c . . - o . ‘ . . . . n . - . . . - . . . . t PROTEOLYSIS AND INHIBITION OF.A BaAMYLASE I. INTRODUCTION Knowledge concerning the catlytic properties of an enzyme is one of the intriguing puzzles of biology. Enzymatic activity undoubtedly relies on the structure, the conformation and the specific chemical groupings which are present in the molecule, and an understanding of enzymatic catalysis will not be complete until these factors are inter- related. Protein chemistry, in the past few years, has made great strides towards a solution of this problem as evidenced by the eluci- dation of certain active sites of certain enzymes, the complete amino acid sequence of another (ribonuclease), and by experiments with model systems. Nevertheless, it appears probable that a solution of the intimate mechanism of action for any enzyme will require much ingenious experimentation. I On reviewing the numerous avenues for obtaining information into the mechanism of enzyme catalysis, two approaches have appeared to warrant study. These are: 1. an investigation of the group(s) es- sential for catalytic activity, and 2. a study of active fragments re- sulting from the proteolytic degradation of a native molecule. The results of such inquiries could lead to the labeling of the active site and to a feasible mechanism which could be tested with the aid of Inodel compounds. The observations reported in this text extend pre— ‘Vious investigations carried out in this laboratory on the enzyme sweet potato B-amylase (l). ..... 2 B—amylases appear to be restricted to heigher plants. They have been crystallized from barley, wheat, soybeans and sweet potatoes. The occurrence of a B-amylase in sweet potatoes was shown in 1920 by Gore (2). Gore observed that slow heating of sweet potatoes in the 60° to 80°C range yielded a very high conversion of the starch into maltose. In l9h6 Balls gt El (3) succeeded in crystallizing this enzyme by ammonium sulfate fractionation; two years later they reported a re- finement in their isolation (h). Balls and co-workers noted that the enzyme is only slowly denatured at 65°C and utilized this property in one of the initial steps in the purification. The pH versus activity curves with citrate and acetate buffers showed that the optimum pH is h.S (h). Singer and England (5) studied the physiochemical properties of the molecule and found the protein to be electrophoretically homo- geneous. Nevertheless, an impurity of approximately 3 percent was detected in the ultracentrifuge. A molecular weight of 152,000 was calculated from these ultracentrifuge data. The work of England and Singer (6) indicated that sweet potato fi-amylase is inhibited by substances known to attack sulfhydryl groups. Low concentrations (lO'6M) of p—chloromercuribenzoate, silver nitrate, and mercuric chloride gave rapid inactivation. A twenty—fold excess of glutathione could partially reverse (to the extent of 50 percent) such inhibition. Oxidation of the sulfhydryl groups by o-iodosobenzoate also led to inactivation. This inactivation appeared to involve no poly— merization, since the sedimentation properties of the inactivated enzyme "were identical with those of the native molecule. Hellerman, Chinard and Ramsdell (7) have studied the reaction between cysteine and o—iodoso- benzoate. 3 Since no reports on the total amount of qysteinyl residues nor on the number of sulfhydryl groups required for enzymatic activity of sweet potato B-amylase are to be found in the literature, investigations into this important feature of the molecule have now been carried out. Other work having to do with this enzyme and described in this text has in- volved l. purification of the enzyme and 2. a quantitative determina— tion of its amino acid content. Mechanism of B-Amylase Action The following discussion will not attempt to differentiate between the various B—amylases since no variations have been noted in their mechanism of action. Contrary to the usual connotation in carbohydrase nomenclature, the name B-amylase should not suggest the presence of B-linkages in the substrate. In general, it may be stated that these enzymes catalyze the hydrolysis of starch (a-l,h-glucosidic linkages) to the disaccharide, B—maltose. It has been demonstrated that hydrol- ysis catalyzed by B-amylase commences from the non-reducing end of the polysaccharide and continues until the entire chain is converted into maltose or until enzyme action is blocked by chain branching. The presence of B—maltose as the end-product has been cited by Kuhn (8) and Freeman(9). These workers noted that the products of the reaction mutarotated upwards. Since maltose was identified as the end— product, B-maltose, which would mutarotate upwards to the equilibrium :mixture, was believed to be the sole initial product. Recently, Thoma and Koshland (10) pointed out that an upward mutarotation need not imply a quantitative release of B-maltose; it merely indicates that the initial products contain more of the B-isomer than does the equilibrium mixture. Accordingly, Thoma and Koshland carried out a study on the extent of inversion. The rotation of a maltotetraose solution immediately after hydrolysis catalyzed by B—amylase indicated that B-maltose is indeed quantitatively released. This observation apparently rules out the possibility of cleavage by a free carbonium ion mechanism. Chemical alterations of the reducing terminus of oligiosaccharides have demonstrated the attack of B—amylase at the non-reducing terminus. Hydrolysis of methyl-a-maltotrioside yielded maltose and methyl-d- glucoside (10). Hydrolysis of maltoheptanoic acid, catalyzed by the enzyme, gave maltose and maltotrionic acid (11). As the hydrolysis of starch and glycogen catalyzed by B-amylase progresses towards branches in the chains, the hydrolysis rate becomes much slower. Hence, amylopectins and glycogens following B-amylase treatment give high molecular weight "limit dextrins". These dextrins, though resistant to hydrolysis catalyzed by B-amylase, are rapidly cleaved under the influence of a-amylases. Chemical and enzymatic in- vestigations have shown that all the branches in the original substrate are found in the limit dextrin (l2). Peat and co-workers treated a limit dextrin with an "R—enzyme" preparation (13) ("R—enzymes" are specific for the hydrolysis of (1-6)— a-glucosidic linkages). The outer "stubs" of the limit dextrins were shown to consist entirely of maltose and maltotriose, indicating that B-amylase removes all but the three innermost glucose units from the odd-membered outer chains, and a single maltose unit from the even- meflbered chains. Studies in which starch or glycogen were hydrolyzed in the presence of H2018 have shown that B—amylase catalyzes the cleavage of the 1—h- glycosidic bond between the Cl and the oxygen bridge (1h,15). Accordingly, Mayer and Larner (1h) proposed a mechanism for the catalytic hydrolysis based on this observation. This mechanism involves the following steps: 1. orientation of the substrate on the enzyme surface, 2. protonation of the bridge oxygen to form an oxonium ion which is cleaved on the Cl side, and 3. stereOSpecific hydration of the resulting carbonium ion. Koshland (16) has explained B—amylase catalysis as an SNZ reaction in which water attacks the potential aldehyde carbon from the backside, diSplacing the R—O group and, inverting the configuration at C1. Thoma and Koshland have put forward evidence that B-amylase catal— yzes hydrolysis through an induced-fit mechanism (10,17). The induced— fit theory postulates that the substrate induces changes in the con- formation of the enzyme and that these changes are required for enzyma- tic activity. In contrast to this, the older template theory proposes that the enzyme, due to its conformation, dictates the type of substrate to be hydrolyzed. The facts used in proposing the induced—fit theory are the following: 1. the Schardinger dextrins, cyclohexa— and cyclohepta- amylose are not cleaved in the presence of B-amylase but are strong competitive inhibitors of the enzyme, and 2. the hydrolysis of maltose catalyzed by B-amylase is very slow, that of maltotriose is slow, whereas that of maltotetraose is quite rapid. Thoma and Koshland's :model postulates that reaction of a group, X, on the enzyme with the substrate, induces the enzyme to fit closely to the substrate. In a reactive enzyme-substrate complex the two enzyme catalytic groups, A sand B, are brought into a favourable proximity with the substrate. It 6 is assumed that the enzyme forms a complex with the Schardinger dextrins, in which the two groups, A and B, are not able to complete the catalytic process. The Exopeptidases Since previous experiments having to do with the action of various endopeptidases on sweet potato B-amylase (1) did not yield active frag- ments, it was decided to test the action of the ex0peptidases on the enzyme. Exopeptidases catalyze hydrolysis of peptide bonds adjacent to terminal a—amino or terminal a—carboxyl groups. The best character— ized of these enzymes are the carboxypeptidases, A and B, and leucine aminopeptidase. It is worth noting that these enzymes have been instru- mental in the determination of amino acid sequences (33,3h,35). Hirs, Stein and Moore (33) elucidated the primary structure of ribonuclease with the aid of both carboxypeptidases A and B. Dixon, Kaufman and Neurath (35) used leucine aminopeptidase to determine the sequence around the phOSphoserine residue of trypsin.modified by reaction with diisoprOpyl phosphofluoridate. It is interesting also that these exo- peptidases have been used to modify native proteins. The action of carboxypeptidase A on various proteins shows a wide range of effects from the liberation of one amino acid as in its action on tobacco mosaic virus protein (36), to many when it acts on glucagon (37). In the hydrolysis of yeast enolase catalyzed by carboxypeptidase A, it has been reported (38) that over 150 amino acid residues are removed with— out 1033 of enzymatic activity. Drechsler and Boyer (39) have reported that carboxypeptidase A catalyzes the hydrolysis of three tyrosyl residues from the aldolase molecule with a concomitant decrease in 7 activity of 93 percent towards the substrate fructose 1,6-diphosphate. Perhaps the most outstanding example of exopeptidase degradation of native proteins is that reported by Hill and Smith (h0,hl,h2). Leucine aminopeptidase degrades approximately two-thirds of the mercuri- papain molecule, liberating free amino acids, without altering the enzymatic activity of papain when it is reactivated with cysteine. A homogeneous active fragment was obtained from the proteolysis mixture. This fragment contains 76 of the original 185 residues and differs from intact papain in ultraviolet Spectrum, amino end group and molecular weight, but shows similar behaviour with respect to denaturation by heat, acid and urea. Malmstrdm (h3) has reported that leucine amino- peptidase releases about 150 amino acids from the N—terminus of enolase without loss of enolase activity. Carboxypeptidase B is a relatively new tool in the hands of the biochemist. Its presence in bovine pancreas was first reported in 1956 by Folk (hh) and, a homogeneous preparation was described in 1960 (hS). The enzyme Specifically catalyzes the hydrolysis of lysine and arginine from the carboxyl terminus of proteins. Folk and Gladner (A6) studied the effect of carboxypeptidase B on trypsin and noted that 3.5 moles of arginine were released in 2 hours. These experiments indicate that it is possible to degrade a native enzyme and obtain a smaller unit which may still contain part or all of the original activity. With this thought in mind, the proteolytic action of these three exopeptidases on sweet potato B—amylase has been examined. 8 Inhibitors Inhibitors of enzyme catalysis have proven to be a useful aid in determining the type of groups essential for catalytic actiVity. They alsb provide a means of labeling amino acids at the so-called "active site" of the molecule. A well known case of inhibition is that by diisopropyl phospho- fluoridate in abolishing completely and irreversibly the proteolytic activity of chymotrypsin and trypsin (18). In each instance a crystal- line inactive derivative containing a single diisopropyl phosphoryl group can be isolated (19). Acid hydrolysis of these protein deriva— tives produces significant amounts of phosphoserine (20,21). Proteolysis of these derivatives followed by amino acid sequence studies have led to the elucidation of the amino acid sequences about these serine residues (22,23). The phosphoserine residues in trypsin and chymotryp“ sin are found in the sequence: glycyl, aspartyl, seryl, glycyl (2h). In the past decade, the inhibition of enzymes by reagents which are known to react with thiols has received much attention. The analytical demonstration of sulfhydryl groups in an enzyme, and the reversible inactivation of the enzyme by a reagent such as p—chloro- mercuribenzoate has been taken as evidence that one or more cysteine residues are involved in the catalytic process. Recently vallee, Combs, and Hock (27) have reported that the activity of Carboxypeptidase .A depends on a zinc mercaptide. One zinc ion appears to be bound to the only titratable sulfhydryl group of the zinc—free protein. Replace- ment of zinc with the sulfhydryl group reagents, p—chloromercuribenzoate or silver ion, renders the enzyme inactive. Removal of these reagents by cysteine and replacement of the zinc restores the carboxypeptidase A activity. These observations indicate that it should be possible to explore the chemical nature of the "active site" of carboxypeptidase A by labeling this sulfhydryl group. The possibility of labeling the "active site" of an enzyme has initiated studies into the effect of various reagents which are known to react with sulfhydryl groups on the molecule sweet potato B—amylase. The observations reported in this text attempt to correlate inhibition of B-amylase activity with the number of sulfhydryl groups undergoing reaction. Inhibitors which attack sulfhydryl groups have been thought to be Specific reagents for thiol compounds. Nevertheless, it is known that ribonuclease which contains no sulfhydryl groups is inhibited by iodo- acetic acid, one of the classical sulfhydryl reagents. Gundlach, Stein and Moore (25) found that inactivation of ribonuclease by iodo- acetic acid resulted in the alkylation of different amino acids at various pH values. At a pH of 8.5 the e—amino groups of lysine were alkylated. At pH 5.5 a nitrogen of histidine (presumably a ring nitro— gen) was alkylated, and at pH 2.8 the sulfur of methionine underwent alkylation. With the aid of C14-1abeled bromoacetic acid Barnard and Stein (26) have demonstrated that the histidine which is alkylated is the one nearest the carboxyl—terminus of ribonuclease. The reaction of N—ethyl maleimide with proteins was also believed to be Specific for sulfhydryl groups, and the spectrophotometric method .for determining thiols with N—ethyl maleimide (28) has been employed in Imahy instances for the quantitative determination of cysteinyl residues in.proteins (29,30). The method of Alexander depends on the decrease in 10 absorbance at 300 mu resulting from a presumed addition of the thiol across the double bond of N-ethyl maleimide. Nevertheless, Smyth, Nagamatsu and Fruton have shown that N-ethyl maleimide reacts with imidazoles and the amino groups of peptides in,possibly, aan-acylation reaction. In the case of imidazoles, reaction proceeds to the forma— tion of a polymer, possibly consisting of N-ethylmaleamic acid. Recently Riggs (32) has shown that a decrease in absorbance is observed when N-ethyl maleimide(lO-3M) is added to 0.1M solutions of various amino acids. These results give rise to doubts concerning the reliability of Alexander's spectrophotometric method. It is true that the condi- tions of Riggs and Smyth 32.21 are much more rigorous (higher concen— trations of N-ethyl maleimide and amino acids) than those employed by Alexander, nevertheless, there is the possibility of acylation reactions in conjunction with the alkylation of sulfhydnyl groups. Portions of the observations reported in this text are attempts to clarify this uncertainty. N—ethyl maleimide reacts with cysteine to form S-cysteino- (N-ethyl)—succinimide (31). On acid hydrolysis, S—cysteino-(N—ethyl)- succinimide is converted to S~cysteinosuccinic acid. Reaction of N-ethyl maleimide with sulfhydryl groups of proteins and acid hydrolysis of the protein derivatives should also lead to the production of S—cysteino- succinic acid also. These reactions are shown in Chart 1. The uptake of N—ethyl maleimide in the presence of various proteins ‘was studied in two ways: (1) by the Spectrophotometeric method outlined Lby Alexander, and (2) by determining the amount of S—cysteinosuccinic zacid produced from an acid hydrolysate of the proteins treated with N- ethyl maleimide. Comparison of the two methods should indicate, within eaxperimental error, the presence 6f reactions other than reaction with cysteine residues. 0 n t CH 2CH 3 NHZ Hci-c/ it 0 I NH 1 O C=0 0 CNCHCH SH + HC " _ 2 _ . . ,3 ll f>NCH 2CH3 HC-C I! 0 O n HOZCCHCHZS-CH- ' NCH CH NH 2 3 2 CHZfi/ O H 20 ii ! NHZ CHZOOZH 1 NH 1 0 (9:0 0 N I ll —CNCHCHZS—CH— ,3 | CC>VCH 20113 2 ll 0 CH CHART I. Formation of S-Cysteinosuccinic Acid. II. EXPERIMENTAL 1. Apparatus Spectrophotometers.-Absorbance measurements in the ultraviolet range were carried out with the Beckman Model DU spectrophotometer. Controlled reaction temperatures were obtained by fitting the instru— ment with a thermospacer assembly and by connection to a circulating bath. A Beckman Model B spectrophotometer was used for measurements in the visible range. This instrument was adapted for test tubes by re- placing the cell compartment with the test tube compartment. Columns.- (a) Protein Chromatograph --Columns fitted with glass wool plugs and packed.with the desired resin were employed in the purification of enzyme solutions. (b) Amino.Acid Chromatography--The separation of the neutral and acidic amino acids required a 0.9 x 165 cm jacketed column. A condenser with an inner diameter of 0.9 cm and fitted with a sintered plate was used for the basic amino acids. This column also proved useful in the preliminary investigations of the compound S-cysteinosuccinic acid. Sterile Apparatus.- Proteolytic digestions of longer than six hours were carried out in a sterile apparatus. The apparatus consisted of a Pyrex bacterial filter attached to a 20 ml filter tube and had a side arm to permit withdrawal of samples during the reaction without contam- ination. The apparatus was sterilized at 15 pounds pressure for 30 minutes prior to each run. 12 13 Standardized Test Tubes.-Soft glass test tubes were standardized according to the procedure of Stein and Moore (h7). 2. Reagents Proteins.— The sweet potato B-amylase was a twice crystalline product obtained from Worthington Biochemical Corp. The enzyme is pre— pared according to Balls 32,31 (h). Carboxypeptidase A (lot 601), prepared according to the method of Anson (h8) was also obtained from Worthington Biochemical Corp. Aldolase (lot B-l89h) which is isolated from rabbit muscle according to the method of Taylor, Green and Cori (A9) was purchased from Mann Research Laboratories. Egg albumin which was crystallized five times (lot F 63) and thrice crystallized B-lacto— globulin (lot F 26) were purchased from Pentex Incorp. Regiflsr Amberlite MB-l (lot 700613) and Amberlite 03-120 Type 2 (lot 785993) were obtained from Fischer Scientific Co. Sephadex G—75 was purchased from Pharmacia. Diethylaminoethyl cellulose (lot 107h18, 0.78 meq. per g) was obtained from the California Corporation for Bio- chemical Research. Chemicals.- N-ethyl maleimide (lot c2282) and L—qysteine hydro- chloride monohydrate (lot B2056) were acquired from.Mann Research Laboratories. p-Chloromercuribenzoate (sodium salt, lot 102735) was purchased from the California Corporation for Biochemical Research. Carbobenzoxy-glycyl-L—phenylalanine (lot 81310) and hippuryl—L—arginine (lot F2911) were products of Mann Research Laboratories. L-leucin- amide hydrochloride (lot 653A) was purchased from the Nutritional Bio- chemical Corporation. 1L1 Ninhydrin Color Reagentr- The reagent was prepared by dissolving 2 g of ninhydrin.(Nutritional Biochemical Corp.) and 0.3 g of hydrindatin in 75 ml of methyl cellosolve (Fischer Scientific Co.). TWenty-five ml of hM acetate buffer (pH 5.25) was then added to this solution. The reagent was always freshly prepared. The hydrindatin.was prepared according to the method of Stein and Moore (50). Twenty g of ascorbic acid in 100 ml of water at h0°C was added to 500 m1 of water at 90°C, and containing 20 g of ninhydrin. 0n standing, the hydrindatin crystal— lized. The product was filtered and dried over phosphorus pentoxide. The yield was 18.5 g. 3. Correlation of Protein Concentration to Absorbance at 280 mu Approximately 10 mg of Bmamylase was dialyzed in the cold for 2h hours against 0.1M phOSphate buffer (four changes of buffer). The enzyme solution was centrifuged to remove any denatured protein and was diluted to 5 ml. Aliquots were removed and diluted for optical density measure- ments at 280 mu. Kjeldahl nitrogen determinations were performed on 0.5-ml samples. Since the dry protein contains 15.hl percent nitrogen (1), nitrogen values were converted to mg of protein. The results are reported in Table 1(see Results). A. Assay of B-Amylase Activity The method of Noelting and Bernfeld (51,52) was employed for assaying B-amylase activity. This procedure is based on the formation of a colored product from the reaction of maltose and 3,5—dinitrosal- icylic acid in alkaline solution. The concentration of this colored material is measured spectrophotometrically at 5h0 mu. y-s——-_————_ 15 Reagentc- Five 9 of 3,5-dinitrosalicylic acid was moistened with a few ml of water and 100 ml of 2N sodium hydroxide was added. The suspension was brought to a volume of 250 ml by the addition of water and stirred until the 3,5-dinitrosalicylic acid dissolved. One-hundred and fifty g of Rochelle salt was then added to this solution and the solution finally diluted to a volume of 500 ml. Assay Procedure.— One ml of a one-percent starch solution (pH h.63 0.01M acetate buffer) was placed in a 25 m1 volumetric flask and equi— librated (at 30°C) in the constant temperature bath. One ml of enzyme solution was then added and the reaction allowed to proceed for 185 seconds. Reaction was stopped by the addition of 2 ml of the 3,5—dinitro- salicylic acid solution, and the flask placed in a boiling water bath for 5 minutes, cooled, diluted to 25 ml and the absorbance read at 5h0 mu against that of a blank. Enzyme concentrations were adjusted to approximately 0.001 mg per m1. In inhibition studies, enzyme con- centrations were raised tenfold when inhibition neared 90 percent. 5. Purification of fi—Amylase on Sephadex G—75 Singer (5) reported that ultracentrifugation of an eight times recrystallized enzyme preparation revealed the presence of a slower moving component. This impurity amounted to approximately 2 to 3 per— cent of the total protein. The commercially available enzyme also con— tains this impurity, as shown by ultracentrifugation, and it was thought that purification was a prerequisite to studies on the enzyme. The following procedure was employed in the purification step. A sample of B—amylase was dialyzed for 2h hours in the cold, against redistilled water (two changes). The solution was added in the cold, to a l x 15 cm -;-- t“!- a 16 column of Sephadex G-75 which had been equilibrated with water. Em— ploying water as the eluting solvent, 2.3 ml fractions were collected. Samples containing B-amylase activity were pooled and perevaporated in the cold to a concentration of approximately 1 mg per ml. This sample was dialyzed against 0.1M phosphate buffer (pH 6.0), centrifuged to remove dust and examined in the ultracentrifuge for the presence of impurities. 6. Quantitative Amino Acid.Analysis This report is a continuation of previous results (1) wherein analysis is reported for 13- and 72—hour hydrolysates. Analyses have now been performed on h8—hydrolysates (impure enzyme) and on 72-hour hydrolysates (purified sample). Preparation of the Samples.— Solutions of sweet potato B—amylase were exhaustively dialyzed in the cold against redistilled water (several changes of water). The enzyme solutions were placed in hydrolysis ampules and lyophillized. Constant boiling hydrochloric acid (h to 5 ml) was added, the ampules were evacuated, sealed and placed in a 105°C oven for either L8 or 72 hours. The samples were removed from the ampules and dried in vacuo over sodium hydroxide. One ml of water was added to the residue and the samples were again brought to dryness. This drying procedure was repeated twice to insure complete removal of the hydro- chloric acid. Ten m1 of buffer (either pH 3.25 or 5.28) was added to the residue, and the resulting solutions were stored in the frozen state until they were analyzed for amino acid content. Operation of Columnsu— (a) One—hundred and Fifty-cm Column. One ml 17 of the protein hydrolysate, in pH 3.25 buffer, was added to the top of the column and allowed to flow into the resin. This was followed by two, l-ml aliquots of buffer which were also allowed to flow into the resin packing. The column was connected to a separatory funnel, con- taining pH 3.25 buffer, and air pressure of h to 5 pounds was applied. The effluent was collected in 2—ml fractions while maintaining the temperature of the jacketed column at 50°C throughout the run. An eluant of pH h.25 buffer was introduced in time to allow valine to emerge with the new buffer. Stein and Moore (53) suggest making this change at an effluent volume 2.15 times that at which aSpartic acid emerges from the column. This procedure resulted in poor resolution of cystien and valine. The best separations between these two amino acids were obtained by introducing pH b.25 buffer at an effluent volume 2.15 times the peak of aSpartic plus hO ml. Upon the elution of phenyl- alanine from the column, 0.2N sodium hydroxide was passed through the column overnight. This operation was followed by re—equilibration of the column with pH 3.2; buffer. This procedure readied the column for the next run. (b) Fifteen—cm Column;— The operation of this column is practically identical to that of the 150—cm column except that the eluting buffer is pH 5.28. Upon the appearance of arginine the column may be used immediately for another sample. Leucine Standard Curveu— For the preparation of this standard curve, 131 mg of L—leucine was dissolved in 10 ml of redistilled water, and aliquots of this solution were diluted to concentrations of 0.05 to 1.0 micromoles per 2 ml. One ml of the ninhydrin reagent was added to 2-ml 18 aliquots of the solutions in previously standardized test tubes. The test tubes were stoppered and placed in a boiling water bath for 15 minutes. 0n cooling, 10 ml of 50 percent ethanol was added to each tube and the absorbance was read at 5h0 mu against a blank. When tubes had an optical density greater than 0.600, an additional 5 ml of 50 per- cent ethanol was added. The amount of amino acid present in each tube of effluent was determined in a similar manner, except for proline which was read at th mu. The amount of amino acid present was calculated in terms of leucine equivalents which when divided by its color yield (50) gave the concentrations of the amino acid. 7. The EXopeptidases Experiments with Carboxypeptidase A Preparation of the Carboxypeptidase A Solutionu—.Approximately 0.1 ml of the carboxypeptidase A suspension was dissolved in a few ml of 0.01N soidum hydroxide, and the resulting solution was diluted to 25 ml with phOSphate buffer (pH 8.0, 0.1M). The absorbance at 280p divided by 2.3 gave the mg of carboxypeptidase per ml (39). The activity of the carboxypeptidase solution was measured on the substrate, car- bobenzoxy-glycyl—L-phenylalanine. Action of Carboxypeptidase A on figAmylase.-Solutions of B—amylase containing approximately 30 mg of the enzyme, were dialyzed in the cold against redistilled water and passed through a l x 10 cm column of mixed—bed resin (Amberlite MB-l). This treatment freed the protein of any absorbed amino acids. Initially, experiments were carried out which involved no purification of the enzyme. The resulting solutions were perevaporated in the cold to a volume of approximately 5 ml, and 19 were dialyzed against phOSphate buffer (pH 8.0, 0.1M). Carboxypepti- dase A was added to the resulting solutions so that the ratio of carboxy- peptidase to protein was 1 to 50, and proteolysis was allowed to proceed at 30°C. At various times 0.1 m1 aliquots of the reaction.mixture were withdrawn for B-amylase activity measurements. At the same time, ali- quots were removed for the determination of amino acids released. This amino acid analysis was carried out in the following manner. To 1 m1 of the protein solution was added 1 ml of 5 percent trichloroacetic acid. The resulting mixture, containing the suspended, denatured pro- tein, was allowed to stand in the refrigerator for 30 minutes before centrifuging. .Aliquots (1.5 m1) of the supernatant solution were then mixed in test tubes with 1 ml of the ninhydrin reagent. The tubes were boiled for 15 minutes, diluted with 10 m1 of 50 percent ethanol, and the absorbance of the solutions measured at 570 mu. Experiments with Carboxypeptidase B Isolation of the Enzymec- The enzyme was prepared according to Folk gt a1 (h5). This procedure involves preparation of an acetone powder from swine pancreas, extraction of the powder with water, and fractionation of the extract with ammonium sulfate. The fraction of protein precipitating between 0.35 and 0.60 ammonium.sulfate saturation was purified on diethylaminoethyl cellulose (0.78 m.e. per g). Fifty g of acetone powder gave 280 mg of protein in a total volume of 15 ml. The activity of the preparation was determined on the substrate hippuryl— L—arginine and contained 1,200 units per mg of protein. Action of Carboxypeptidase B on B-amylase.-Samples of B-amylase which were purified as outlined in the carboxypeptidase A experiments 20 were dialyzed against 0.1M phOSphate buffer (pH 8.0, 0.1M NaCl). Solu- tions of carboxypeptidase B were added so that the ratio of carboxy- peptidase B to protein was 1 to 25, and proteolysis was allowed to pro- ceed at 30°C. At various times, aliquots were removed for]; B—amylase activity measurements, and 2. ninhydrin color yields of the trichloro- acetic acid soluble fraction. The results of this investigation are reported in Table VI (see Results). One experiment was performed in which the cummulative effect of carboxypeptidases A and B was studied. Carboxypeptidase B was added to 5 ml of a B-amylase solution in pH 8.0 phOSphate buffer (0.1M) containing 20 mg of protein. The reaction was allowed to proceed for h hours at 30°C and then carboxypeptidase A was added. Experiments with Leucine Aminqpeptidase Preparation of the Enzyme.— The enzyme was prepared according to Smith at 31 (5h). The procedure involves : 1. preparation of an acetone powder from swine kidney, 2. extraction with water and ammonium sulfate fractionation of the extract between 0.50 and 0.70 saturation, 3. a heat treatment at 70°C, and h. electrophoresis on a starch column. The enzyme had a Specific activity of 51 as measured on the substrate leucin— amide hydrochloride. Action of Leucine.Aminopeptidase on.fi-Amylase.—-A sample of sweet potato B—amylase (10 mg in h ml) was dialyzed against pH 8.5 tris(hydroxy- methy1)aminomethane buffer (0.005M). Leucine aminopeptidase was added to the B-amylase solution so that the ratio of the leucine aminopepti~ dase concentration to that of B—amylase was 1 to 50. At various times, aliquots were removed for B-amylase activity measurements. At the same 21 “time, aliquots were removed and examined for released amino acids. This amino acid analysis was carried out in an identical manner to that described in the carboxypeptidase A experiments. 8. Inhibitors Inhibition of B-Amylase,by:p-Chloromercuribenzoate.— The spectro- photometric determination of sulfhydnyl groups in proteins as developed by Boyer (55) was used in these studies. This method is based on the increase in absorbance at 250 mu of a mixture of the protein and p— chloromercuribenzoate as a consequence of reaction of p-chloromercuibenz— oate with sulfhydryl groups. Solutions of the enzyme were prepared by dialyzing the samples against the desired buffer. These dialyzed solu— tions contained approximately 1 mg of protein per ml and the reaction was studied at three pH values, h.5 (0.1M acetate), 6.8, and 8.0 (0.1M phosphate). The p—chloromercuribenzoate solutions were prepared by dis— solving 50 to 60 mg of the solid in the least amount of 0.1M sodium hydroxide, and diluting to approximately 5 x 10—4M with the appropriate buffer. In a typical experiment, 0.05 ml of the p—chloromercuribenzoate solution was added from a microburette to a 3—ml aliquot of-the B—amylase solution. The solution was carefully mixed and allowed to stand for 30 minutes in the sample compartment of the Beckman DU Spectrophotometer before determining the increase in absorbance. The sample compartment of the Spectrophotometer was maintained at 30°C during the run by means of a circulating thermostat. The addition of 0.05-ml aliquots of p— chloromercuribenzoate was repeated until the increase in absorbance at 250 mu was constant. Reactions were carried out under identical condi— tions of concentration and temperature for the determination of B-amylase activity. 22 Reactivation of B—Amylase Inhibited by p—Chloromercuribenzoate and Mercuric Chloride.-A B-amylase solution, which contained 1.68 mg of the enzyme per ml was dialyzed against pH 8.0 phosphate buffer. One ml of either a p—chloromercuribenzoate or mercuric chloride solution (0.075 umoles per ml) was added to 1 ml of the enzyme solution, and the mixture was allowed to stand at 30°C. Thirty minutes later, either 1 ml of versene (0.75 umoles per ml), cysteine (7.5 umoles per ml) or a combina- tion of both was added. These solutions were allowed to stand another 30 minutes before B-amylase activity was determined. Inhibition of_§:Amy1ase by N—Ethyl Maleimide.— Preliminary investi~ gations involved studies, over a wide pH range, into the effect of 10‘3M.N—ethyl maleimide on the catalytic activity of B-amylase. Samples of sweet potato B—amylase, containing approximately 1 mg per ml were dialyzed overnight against the desired buffer. Studies were carried out in pH h.5, 0.1M acetate buffer and 0.1M phosphate buffer pH 6.8, 7.0, 7.5, 8.0, and 8.5. An equal volume of 2 x 10-3M N-ethyl maleimide solu- tion, in the appropriate buffer, was added to the protein solution and the reaction was allowed to proceed at 30°C. At various times, ali— quots of the reaction were removed and tested for B-amylase activity. Spectrophotometric Determination of Sulfhydryl Groups by N-Ethyl Maleimideu- The procedure of Alexander (28) was employed in these studies. A sample of the purified enzyme was perevaporated, in the cold, to ap- proximately 6 to 8 mg per ml and dialyzed against phosphate buffer (pH 8.0, 0.1M). An aliquot of N—ethyl maleimide was added to the enzyme solution, making the final concentration of the N-ethyl maleimide 1 x 10-3M, and the decrease in absorbance at 300 mu was followed 23 Spectrophotometrically in the Beckman spectrophotometer. The sample compartment of the spectrophotometer was maintained at 30°C by means of a circulating thermostat. At various times, aliquots of the reaction were removed and tested for fi-amylase activity. From a knowledge of the amount of enzyme present, the decrease in absorbance at 300 mu, and the percent inactivation, Figure h was plotted (see Results). N-ethyl maleimide uptake in the presence of 8M urea was studied by adding 0.5 ml of the enzyme solution to 3 ml of 9.3M urea which was 1 x 10‘3M in N-ethyl maleimide. Again the decrease in absorbance at 300mu was measured. The urea solutions were always freshly prepared in the desired buffer, tested for cyanate (56) and contained 1 x 10’4M ethylenediaminetetraacetic acid. Action of Other Inhibitors on B-Amylase.—-B-Amy1ase solutions con- taining approximately 1 mg per ml were dialyzed against the desired buffer and an equal volume of either iodoacetic acid (1 x lO‘ZM, pH h.5, 0.1M acetate and pH 7.5, 0.1M phosphate), maleic acid (10'3M, pH 8.0, 0.1M phOSphate), or sodium arsenite (l x lO'ZM, pH 6.8, 0.1M phosphate) was added. At various intervals, aliquots were removed for activity measurements. 9. Quantitative Determination of S-stteinosuccinic Acid on the Stein-Moore Column Preparation of S-Cysteinosuccinic ACIdT— This compound was prepared according to the procedure of Morgan and Friedman (57,58). To 200 m1 of water, 9.3 g of maleic acid was added, and the pH was adjusted to 7.h. Cysteine (2.h2 g) was added to the resulting solution, and the 2h flask containing the mixture was evacuated and placed in a 37°C oven for 8 hours. The solution then was made acidic to congo red by the addition of dilute sulfuric acid, and 6 grams of mercuric sulfate was added. The precipitate which formed was removed by centrifuging, de- composed with hydrogen sulfide, and the resulting mixture was filtered. Cadmium acetate (2 ml of a 10 percent solution) was added to the filtrate to precipitate excess cysteine. The mixture was filtered. The mercuric sulfate treatment was repeated on the filtrate. The precipitate was decomposed again with hydrogen sulfide, and the mixture was filtered. The sulfate ion was quantitatively removed from this filtrate by the addition of a h percent solution of barium hydroxide. The barium sul— fate was filtered off, and the filtrate was reduced in a flash evaporator to approximately 50 ml. The remaining water was removed by lyophilliza- tion. The residue which was a white powder was washed in ethanol and dried in vacuo over phOSphorus pentoxide. The yield was 3.93 g (83 per- cent of theory). The specific rotation, [algo in water solution (c = 1 percent) was —29.h0. Recrystallization of 0.h g of this product from 10 ml of glacial acetic acid yielded 195 mg (h8 percent of theoty). The melting point was 135-136°C and the specific rotation, [d]fi° in water solution (c = 1 percent) was -.13. The percent nitrogen found was 5.87i0.13, calculated for C7H1106NS N was 5.87. To 100 ml of citrate buffer (pH 3.25) was added 23.7 mg of the re— crystallized S-cysteinosuccinic acid. One ml of this solution was placed on the l50—cm Stein—Moore column (this reporesents l uncle of the compound), and its position of emergence from the column was ob- served. Since diastereoisomers should be formed on the addition of qysteine to the double bond of maleic acid, attempts were made to resolve 25 the isomers. This was performed by lowering the pH of the eluting buffer to pH 3.0. The amount of color obtained when S-cysteinosuccinic acid is re— acted with ninhydrin, in a manner identical to that described for amino acid analysis is shown in the Results in Table XII. Preparation of S—Cysteino-(N—Ethyl)—Succinimide.— This procedure differed from that reported by Smyth and co-workers (31). L—cysteine hydrochloride monohydrate (875 mg) was added to 25 m1 of water and the pH was adjusted to 6.0 by the addition of 1N sodium hydroxide. To this solution was added 625 mg of N-ethyl maleimide. This reaction mixture was allowed to stand at room temperature for 1 hour and then brought to dryness in a flash evaporator at 30°C. The solid residue was crystal- lized from 90 percent ethanol. The yield was 950 mg (77 percent of theory). Five—hundred mg of this product was twice recrystallized from 80 percent ethanol. The recovery was 300 mg. The percentage composi— tion found for this substance was: C, hh.18; H, 5.60; 3, 12.93; N, 11.20. That calculated for C9H1404NZS was C, h3.90; H, 5.69; S, 13.01; N, 11.38. Conversion of S-gysteino-(N-Ethyl)—Succinimide to S-stteino— succinic Acid.— To 10 ml of water was added 2h.6 mg of S—cysteino—(N- ethyl)—succinimide. One ml aliquots of the resulting solution were placed in Pyrex hydrolysis tubes and lyophillized to dryness. Three ml of constant boiling hydrochloric acid was added to each residue, and the tubes were evacuated, sealed and placed in a 105°C oven for varying lengths of time. On completion of the hydrolysis, the solutions were evaporated to dryness in vacuo over sodium hydroxide. These samples were dissolved in 10 ml of pH 3.25 citrate buffer. One ml samples 26 then were placed on the Stein—Moore column and the amount of S—cysteino— succinic acid determined from the elution patterns. Yields of S~Cysteinosuccinic Acid from N—Ethyl Maleimide—Treated Sweet Potato B—Amylase.— The enzyme solutions were dialyzed against phosphate buffer (pH 8.0, 0.1M). To the enzyme solutions (15 to 20 mg in 5 to 10 ml of solution) was added an equal volume of 2 x 10'3M N- ethyl maleimide. The reactions were allowed to proceed to approximately 95 percent inhibition. The reaction period was then followed by either: 1. Dialysis against redistilled water, 2. passing the solution through a l x 10 cm column of mixed—bed resin (amberlite, MB—l), or 3. adjust- ing the pH of the solution to h.5, followed by dialysis against 0.1M acetate buffer (pH h.5) and then dialysis against redistilled water. The resulting solutions were placed in hydrolysis tubes and lyophillized. Constant boiling hydrochloric acid then was added to the residues, and the tubes were evacuated, sealed and placed in a 105°C oven for 72 hours. The hydrolysates were brought to dryness in vacuo over sodium hydroxide and made to a known volume with pH 3.25 citrate buffer. A sample of these solutions was placed on the Stein—Moore column and another frac— tion was used for micro—Kjeldahl nitrogen determinations. The reaction mixtures from the spectrophotometric analysis of N— ethyl maleimide and B-amylaSe in 8M urea were exhasutively dialyzed against redistilled water and treated in a similar manner for amino acid analysis. Experiments with N-Ethyl Maleimide on other Proteins Experiments with Aldolase.— Approximately 1 ml of the aldolase 27 SuSpension was dialyzed against a pH 7.8 ethylenediaminetetracetic acid buffer (lo—3M) and passed through a 1 x 10-cm column of mixed-bed resin (Amberlite, MB-l) (39). The effluent protein solution was perevaporated in the cold to 3 or A ml, and dialyzed against a pH 6.8 phosphate buffer (0.1M). To 2 ml of the enzyme solution was added 1 ml of a 3 x lO-SM N—ethyl maleimide solution, and the decrease in absorbance at 300mu and 30°C was followed in the Beckman DU spectrophotometer. The concentra- tion of aldolase was determined from its absorbance at 280 mu since the absorbance at 280 mu divided by 0.91 equals mg of aldolase per ml (39). Each reaction mixture of 3 ml volume contained approximately 10 mg of aldolase. Experiments in 6M urea were performed by adding 1 ml of the enzyme solution to 2 m1 of 9M urea which was 1.5 x 10’3M in N—ethyl maleimide, and the reactions followed in the Beckman DU spectrophotometer. On completion of the reactions as determined spectrophotometrically, the protein solutions were exhaustively dialyzed against redistilled water and lyophillized. Constant boiling hydrochloric acid (3 to h ml) was added to the residue and the tubes were evacuated, sealed, hydrolyzed for 72 hours in a 105°C oven and the samples diluted to a known volume with pH 3.25 citrate buffer. Aliquots of these solutions were placed on the Stein—Moore column and the amounts of S-cysteinosuccinic acid determined. The protein content of these solutions was calculated from: 1. the amino acid analysis and correlation of these values to the values reported by Rutter (59), and 2. micro—Kjeldahl nitrogen determinations. A value of 16.8 percent nitrogen.was used in calculating mg of protein from mg of nitrogen (60). .‘ -___&<-i_ 28 Experiments with 0va1bumin.— Samples of ovalbumin (10 to 20 mg in 5 ml) were dialyzed in the cold against the desired buffer. To 2 ml aliquots of the protein solutions were added 1 ml aliquots of 3 x 10'°M N—ethyl maleimide and the reactions followed in the Beckman DU spectro— photometer at 300mu. Experiments were performed at pH h.5 in 0.1M acetate buffer, and at pH 6.8 in 0.1M phosphate buffer. Experiments in BM urea were performed by first adjusting the pH of the urea solution to pH h.5. Approximately 25 mg of ovalbumin was diSSolved in 10 m1 of the urea solution. To 3 m1 of this protein solu— tion was added 0.5 m1 of 3.5 x 10‘3M N—ethyl maleimide and the reaction was followed spectrophotometrically at 300 mu in the Beckman Spectro- photometer. A standard curve of protein concentration versus absorbance at 280 mp was prepared and served for determining the amount of protein present in a reaction. The method for determining the amount of S— qysteinosuccinic acid was identical to that followed in the aldolase experiments. Protein concentrations of the hydrolysates were calculated from the amino acid concentrations and the amino acid composition reported in the literature (61). A molecular weight of hh,500 for ovalbumin was used in determining the moles of sulfhydryl that reacted per mole of protein. Experiments with fl-Lactoglobulin.— Experiments were carried out in 8M urea, pH 6.8, 0.1M phosphate, and the conditions were identical to the ovalbumin experiments. The molecular weight was taken to be 35,500, the absorbance at 280 mu of a 1 percent solution as 9.6 (55), and the percent nitrogen as 15.6 (62). 29 One experiment was performed in which the concentration of N-ethyl maleimide was 10‘1M. Approximately 25 mg of protein was dissolved in 10 m1 of phosphate buffer (0.2M, pH 8.0) and 125 mg of N—ethyl maleimide was added. The reaction was allowed to proceed at 30°C for 2h hours. After this the solutions were dialyzed against redistilled water and treated as previously described for amino acid analysis. Experiment with Polylysine.— A sample of polylysine (lh6 mg in 10 m1 of phosphate buffer, 0.2M, pH 7.5) was added to 62.5 mg of N—ethyl maleimide and the reaction was allowed to proceed at 30°C for 2h hours. The solution was exhaustively dialyzed against redistilled water, lyophillized, hydrolyzed for 72 hours and an aliquot of the hydrolysate placed on the Stein—Moore column. III. RESULTS AND DISCUSSION 1. Correlation of Protein Concentration to Absorbance at 280 mu Table 1. Correlation of Protein Concentration to Absorbance at 280 mu Mg of Protein per m1 Absorbance at 280 mu 0.057 0.082 0.096 0. 1112 0.192 0.276 0.38h 0.56h From these results it was calculated that the absorbance of a 1 percent solution is lh.h. This figure is not in agreement with that reported by Englard and Singer (5). These workers obtained a value of 17.1 based on a value of 13.5 for the percent nitrogen of the enzyme. When Englard and Singer's data is recalculated using 15.hl as the value for the percent nitrogen, the absorbance value for a 1 percent solution becomes lh.9. Balls e2 31 (h) reported that sweet potato B—amylase contains 15.1 percent nitrogen. This figure agrees favorably with the value of l5.h1 which was employed in these studies (1). The correla— tion of protein concentration to absorbance at 280mu provided a rapid and accurate method of determining protein concentrations, and was em- ployed throughout as a means of determining protein content. 2. Purification of firAmylase 0n.Sephadex G-75 Figure 1 shows that a purification of the enzyme is obtained on elution from the dextran gel, Sephadex G-75, with a small, inactive 30 31 ‘peak.appearing after the active component. This operation increases the activity of the B-amylase approximately 15 percent and yields a preparation which is homogeneous in the ultracentrifuge. 3. Results of the Amino Acid Analysis The results of the amino acid analysis are reported in Tables II and III. varying the length of hydrolysis indicated that threonine, serine, and tyrosine undergo a significant amount of decomposition. Therefore, the amounts of these amino acids were calculated by extrapo- lation to zero time. On the other hand, valine and isoleucine in— creased with time, and the true values were taken to be the 72—hour values. - Half-cystine and histidine are present in the lowest amounts. The molecule contains 22 half-cystine and 19 histidine residues. The acidic amino acids, aSpartic acid and glutamic acid, comprise 20 percent of the molecule. Assuming that the ammonia arises from hydrolysis of the amides, glutamine and aSparagine, it can be stated that approxi- mately one-half of glutamic acid and aSpartic acid is present as these amides. A. Action of the Exopeptidases on Sweet Potato B-Amylase Carboxypeptidase A.— Carboxypeptidase A is a pancreatic exopeptidase which catalyzes the hydrolysis of peptide bonds from the carboxyl terminus of proteins. The enzyme shows maximal activity towards carboxyl— terminal aromatic amino acids but is completely inactive toward sub- strates containing carboxyl—terminal arginine, lysine, or proline. It was reported previously (1) that proteolytic action of carboxypeptidase A F! 32 >._._>_._.o< J<2_o_mo hzmo mud nu nu nu nu nu 2 w w 6 4 2 Hr h»..b_ .— F. no N A Y 8.“ mw mm AA . _ . O 0 mu 160mm ._.< wozu50 compsfim .m ouomwm mmmZDZ wmnh o... . 8 on os on - _ 1 r mom n I l I L am< _ P . _ _ NAu Y C) aonvaaosevn ‘9 0 any A8 'were placed in a graph and all unknown amounts of S-cysteinosuccinic acid were calculated from this curve. Table XII. Color Yield from the Reaction Between Ninhydrin and S- cysteinosuccinic Acid Micromoles of Absorbance at 570 mu S-Cysteinosuccinic Diluted with 10 ml of Diluted with 15 m1 of Acid 50 percent Ethanol 50 percent Ethanol 0.1 .187, .190 0.2 .365, .375 0.3 .5A0, .5A5 .385, .385 0.6 .775, .780 Studies on.Sjgysteino-(N-ethyl)-succinimide.- Elution patterns of one umole of S—cysteino—(N—ethyl)-succinimide from a 30-cm column of Amberlite CG-l20 show the presence of two ninhydrin positive peaks. Hence it is postulated that the diastereoisomers resulting from the addition of dysteine to the double bond of N—ethyl maleimide are separ- able. These peaks are approximately of equal size. Table XIII. Conversion of S-Cysteino—(N-ethy1)-succinimide to S-Cysteino- succinic Acid Time of Hydrolysis Percent Conversion to (Hours) S—Cysteinosuccinic Acid 2A AA 72 87 120 9A A9 A study of the conversion of S-cysteino-(N-ethyl)-succinimide to S-cysteinosuccinic acid is reported in Table XIII. These data indicate that S—cysteino—(N-ethyl)-succinimide is quite stable to acid hydrolysis. An elution pattern from a 30—cm column of Amberlite CG-12O supported this fact. Figure 6 is an elution.pattern of a 72 hour hydrolysate of l umole of S-cysteino-(N-ethy1)—succinimide. The first ninhydrin positive compound to appear is S-cysteinosuccinic acid and it repre- sents 87 percent conversion from S-cysteino-(N-ethy1)-succinimide. ASpartic acid, which was added as a tracer, follows S-cysteinosuccinic acid, and finally the diastereoisomers of S-cysteino-(N-ethy1)-succin- imide are eluted. Since the protein hydrolyses were carried out for 72 hours, all S-cysteinosuccinic acid values were divided by 0.87 to correct for incomplete hydrolysis of S-cysteino—(N-ethy1)—succinimide. Reaction of N—Ethyl Maleimide with ProteinSa— Table XIV reports the results of the Spectrophotometric method, and the yields of S- cysteinosuccinic acid from hydrolysates of N-ethyl maleimidewtreated proteins. In evety instance the two methods compare favorably, indi- cating that under conditions which are suited for the spectrophotometric analysis, the reaction.betweean—ethy1 maleimide and proteins appears to be limited to reaction.with sulfhydryl groups. Complete acidic and neutral amino acid elution patterns were performed on all of the N- eathyl maleimide treated proteins. In each case the only new peak was fS—cysteinosuccinic acid. The sulfhydryl content of aldolase as calculated with this reagent (:ompares favorably with the values obtained by the action of p-chloro- inercuribenzoate on this molecule (55). B-Amylase, in 8M urea, shows the IDresence of 15 to 16 cysteine residues. Amino acid analysis indicates .\ 6 rl\ Kw~.r o.mH m.mH o.m mo m.mH «.ma .nowuse senennoed 2H.o .nons an onnasa<.a m.m w.m o.m an H.: s.m .noumsn sentenced 2H.o encase<-a -- m.nm m.c an N.cm H.5N .nomwsn sentenced 2H.o .nons so onnaocna as a; as an m.n a.s .nouosn onsenmoed 2H.o onnaooa< mw m.H H.N w.o ma H.“ o.~ .nooosn canenmoed 2H.o .nons 2m caflsnoflmonona-a o.m a.“ m.a an d.m m.m .noousn chanson 2H.o .nsns aw cassnan>o -- o m.e an .nonmsn sentenced 2H.o cassnan>o o o m.: an .nooosn oneness 2H.o nassnan>o nuns oficfioosmocfiopm>01m Ho mpfioww hm hfifimofippoaopocmoppooam «oopnmmoz mm cofipomom mcfiomuooc: mdbouo thoxdmasw Mo uoaadz mcofiofiocoo mcowpomom cfiopowm ofio¢ oficfiooSmocfiopm>Uum mo moan“? an“: compo: paupoaopoemoupooam oAp Mo aomwhwaaco 5% 2.3 ABSORBANCE C>8 (345 04 (>2 51 I. coon H Mini CHZSEMCOOH _ CH COOH \i IHiP - coon H2 hICH _ ZCHZSCI-IaNE _ (0‘20 3. l l A l 1 l l l 4 8 l2 IS 20 TUBE NUMBER Figure 6. Elution curve of a 72—hour acid hydrolysate of S—cysteino-(N-ethy1)-succinimide from a 30-cm Stein-Moore column. 52 the presence of 22 half-cystine residues. Hence, it can be concluded that the B-amylase molecule contains 3 disulfide linkages. Reactions in 0.1MZN-Ethyl Maleimide. Chromatography on an.Amber- lite CG—l20 column of acid hydrolysates of B—lactoglobulin which were treated with O.lM.N-ethy1 maleimide showed the appearance of a new peak which was eluted directly after proline. Polylysine, treated in an identical manner also gives rise to this peak. It is postulated that this entity arises from the alkylation of the e-amino group of lysine; however, further investigation will be required before this can be established. IV. SUMMARY 1. Purification of sweet potato B-amylase has been accomplished by elution of the enzyme from a Sephadex G—75 column. 2. A complete amino acid analysis of B-amylase is reported. 3. Sueet potato B-amylase is resistant to the proteiolytic attack of carboxypeptidase A and leucine aminopeptidase. This is illustrated by the complete retention of B—amylase activity and by the absence of free amino acids when these enzymes are allowed to react on B—amylase. A. Carboxypeptidase B catalyses the hydrolysis of approximately A amino acids from the carboxyl—terminus of fi—amylase. The combined action of carboxypeptidases A and B leads to a release of approximately 30 amino acids. Nevertheless, the activity of the B—amylase molecule is not affected. 5. p—Chloromercuribenzoate inhibits sweet potato B—amylase activ- ity. This inhibition may be reversed by the addition of a mixture of cysteine and versene. A spectrophotometric analysis of the reaction between p—chloromercuribenzoate and B—amylase in aqueous solution, indicates that six sulfhydryl groups are attacked. These results also suggest that only one of these sulfhydryl groups is required for activity. 6. N—ethyl maleimide also inhibits the catalytic activity of F3-aflw1ase. Fifty percent inactivation in aqueous solution is charac— 't€31?ized by the reaction of one sulfhydryl group. N-ethyl maleimide ITEBEtcts with four sulfhydryl groups in aqueous solution, and 15 to 16 slllihydryl groups in 8M urea. 53 5A 7. The possibility of other reactions between N—ethyl maleimide and proteins, under conditions which are suited for the spectrophoto- metric analysis of sulfhydryl groups, has been.excluded in the case of aldolase, E-amylase, ovalbumin, and B-lactoglobulin. In each in— stance the number of sulfhydtyl groups undergoing reaction as deter— mined Spectrophotometrically compares favourably with the yields of S-cysteinosuccinic acid produced from acid hydrolysates of N-ethyl maleimide treated proteins. (1) (2) (3) (A) (5) (6) (7) (8) (9) (10) (11) (12) (13) (1A) (15) (16) ('17) ( 18) (<19) (20) V. REFERENCES Evard, Rene. Ph.D. Thesis, Michigan State University, 1959. Gore, H. C. J. Biol. 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