SONICATION STUDIES ON. THE HYDROLYSIS 0F TRIGLYCERIDES AND EGG PHOSPHOLIPIDS BY LIPOLYTIC ENZYMES Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY LOUIS PAUL GOODMAN 1968 W“' A 1 D l J 3 I This is to certify that the thesis entitled SONICATION STUDIES ON THE HYDROLYSIS OF TRIGLYCERIDES AND EGG PHOSPHOLIPIDS BY LIPOLYTIC ENZYMES presented by Louis Paul Goodman has been accepted towards fulfillment of the requirements for Ph.D. Food Science degree in fifi/Q mM Major érofessoéo Date /{9 MAA/j/ /?éf 0-169 gilfik RY s." Q :V‘Aj\}- ‘ jl.‘ Stdkc dllllVVA.lty o‘— 1/ V x ' HUAB & SENS RflnK nmnrnv mn LIBRARY BINDERS Lil-IIIGPOIII. ”all“: mm 3] .rl... ..II ABSTRACT SONICATION STUDIES ON THE HYDROLYSIS OF TRIGLYCERIDES AND EGG PHOSPHOLIPIDS BY LIPOLYTIC ENZYMES by Louis Paul Goodman Crude pork pancreatic lipase was subjected to sonica- tion from 1—30 minutes using a Branson model 8-75 "Sonifier" at a frequency of 20,000 cps and intensity of 23 watts/cma. 0, 40°, Sonication was performed at temperatures of 100, 50 and 500C. The sonicated enzyme was then tested for activity by reacting it with an olive oil-gum arabic emulsion at 580C. Sonication at 100 and 300C affected the original lipase activity very little even after treatment for 30 minutes. However, during the first 5 minutes of sonication at 400C, there was a small but definite increase in activity. At 500C, a progressive decrease in lipase activity was noted as the sonication treatment was increased. Sonication of a semi- purified lipase preparation at 500C for i, 3, 5, and 10 minutes again produced a small increase in activity during the first 3 minutes of treatment. The crude lipase preparation and olive oil—substrate were sonicated at 580C for 4.5 minutes and the lipolysis compared to that in another sample which was stirred at 600 Louis Paul Goodman rpm for 4.5 minutes. Sonication increased the rate of hy- drolysis by a factor of 2.7. A tripalmitin emulsion, containing methyl myristate as a carrier, was sonicated in the presence of crude lipase for The degree of hydrolysis was compared 9.5 minutes at 450C. No hydrolysis of to a duplicate sample stirred at 600 rpm. tripalmitin occurred during the stirring experiment while 14 u equivalents of palmitic acid were liberated during soni- cation. aThe increased hydrolysis was attributed to the fact that a better emulsion was obtained during the lipolysis of the sonicated samples and to rapid renewal of surfaces for new complex formation. Various lipase preparations were assayed for their phospholipase A:lipase activity ratios using egg lecithin as a substrate. The percentage of saturated fatty acids in the residual lysolecithins was used to indicate the degree of phOSpholipase A activity in the enzyme preparation. Hydrolysis of various phOSpholipids by a semi—purified lipase preparation indicated the following order of re- phosphatidic acid > phosphatidyl ethanolamine > activity: B-arachidoyl lecithin. ' phosphatidyl choline > synthetic No hydrolysis occurred when lysolecithin was reacted for 4.5 hours with the semi-purified lipase preparation. This indi- cated the absence of any lysolecithinases in the preparation and that lipase did not react with lysolecithin to produce glycerOphosphoryl choline. Louis Paul Goodman The sonication for 15 minutes at 500C of phOSphatidyl ethanolamine or phOSphatidyl choline in the presence of lipase resulted in decreased hydrolysis of phOSphatidyl ethanolamine and no apparent hydrolysis of phOSphatidyl choline. To determine whether increases in the duration of soni— cation would produce corresponding decreases in the amount of lysocompounds produced, phOSphatidyl ethanolamine and phosphatidyl choline were sonicated for i, 3, 5, and 10 minutes with the semi—purified lipase preparation. This treatment was followed by a period of no sonication for 29, 27, 25, and 20 minutes,respectively. Analysis of the re— sults showed that the highest amount of lySOphOSphatidyl ethanolamine (59.56%) was obtained from the treatment in— volving no sonication, while in the case of lysophosphatidyl choline, the highest amount (51.91%) was obtained after a 5 minute sonication treatment. Lysocompound formation for both phOSphatidyl ethanolamine and phOSphatidyl choline decreased after 5 minutes of sonication. After 10 minutes of sonication, the amount of lysophOSphatidyl ethanolamine formed was slightly higher (29.44%) than for the 5 minute treatment, while the amount of lysophOSphatidyl choline formed decreased very rapidly to 5.5%. The results cited above may be explained partially by the preferential inactivation of the phOSpholipase activity Louis Paul Goodman in the enzyme preparation during sonication or by the con- tinuous changes in the type of micelles which are formed during the sonication. It is proposed that the hydrolysis is more rapid when the micelles are larger than when they are broken up into many smaller units. Sonication of phOSphatidyl choline or phOSphatidyl ethanolamine for periods of 10—15 minutes or longer, at 300C, produced many unknown peaks as evidenced by the GLC traces of the fatty acid methyl esters. Many of the peaks appear- ing immediately after the solvent front were due to oxida- tion products. It is suggested that some of the unknown peaks appearing downscale from the solvent front were due to hydroxy compounds which were formed at the site of the double bonds of the unsaturated fatty acids during the sonication treatment. Other products may be free radical induced polymers. SONICATION STUDIES ON THE HYDROLYSIS OF TRIGLYCERIDES AND EGG PHOSPHOLIPIDS BY LIPOLYTIC ENZYMES BY Louis Paul Goodman A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1968 DEDICATION This dissertation is dedicated to the memory of my mother and to my wonderful father whose long standing encouragement and inspiration is greatly appreciated. ii ACKNOWLEDGEMENTS My sincere appreciation is extended to Dr. L. R. Dugan, Jr. for his suggestion of the problem, effective guidance, good humor, and for his critical review of this disserta- tion. The author is grateful to Dr. B. S. Schweigert, Chair- man of the Food Science Department for his support and interest in this study. Appreciation and thanks are extended to the members of the guidance committee: Drs. C. L. Bedford, A. M. Pearson, and L. L. Bieber for their 0. Mickelson, C. H. Suelter, advice and effort in reading this dissertation. The assistance of Dr. S. Lande of the Department of Chemistry, Michigan State University, for performing and interpreting the NMR Spectra is greatly appreciated. I offer my thanks to my fellow graduate students, for their companionship and suggestions during the period this research was conducted. Lastly, the author is especially grateful to his wife, Eileen, for her understanding and encouragement throughout his graduate program. iii DEDICATION . . . . . . . ACKNOWLEDGMENTS. . . . . . . LIST OF TABLES . . . . . . . LIST OF FIGURES. . . . . . . . LIST OF APPENDICES . . . . . . INTRODUCTION . . . . . . LITERATURE REVIEW. . . . . Sonication. . . . . . Lipase. . . . . . . . Phospholipases. . . . . EXPERIMENTAL . . . . . . . Materials . . . . . . Methods . . . . . . . TABLE OF CONTENTS Sample Preparation . . . . Chemicals and Reagents . . . . O C O O O O O C O C C . O O O C I O O Extraction of Egg Phospholipids. . . Silicic Acid Column Column Chromatography. Diethylaminoethyl (DEAE) Cellulose Column Chromatography. . . . . . . . . . Thin-Layer Chromatography (TLC) . PhOSphorus Determination . . . . . . . Preparation of Methyl Esters for Gas—Liquid Chromatography. . . . . . . . . Gas—Liquid Chromatography. . . Preparation of PhosPhatidic Acid and Methyl— ated Phosphatidic Acid. . . . . Sonication Experiments Using Crude Hog Pancreatic Lipase . . . . . . . . . . iv Page ii iii vi vii ix 11 52 58 58 58 58 41 41 41 47 49 52 55 54 55 58 TABLE OF CONTENTS -Continued Page Preparation of Hog Pancreatic Lipase Frac- tions with Increased Activity. . . . . 67 Activity Measurement of Lipase Fractions. . 68 Synthesis of 8 Arachidoyl Lecithin from Egg Lysolecithin . . . . . . . . . . . 69 Egg Phospholipid Hydrolysis by Various Lipase Preparations. . . . . . . . . . 72 Sonication of Egg Phospholipids and The Sephadex G-200 Lipase Fraction . . . . 76 RESULTS AND DISCUSSION. . . . . . . . . . . . . . . . 79 Preliminary Experiments with Lipase and Its Reaction with Lecithin, PhOSphatidic Acid and Methylated Phosphatidic Acid. . . . . . 79 Sonication Experiments Using Crude Hog Pancre- atic Lipase (Experiments 1-6) . . . . . . . 87 Preparation of Hog Pancreatic Lipase Fractions with Increased Activity . . . . . . . . . 97 Synthesis of 8 Arachidoyl Lecithin from Egg Lecithin. . . . . . . . . . . . . . . . . . 105 . . . 104 Reactions of Lipase and Egg Phospholipids. Sonication of Egg Phospholipids and The Sephadex G-200 Lipase Fraction . . . . . . . . . . . 127 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . 158 PROPOSALS FOR FURTHER RESEARCH. . . . . . . . . . . . 140 LITERATURE CITED. . . . . . . . . . . . . . . . . . . 145 166 APPENDICES. . . . . . . . . . . . I I II III Ill ll , II LIST OF TABLES Table 1. 2. 10. 11. Percent of original lipase activity after 5 nica— tion treatment at constant temperature (1 2 C). . Starting and final temperatures of sonication at 1, 5, 5, 10, 20, and 50 minutes . . . . . . . . . Effect of sonication and stirring of enzyme and olive oil-gum arabic emulsion for 4.5 minutes at 580C 0 O O O O O O O O O O O O O O O Effect of sonication and stirring of enzyme and tripalmitin for 9.5 minutes at 450C . . . . . . Purification of hog pancreatic lipase by three different procedures. . . . . . Fatty acid composition of various phOSpho- O O O O O O O O lipids. . . . . . . Fatty acid composition of the lySOphOSphatidyl choline produced by the action of various lipase fractions on egg phosphatidyl choline . . . . . . Fatty acid composition of phosphatidyl choline and phosphatidyl ethanolamine hydrolysis products after reaction with the Sephadex G-200 lipase fraction (Experiment 9) . . . . . . . O O O O O 0 Fatty acid composition of egg phOSphatidic acid and lysophosphatidic acid formed after reaction with the Sephadex G-200 lipase fraction for 0.5 and 4.5 hours (Experiment 12) . . . . . . . . Percent lysocompound formed after reaction of various egg phospholipids with the Sephadex G-200 lipase fraction . . . . . . . . . . . Fatty acid composition of synthetic 3 arachidoyl phosphatidyl choline and lySOphOSphatidyl choline formed after reaction with the Sephadex G—200 lipase fraction (Experiment 15) . . . . vi Page 92 92 96 96 98 109 115 117 121 124 125 Figure 1. 2. 10. 11. 12. LIST OF FIGURES Arrangement of sonifier probe, water bath, ring motor, and metal beaker. . . . . . . Closeup View of the sonifier probe and metal beaker. . . . . . . . Infrared spectra of phosphatidic acid and methylated phosphatidic acid. . . . . Nuclear magnetic resonance Spectra of phOSpha— tidic acid. . . . . The effect of heating and sonicating for 1 m1n- on lipase activ- at various temperatures, ute, ity 0 O O O O O O O O The effect of sonication time and temperature on lipase activity. . . . Chart recording temperature of water bath 8nd enzyme during a sonication run at 50°C 1 2 C. Sephadex G-200 chromatography of the ammonium sulfate precipitate of hog pancreatic lipase. Linear relationship (Up to 0.15 ml) between lipase concentration and total units of activ— ity produced during lipolysis at 570 C . Electrophoretic separation of the Sephadex G—200 lipase fraction from hog pancreas . Preparative TLC plate of the phosphatidyl ethanolamine mixture after reaction With 0.5 ml of the Sephadex G-200 lipase fraction . Preparative TLC plate of the phosphatidyl choline mixture after reaction with 0.5 ml of the Sephadex G-200 lipase fraction. . . . . . vii stir- Page 59 59 82 84 88 95 99 101 102 106 106 LIST OF FIGURES - Continued Figure 15. 14. 15. 16. Preparative TLC plate of the phosphatidic acid mixture after reaction with 0.5 ml of the Sephadex G—200 lipase fraction. . . . . . . Preparative TLC plate of sodium deoxycholate and serum albumin used in the reactions of phos- pholipids (Figure 11-15) with the Sephadex G-200 lipase fraction . . . . . . . Percent lysocompounds formed after hydrolysis of phOSphatidyl choline and phOSphatidyl ethanolamine by the Sephadex G-200 lipase frac— tion (Experiment 15). . . . . . . . . . Activity of the Sephadex G-200 lipase fraction after sonication for 1, 5, 5, and 10 minutes. . viii Page 107 107 155 155 Appendix A. B. C. LIST OF APPENDICES Page Effect of sonication treatment on various enzymes mentioned in the literature. . . . . . 167 Standard curve for phOSphorus determination. . 171 Standard curve for protein determination . . . 172 ix INTRODUCTION During the 20 years from 1948-1968 ultrasonics, or the production of sound waves above 20 kc/s, has been applied to a variety of uses in many fields of interest (80,176). It is used in the food industry for cleaning and degreasing, mixing and emulsification, defoaming, density and viscosity measurements, and liquid level measurements. Uses of ultra- sonics in the biological field include rupturing of bacterial cells, virus extraction, emulsification, solubilization of lipids in aqueous suspensions, fat extraction, and studies on the activity of various enzymes after sonication. -Sonication of enzymes has resulted in both their acti- vation and deactivation depending upon the nature of the particular enzyme, and the frequency, intensity, duration, and temperature of sonication (See Appendix A). The only two papers which have been published on the sonication of lipase were by Kasahara and Yoshinare (119) who sonicated milk lipase and Buonsanto §§_§l, (59) who sonicated plasma lipase. Due to the fact that hog pancreatic lipase is com- monly used as a tool for triglyceride and phospholipid analysis, it was of interest to determine the effects of sonication on this enzyme. The literature also revealed that sonication was usually performed either on the enzyme or on the substrate, and they were then combined to measure enzyme activity. However, in this research the enzyme was soni- cated alone in addition to sonicating the combined enzyme and substrate. The action of pancreatic lipase on triglycerides is specific in that only the a, d' positions of a triglyceride are hydrolyzed. In addition, lipase will hydrolyze the primary ester sites of various other substances such as 1-monoglycerides, 1,2 or 2,3-diglycerides and esterified primary alcohols. Since many phospholipids of the glycero- phOSphatide type contain two acyl ester positions, it was reasoned that pancreatic lipase should act on these compounds to produce their respective Z-acyl ester lysocompounds. The problem, however, is complicated by the fact that the pan- creas contains many proteolytic and lipolytic enzymes. Therefore, a purified lipase is necessary to establish whether only the 1—acy1 ester is being hydrolyzed. Fortunately, many of the proteolytic and lipolytic enzymes may be separated from lipase by the standard techniques used in enzyme puri- fication, i.e., ammonium sulfate precipitation, extraction with various solvents, various types of column chromatography and various methods of electrophoresis. The lipolytic enzymes, aside from lipase, which are of importance during a purification procedure are phospholipase A and phospholipase B. Phospholipase A hydrolyzes the 2-acy1 ester position of a phospholipid while phospholipase B hydrolyzes the 1-acyl ester of a lysophOSpholipid. The methods required for testing the various enzymes that may be present in a particular preparation usually involve a combi- nation of thin-layer chromatography and gas liquid chroma— tography in addition to testing the enzyme on the standard olive oil emulsion and with a known synthetic phOSpholipid. The main objectives of this study were: 1. To determine the effects of sonication on the enzymic activity of hog pancreatic lipase for various time and temperature treatments. 2. To sonicate with the enzyme and substrate (either triglye cerides or pure egg phospholipid fractions) combined, and to compare the relative rates of hydrolysis with the rates in a non-sonicated system. 3. To establish whether sonica- tion may be used as a means of increasing the rate of lipase hydrolysis of the higher melting triglycerides. 4. To de- termine the ratios of phospholipase A: lipase activity in the various lipase preparations used for the hydrolysis of sonicated and unsonicated phOSpholipids. LITERATURE REVIEW Sonication History of Sonication (Ultrasonics) Brandish (32) stated that the study of ultrasonics actually began with the work of Langevin who obtained a British patent for a system of submarine signalling. Boyle and co-workers (31) followed Langevin in the physical field. Wood and Loomis, as cited by Brandish (32), conducted re— search in the biological field. Definition Sound waves which are produced above 20 kilocycles per second (kc/s) are considered to be in the ultra—high fre- quency classification (32). This definition is somewhat arbitrary since there is no set frequency which separates low frequency from high or ultra-high frequency sound waves. The basis for choosing 20 kc/s is that most humans can hear sound waves up to that frequency. Generation of Ultrasonic Vibrations Two methods are generally employed in ultrasonics to produce high frequency vibrations: the magnetostriction oscillator and the piezoelectric oscillator (32). The 5 former is produced by applying an axial magnetic field to an unannealed nickel rod. The rod will then undergo con— traction as the field strength is increased. By clamping the rod at its mid-point and exposing it to a suitably alternating axial magnetic field, it is possible to excite resonant longitudinal vibrations at a frequency in inverse proportion to the length of the rod. For a 12.5 cm. nickel rod, a fundamental resonance of 20.4 kc/s may be obtained. The piezoelectric oscillator uses the principle that the compression or extension of many crystals results in the development of an electrical charge on certain of the crystal faces. This so-called piezoelectric effect, occurs in quartz tourmaline, Rochelle Salt, and certain other crystals. The inverse piezoelectric effect, contraction and expansion in an applied electric field, is the basis of the piezoelectric oscillator. Ultrasonic vibrations of frequency as high as 7x105 kc/s may be obtained by the application of an alternating voltage of the correct fre- quency to the faces of a quartz disc of the required thick- BESS. Elfiquency and Intensity of Sonication Frequency as previously described is measured in cycles per second. It has been shown that in many cases the fre- quency of the sound wave has little or no effect on the chemical reaction rate (221). Buonsanto §£_§l, (39), while at 0.2 watts/cm2 intensity, and 5 second exposure, only slightly increased the amount of butyric acid produced from tributyrin by lipase. On the other hand, the same workers 4,000 kC/s, with an intensity of 0.5 watts/cma, and an ex- posure of 20 seconds. Intensity of sonication is usually rated in watts/cm2 of energy emitted from the surface of the sonicating probe tip. Most workers agree that by increasing the intensity of sonication the enzyme becomes more susceptible to inactiva- tion. Usually at very low intensities which are below the threshold of cavitation (the formation and violent collapse of small bubbles or cavities in the liquid as a result of pressure changes), no change is observed. Above this threshold the reaction rate increases more or less linearly (221). Cavitation is actually the cause for practically all of the observed chemical effects of ultrasonics in liquid sYstems (221). Chambers and Flosdorf (43) state that to cause denaturation of an enzyme, the intensity of the sonic waves must be sufficient to promote vigorous cavitation of the solution. Buonsanto 23.3l. (39) sonicated plasma lipase at an exposure time of 15 seconds and a frequency of 1000 kc/s increasing the ultrasonic energy from 0.5 to 2.5 watts/cme. They reported increasing inhibition of the en- zyme as the intensity was increased. Similar results were reported by the same group (38) using serum cholinesterase. Side Effects Caused by Sonication Besides cavitation,which is largely responsible for many chemical changes in sonicated systems, Thompson and Queirolo (213) mentioned various other phenomena which may occur during sonication of enzymes: 1. Rupture of the enzyme molecule, as indicated in the case of arginase by a lowering of the viscosity of the enzyme° 2. Distortion of the molecule, also indicated by a lowering of the viscosity. 3. The energy may be insufficient to disrupt or seriously distort the molecule and yet sufficient to weaken some of the many intramolecular and peripheral bonds thus facilitating chemical reactions with the products due to cavitation. + ++ . . . . 4. Detachment of Co+ and Mn activating ions in the case of arginase. Other chemical effects caused by intense ultrasound vibrations are reactions of hydrolysis, addition, oxidation, polymerization, depolymerization, and molecular rearrange- ment. The irradiation of water containing dissolved air produces hydrogen peroxide, nitrous acid, and nitric acid (221). The addition of oxygen to the olefinic linkage of oleic acid and certain oils results after sonication (89). Temperature A direct effect of sonication is an increase in tempera- ture of the sonicated material. Precise temperature control is difficult to achieve in a liquid undergoing irradiation, because the absorption of many watts of ultrasonic energy represents a heat source of considerable magnitude (221). In addition to this overall heating, diSpersed particles if present are raised to a temperature several degrees above the average temperature of the suspension (78,79). It is there- fore very important to cool the material being sonicated. Various workers have invented different devices to accom— plish cooling of the material during sonication (100,177,213). Duration of Exposure Duration of exposure to sonication is the next most im- portant parameter to consider after having chosen the desired frequency and intensity. There is general agreement among workers (38,55,97,160,185) that the longer sonication is performed, the more enzyme inactivation will occur. Under conditions where little or no cavitation is produced however, the above statement may not always hold true. For example, El‘piner gt__l, (82) sonicated a 0.01% aqueous solution of ribonuclease in air, at a frequency of 670 kc/s, intensity 8-10 watts/cma, 30 minute duration time, and found no losses in enzyme activity. The activation of certain enzymes at short durations of exposure, i.e., 10—300 seconds, has been reported (37-39, 42,117,174,175,214,234). However, if sonication was con- tinued there was usually a loss in activity. Methods of Controlling Enzyme Inactivation Since cavitation is necessary to cause enzyme denatura- tion and other chemical changes, it is desirable to prevent or suppress the amount of cavitation produced. This may be accomplished by using pressure (42,43), or working at condi- tions which are above or below the cavitation threshold (136). Cavitation is usually absent in a completely degassed liquid because the disappearance of the nucleating gas bubbles great- ly increases the strength of the liquid (80). Other methods used for reducing enzyme inactivation are: vaccuum (43), reducing substances (185) or blanketing the reaction vessel with such gases as nitrogen (18,42,43,46, 185) hydrogen (18,42,43,46,55,81,82,97,185,213), or argon (81). However, various workers reported that the inert gases did not prevent the inactivation of their enzymes (46,97,213). Enzyme Concentration and Purity Sonication of dilute enzymes usually results in their inactivation more rapidly than at higher concentrations. Oparin gt El- (160) studied the effect of ultrasonics on yeast invertase in aqueous solutions ranging from 0.001% to 10 0.01%. Results indicated that greater inactivity is achieved by long exposures and the use of very dilute solu- tions. Naimark and Mosher (158) related purity of the enzyme to resistance against sonication inactivation, even in dilute solutions. Using a 0.013% pepsin preparation, they sonicated at a frequency of 9 kc/s using a constant intensity of 200 volts, for time periods up to 25 minutes, at 13-160C. No inactivation of the pepsin was reported. In contrast, Chambers (42) found that there was a definite inactivation of pure pepsin. ‘ Sonication at 540-960 kc/s applied to whole milk first slightly activated the xanthine dehydrogenase and then slowly inactivated it. When applied to a su5pension of washed fat globules from cream, or to partially purified xanthine dehydrogenase, it rapidly inactivated the enzyme (174). Robert and Palonovski (175) found that xanthine oxidase in fresh milk is attached to the surface of the fat globules as lipoprotein cenapses. The enzyme is intensely activated before denaturation by ultrasonics and other physical means which break these cenapses. From these studies it appears that the enzyme is protected from inac- tivation when impurities or endogenous materials are in the sample. After these materials are detached from the enzyme by sonication, inactivation may then occur. 11 Lipase Historical Wills (226) in discussing the historical background of lipases, stated that one of the first observations of lipase activity in the pancreas was made in 1846 by Claude Bernard. Marcet, as cited by Sumner and Somers (209), described gastric lipase in 1958. The presence of lipases in plant seeds was demonstrated by Muntz (154) in 1871 and Green (98) in 1890. Although many researchers demonstrated the presence of lipase in various sources, there was relatively little advance in the methods of purification or the knowledge of the properties of lipase until the early 1920's when Willstatter and his colleagues began their investigations. For the next 30 years lipases received only minor attention and very little progress was made in this field. However, beginning with the mid 1950's, Desnuelle and his school have extensively studied the enzyme with regard to its purifi- cation and substrate specificity (65-74,83,84,139-142,187- 195). They were responsible for much of the knowledge we now have concerning lipase. Definition of Lipase Esterases comprise a broad and rather ill-defined group of enzymes hydrolyzing ester bonds in various substrates. Lipases are customarily classified in the esterase family, 12 but the borderline between esterases prOper and lipases has never been established with absolute certainty (4,113,155). Lipases are usually associated with the degradation of typical triglycerides. Another definition states that lipase acts upon longer-chain fatty acids, while esterases act preferentially on the shorter—chain fatty acids. Desnuelle and Sarda (72) suggested that the differentiation between an esterase and lipase be based upon the physical state of the substrate, i.e., lipases act primarily on emulsified sub— strates, while esterases act on soluble substrates. The latter definition, however, is in disagreement with many workers who used water-soluble esters, e.g., p—nitrophenyl acetate or Tweens as "Lipase substrates." Whenever water- soluble substrates have been used, it appears that an ester- ase and not a lipase was actually under investigation (226). The official name of lipase as designated by the International Union of Biochemistry is glycerol ester hy- drolase and is given the number E.C.3.1.1.3 (77). Distribution of Lipases Lipases are found in animals, plants, and microorgan- isms. Hog pancreatic lipase has received much attention in the past, but more recently the lipases of microorgan- isms, castor bean, and milk have also been studied. A de- tailed review of the distribution of lipase is given by Wills (226) and Desnuelle and Savary (74). Ill (I. II 'I) [ll- I'I l I- ll] III | Ill. [ I‘ll {I 13 Specificity of Pancreatic Lipase Alcohol Type ang_Sequence of Fatty Acid Splitting from Triglycerides. Pancreatic lipase is specific in hydrolyz- ing the esters of primary alcoholic groups (13,147). Thus, in a triglyceride, the 1 and 3 positions or the d and a‘ positions would be attacked. The hydrolysis of a trigly— ceride by pancreatic lipase is now assumed to follow a three step process as shown in the diagram below (226) HgOg-Rl HOE-R2 + R3C00H 1 o. H20C—R1 ‘ CH20H 0 Lipase CHeoH E H20H 2 B CHot-Ra a=- H0 —R2 g Emulsion HOH , System H20H H20H -—+ 3 (I CHZO ‘R3 8 // + CHQOH CHO -R2 R1COOH + L + RgCOOH H2 0423 + R1C00H R3C00H Actually very little glycerol is ever formed either in vitro (9,68-70,90) or in vivo (3.23.27.65.147). Both the in vitro and in vivo studies have shown that a large accumulation of mono and diglycerides are present even after prolonged hydrolysis. Although lipase is known to act exclusively on the 1,3-positions of a triglyceride resulting in the forma- tion of the 2-monoglyceride, Savary and Desnuelle (193) have found only 70-80% of the 2—isomer and 20-30% of the 14 1—isomer. The presence of the 1—isomer may readily be ex- plained by the fact that the 1,2-diglyceride and 2-mono— glyceride may isomerize to yield a 1,3-diglyceride and a 1-monoglyceride respectively. The studies of Mattson and Volpenhein (150) showed that the 2-monoglycerides are very unstable when mixed with water at alkaline pH. Desnuelle and Savary (74) have cited unpublished work by Entressangles, where the latter author observed that in the presence of free acids, isomerization of 2—monoglycerides is slowed down, and it stops when about 20—30% of the 1-isomer is formed. Borgstrom (26) showed pancreatic juice free of ester- ase activity did not hydrolyze the 2-ester bond if the 1 and 3-ester bonds were substituted by ether linkages. Levizky (129) also using a molecule containing ether substituted linkages for the 1 and 3-ester bonds, concluded that pan— creatin, which is a crude lipase preparation, hydrolyzed the 2-ester bond while a purified preparation did not. Entressangles g; 3;. (84) synthesized a lecithin (1,3-di- palmityl 2—[9, 10~3H]oleylglycerol) labeled in the 2-ester position to prove the specificity of pancreatic lipase. They showed that a large isomerization of the labeled oleic acid takes place, eSpecially in the monoglyceride fractions, and concluded that the positional specificity of pancreatic lipase appeared to be practically absolute. These studies were carried out using a highly purified hog lipase prepara- tion. 15 Coleman (49) postulated that the positional Specificity of lipase was improved by purification. However, Desnuelle and Savary (74) took issue with Coleman and stated that so far, better results have not been obtained with a purified lipase preparation than with a crude preparation containing the same number of lipase units. Pancreatic lipase hydrolyzes esters of different alco- hols, as shown by the studies of Balls and Matlack (13,14). They concluded that only esters of primary alcohol groups were split, and the rate of hydrolysis depended upon the length of the fatty acid chain. (Methyl butyrate was hydro- lyzed by pure pancreatic lipase, provided the ester was present in an emulsion, but the hydrolysis was slower than that of tributyrin (187). Methyl oleate was hydrolyzed by lipase at only one thirtieth the rate of triolein (187). Stereochemical Specificity of Pancreatic Lipase. Since most enzymes possess a stereochemical Specificity, it was thought that perhaps lipase may be Specific for only certain forms of triglycerides. Therefore, Tattrie §t_§l, (212) prepared D, L, and D L 1,2—di-palmityl-3-olein by acylation of the corresponding stereoisomers of 1,2-dipalmitin and subjected them to lipase hydrolysis. The diglycerides iso- lated from the lipase digestion had no detectable rotatory power. The authors concluded that lipase is not a stereo- specific enzyme. 16 Jensen e al. (116) investigated the Specificity of pancreatic lipase on a series of trans fatty acids. They prepared glyceryl—i-elaidate-Z,3-dioleate, glyceryl—1-laurate- 2,3-dielaidate, glyceryl-l—elaidate—Z,3-dilaurate, glyceryl- 1-elaidate-2,3-dilinoleate, and glyceryl~2-elaidate-1,3— dilaurate by acylating pure 1-monoglycerides and 1,3—dilaurin. Pancreatic lipase did not differentiate between elaidic acid and its cis-isomer oleic acid, or linoleic and lauric acids when each respectively was at the primary ester positions of the same molecule. Thus, there was no fatty acid specificity and positional specificity was also maintained. Effect of Fatty Acid Chain Length On Hydrolysis Rates and Intramolecular and Intermolecular Specificity of Lipase. Workers using various triglycerides ranging from triacetin to triolein showed that the triglycerides containing short chain fatty acids are hydrolyzed much faster than those con- taining longer chain ones. Most investigators agreed that either tripropionin (220,225) or tributyrin (197,206) gave the maximum rate of hydrolysis with lipase. Entressangles gtflgl. (83) using synthetic 1-palmityl-3-butyryl-glycerol showed that pancreatic lipase hydrolyzed the shorter chains on the molecule more rapidly than the longer ones. When the glyceride was 10% hydrolyzed, the free fatty acid mixture was found to contain 72% butyric and 28% palmitic. 17 Jensen gg El. (115) stated that if pancreatic lipase differentiated between long and short-chain acids attached to the primary positions of the same triglyceride there was an intramolecular Specificity of the enzyme. However, if lipase differentiated between classes of triglycerides there was an intermolecular specificity of the enzyme. The authors carried out a series of experiments using glyceryl 1-palmiw tate 2,3-dibutyrate (PBB) and glyceryl 1—myristate 2,3- dioleate (M00) as substrates for the enzymatic hydrolysis. Pancreatic lipase released butyrate and palmitate in equi— molar quantities from PBB which meant there was an absence of intramolecular Specificity. However, when equimolar quantities of M00 and PBB were mixed, the enzyme hydrolyzed the latter more rapidly, indicating an intermolecular specificity. When milk fat was used as a substrate for pan- creatic lipase, the enzyme digested some classes of milk triglycerides more rapidly than others. Brockerhoff (33,233) faced the same problem in tri- glyceride analysis of marine fats that Jensen did with milk fats. He found that a meaningful stereOSpecific analysis of a triglyceride could not be performed unless a truly random d,8-diglyceride was obtained. Although such truly random d,8-diglycerides may be prepared from many fats with pancreatic lipase, they have not been prepared from lard or marine oils. The reason for this was that the diglycerides Obtained from lard by pancreatic lipase hydrolysis contained 18 an excess of stearic acid (50) while the diglycerides of marine fat contained an excess of long chain fatty acids (33). In analyzing human depot fat Brockerhoff (33) found that a random lipolysis with pancreatic lipase may sometimes be achieved by diluting the fat with hexane. The author proposed that neither the enzyme nor the substrate, molecu— lar triglyceride, are responsible, but that the effect was due to a structural property of the fat of glycerolmester groups with the fatty acid chains oriented toward the inside in a more or less parallel fashion. The van der Waals forces in the droplet will be most effective on chains that are straighter and longer than the others. As a result, there is a different pull on different fatty acids toward the center, and in consequence a partial withdrawal of certain ester groups from the surface; these grOUps thereby become less readily available to the hydrolytic enzyme. Aside from this effect, the nature of the fatty acid is of no importance in lipolysis. This hypothesis (which would also explain the preferential hydrolysis of short chain acids) is supported by the finding that inclusion of a solvent, for instance hexane, can abolish discrimination. The simplest explana- tion is that the solvent interrupts associations between fatty acid chains, thus promoting a diSpersion of van der Waals forces and a more random structure of the fat droplets. However, the inclusion of hexane in lipolysis is not always 19 effective. For instance, in a cod liver oil and a seal oil, both containing high percentages of 20:5 and 22:6, these acids accumulate in the diglycerides with or without added hexane. In such cases a stereo Specific analysis cannot be performed until conditions are found which allow random lipolysis. These conditions were finally achieved by Yurkow— ski and Brockerhoff (233) with the use of methyl magnesium bromide. Bottino gt al, (28) reported on the resistance of cer— tain long-chain polyunsaturated fatty acids of marine oils to pancreatic lipase hydrolysis. When whale oil trigly— cerides were subjected to pancreatic lipase hydrolysis, eicosapentaenoic and docosahexaenoic acids were found mainly in the di- and triglyceride products, suggesting that they are in the 1,3—positions but resistant to the action of lipase. Their presence in the 1,3-positions and resistance to pancreatic lipase hydrolysis were confirmed. Docosa- pentaenoic acid was also shown to be present in the 1,3- position of whale oil triglycerides but was not lipase re— sistant. The authors postulated that the presence of a double bond near the carboxyl group exercised an inhibitory effect, or that the location of the double bonds in the resistant acids (20:5 and 22:6) placed their terminal methyl groups close to the carboxyl, producing a steric hindrance effect. These arguments were supported by the fact that the C acid of synthetic 1,2-di-octadecenoyl—3-eicosa— 20:5 pentaenoyl glycerol was resistant to pancreatic lipase. 20 The views presented by the above authors offers an explanation to why Brockerhoff's (33) experiments with marine fats did not give random hydrolysis with pancreatic lipase, even in the presence of hexane. It appears that Brockerhoff's theory is correct for only Special cases. Specificity of Lipase Towards PhOSpholipids. Recently De Haas §£_§l, (62) demonstrated that a highly purified hog lipase preparation can hydrolyze the saturated fatty acid esters from the 1 position of egg lecithin. To prove that the positional attack of the enzyme was not governed by the position of the saturated fatty acids in the molecule, they synthesized 1-oleoyl-2-stearoyl-glycero-3—phOSphoryl choline and reacted it with lipase. Upon analyzing the lysolecithin formed by the reaction, only stearic acid was found. This indicated that the site of attack of the enzyme was the same for triglycerides and lecithins, independent of the degree of saturation of the fatty acid constituents. Two phOSpholipases isolated from homogenates of rat pancreas have been studied by Van den Bosch gtflgl. (217). By using a doubly—labeled synthetic lecithin, the authors found that both the 1-acyl and 2—acyl lysolecithins were formed when the lecithin was reacted with the enzyme. The ratios of the two lysolecithins formed could be varied by the absence or addition of deoxycholate and by heat treat— ment of the enzyme. They designated the enzyme producing the 2-acyl lysolecithin as phosPholipase A1 and that 21 producing the 1—acyl lysolecithin as phOSphOlipase A2. PhOSpholipase Al was found to be heat labile. In view of the fact that De Haas _t_§l, (62) showed that lipase will act on the 1-acyl ester position of lecithins, it could very well be that phOSpholipase A1 may actually be a lipase. The hydrolysis of labeled phospholipids and glycerides by rat-liver preparations was reported by Waite and Van Deenen (219). They performed a subcellular fractionation of the homogenate and found that phOSpholipase A1 activity (Specific for the 1-acyl ester) was located mainly in the microsomes, phospholipase A2 activity (Specific for the 2- acyl ester) was in the mitochondria, and lysophOSpholipases were in the soluble fraction. Lipase activity was found mainly in the mitochondrial fraction. These reports tend to indicate that phospholipase Al was not a lipase since Al was found in the microsomal fraction rather than the mitochondrial fraction. The authors stated,however, that the above results were not conclusive since there could have been contamination of the fractions by one another. Effect of Fatty Acid Unsaturation On Hydrolysis Rates. Early investigations gave evidence that pancreatic lipase preferentially Split the unsaturated fatty acids on the triglyceride molecule. Later work failed to confirm these findings. The controversy was finally settled by a series of studies conducted by Savary and Desnuelle (191—193). 22 Using synthetic triglycerides e.g., palmityldiolein and oleyldipalmitin (shown below) they found that the fatty acids in the 1 or 3 positions were readily Split, and that oleic acid in the 1-position was hydrolyzed only slightly faster than palmitic acid in the 1-position. They did not regard the faster splitting of oleic to be Significant and ~concluded that as a general rule the existence of one or two double bonds in the fatty acid did not affect the rate of hydrolysis. (1) CHgo-oleic (1) IHgo-palmitic (1) Hgo-oleic (2) HO-oleic (2) HO-oleic (2) HO-palmitic (3) CH20-palmitic (5) CHgo-palmitic (5) H20-palmitic The Use of Lipase As A Tool For The Analysis of Lipids Distribution of Fatty Acid Chains in Triglycerides. Hydrolyzing triglycerides with pancreatic lipase offers one of the most effective methods for determining the fatty acid distribution of natural fats and oils (29,30,34-36,48,49,51, 115,132,146,148,149,183,193—195,218). Since lipase is Specific for the 1,3-positions of a triglyceride, these fatty acids may readily be determined. Usually the hydrolysis lasts only a few minutes (5~15 min) to avoid the possibility of isomerization of the 2-monoglycerides and the 1,2-digly- cerides which are formed (74). If isomerization occurred, the 8 fatty acids would migrate to the a positions of the 23 glycerol moiety and would then be subjected to hydrolysis by lipase. Structure of Phospholipids. The positions of the fatty acids on purified egg phosphatidyl choline were determined by treating the preparation with a phosphatidase from 9;, perfringens which removed the phOSphoryl choline group (211). The resulting 1,2-diglyceride was prevented from isomerizing by acylating the free hydroxyl groups with myristic acid, an acid which was not present in the original egg lecithin. Lipase was then used to liberate fatty acids from the 1 and 3 positions. Only myristic, palmitic, and stearic acids, were found. Thus, it was concluded that the 1 position of egg phOSphatidyl choline contained saturated fatty acids while the 2 position contained unsaturated fatty acids. Privett and Nutter (167) determined the structure of lecithins from milk, soybean, egg, wheat germ, and safflower via the formation of the acetylated 1,2—diglycerides. The 1,2-diglycerides were first prepared either by phospholipase C degradation of lecithin or acetolysis with acetic acid— acetic anhydride. Fractionation of the acetylated 1,2- diglycerides was accomplished by TLC plates impregnated with silver nitrite. .After GLC analysis of a portion of the acetylated 1,2-diglyceride fractions, the remainder of the fractions were subjected to pancreatic lipase hydrolysis. 24 The products of lipase hydrolysis were chromatographed on TLC plates and monoglycerides isolated and subjected to GLC analysis. From this analysis, the authors determined the isomeric species of the various lecithins. The results obtained with egg lecithin in these experi— ments are not confirmatory to those obtained by Tattrie (211) who stated that only saturated fatty acids are present in the d' position and unsaturated fatty acids are present in the 8 position. Kuksis and Marai (125) also determined the complete structure of egg lecithin. Although working independently from Privett and Nutter (167), they used almost exactly the same techniques as reported by the latter authors. The re- sults on egg lecithin reported by the two groups are in very good agreement. Thus, it may be concluded that there is. between 9-10% of unsaturated fatty acids in the d' position of egg lecithin and 0.9—1.3% saturated fatty acids in the 6 position. .Evidently Tattrie's (211) finding that there are no saturated fatty acids in the d' position of egg lecithin is undoubtedly incorrect. Factors Affecting The Velocity of Hydrolysis Physical State of the Emulsion. Glick EE.E$3 (94) and Frazer gt al. (91) in the 1930's suggested that the velocity of lipase action depended upon the surface area of the 25 emulsion. The fact that lipase is adsorbed at the fat: water interface and acts at the interface was theorized by Sym (210) and Bayliss 23 gig (17). However, these observa- tions were ignored until they were clearly demonstrated by Desnuelle and colleagues. Benzonana and Desnuelle (21) enhanced the original study (187) and showed that lipase is adsorbed by its emulsified substrate and the initial rate of the reaction is a function of the number of enzyme mole- cules adsorbed at the interface. Activators and Inhibitors. Bile salts have long been known to serve as activators for pancreatic lipase (168). Willstatter'gtlal. (227,228) found that bile salts activated hydrolysis of triglycerides only in an alkaline medium while Glick and King (94) noticed that tributyrin hydrolysis was inhibited if bile salts were present at pH values below 7. Wills (222) found that although triolein hydrolysis by lipase was activated by sodium taurocholate in an alkaline medium it was inhibited at pH values below 7. The effect of bile salt may also depend upon the length of fatty acid chain of the triglyceride under investigation as shown by Glick and King (94) and Wills (222). Maximum activation of lipase hydrolysis was obtained when the OH groups in the bile salts were located on the 3 and 12 positions of the sterol ring (201,204). Wills (226) hypothesized that bile salts may play a role in 26 aligning the enzyme molecules in the interfacial layer of the substrate emulsion. In trying to elucidate the activation mechanism of hog pancreatic lipase by sodium taurocholate, Fritz and Melius (92) stated that it appeared as though the hydrolysis of triglyceride to diglyceride was facilitated and the hydroly- sis of diglyceride to monoglyceride was depressed in the presence of the salt. They also concluded that sodium taurocholate acted to Split the diglyceride-enzyme complex and this increased the action of the enzyme on the trigly- ceride ester. The latter is due to the fact that the hydroly- sis of the triglyceride is a much faster reaction than hy- drolysis of either the mono- or diglycerides. The fact that calcium ions activate lipase activity was originally described by Willstatter 23 al. (227,228). Clement gt 3;, (47), found that lipase activity of pancreatic juice was activated by the addition of Ca++ in the presence of sodium taurocholate and the replacement of Ca++ by Na+, K+, Li+, or Mg++, led to a reduction in the rate of hydroly» sis. Shimiza (203), however, stated that lipase was acti- + + +++ ++ + ++ ++ vated by Al+ +, Fe++ , Cr , Fe . Na I Sn I M9 , Pb++. Co++, Zn++, Ba++, NH4+, and K+ in a 0.01M solution. Although it was suggested by Schdnheyder (198) that the primary role of calcium ions was to remove fatty acids formed in the hydrolysis as insoluble calcium soaps, Wills (224) suggested that calcium ions had the additional function of maintaining lipase stability. 27 While testing the effect of several amino acids on lipase activation, Yamamoto (230,231) found that histidine was a more effective activator than histamine. Vitamin P, pantothenic acid, nicotinic acid, and penicillin have also been found to be activators of pancreatic lipase (122). The maximum rate of hydrolysis will occur only when an adequate interfacial area is maintained; therefore, any sub- stance destroying the emulsion and reducing the interfacial area of the substrate may be classified as a lipase "inhibitor." However, true lipase inhibitors must combine with the enzyme and affect it in some manner (226). Lipase is inhibited by several heavy metal ions such as Cu++, Hg++, and Zn++ (162,224), but the inhibition actually depends upon experimental conditions used, and especially the buffer and emulsifying agents. Oxidation of lipase with H202 or other oxidizing agents destroys or reduces lipase activity (114,202). Benthaus (20) found that high pressure inacti— vated lipase. Lipase has been found to be inhibited by certain —SH reagents but to be resistant to others. Inhibition also varied with the substrate used. It is now considered un- likely that -SH groups are part of the active center but that they may be adjacent to it (225). Pancreatic lipase is also inhibited by thiourea, chloroethanol, formaldehyde, quinine, atoxyl, chaulmoogric acid, cinnamate, ricinoleate, fatty acids, vitamin C, chlortetracycline, isoamyl alcohol, isoamyl isobutyrate, sulfa drugs such as sulfadiazine or 28 sulfathiazole (226). Di—iSOprOpyl fluorophosphate (DFP) does not affect pancreatic lipase, but it is inhibited by diethyl-p-nitro—phenyl phOSphate (66,73). DFP inhibits chymotrypsin and other proteinases and is therefore used during lipase purification to prevent digestion of the lipase by other enzymes (190). Temperature and pH. The range for optimum activity of pancreatic lipase is usually described as 55-400c (178,196). Wills (224) showed that pancreatic lipase loses 36% of its activity after 10 minutes at 500C, and 43% after 14 hours. The pH plays a dual role in affecting both activity and the emulsified substrate. The latter is directed towards the substrate: aqueous phase interface. The pH optimum often depends on the type of substrate used (226). Values of pH ranging from 8.2 to 9.2 have been described as optimum for pancreatic lipase (67,96,112). According to Borgstrdm (24), the presence or absence of bile salts will change the pH optimum of lipase activity. Purification of Lipase In 1923 Willstatter g£_§l, (228) attempted to purify lipase but succeeded in obtaining only a few fold purifica- tion. A 10 fold purification of the enzyme was obtained by Glick and King (95) by extracting pancreatin with sodium chloride solution and precipitating the lipase with magnesium 29 sulfate. Wills (223,225) modified Glick and King's procedure and was able to obtain a final purification of 70 fold. Desnuelle and co~workers in France have made the most significant contribution to the purification of pancreatic hog lipase (139-142,188-190). From 1957 to the present (1968), they have continuously improved purification techniques so that both a high Specific activity and yield are obtained simultaneously. In 1964, Sarda _£__;, (190) succeeded in obtaining a lipase preparation with a Specific activity of 7000. Ultra- sedimentation studies of this preparation showed it to be a protein more than 95% homogeneous, sedimenting with a value of Sgo=4.1 (62,186). The main purification steps of their technique involved column chromatography on Sephadex G-200 two times, and once on DEAE. This preparation was free of phospholipase A activity. Fritz and Melius (92) used a 7 step procedure for their purification scheme involving extraction and precipitation with salts and solvents, carboxy methyl cellulose, chroma- tography, and electrophoresis. Their final Specific activity was 7,700. Basky's §£_§l, (16) purest preparation had a specific activity of 10,090 in the presence of sodium deoxycholate. Melius and Simmons (152) prepared a lipase preparation by using only saline solution, ammonium sulfate, butanol, and acetone for extraction and precipitation. They measured 30 the specific activity of their enzyme against olive oil (18,000) and tributyrin (36,000) as substrates. Sarda (186) stated that lipase preparations completely devoid of phos— pholipase A activity can be obtained by the above (152) procedure by using an additional step of chromatographing the enzyme on DEAE—Sephadex. Methods of Measuring Lipase Activity Since lipase hydrolyzes only water-soluble substrates in a heterogeneous system (187), any method proposing the use of a water-soluble substrate must be rejected. Water- soluble systems that have been used are the Tweens (7,22), p—nitrophenyl acetate, and p—nitrophenyl butyrate (93). Lipase activity may be determined by a number of techniques which involve the use of a triglyceride emulsion and the subsequent determination of ester bond hydrolysis. The hydrolysis of the ester bond may be followed by titrimetry to a phenolphthalein end point (87) or potentiometrically with a pH meter to a constant end point (141). When kinetic studies are involved, the latter technique may be fully automated by the use of a recording pH stat (71,92,141,163, 170). The use of nephelometry (25,99), where the Optical density at 650 mu is measured, provides an indirect method of measuring lipase activity. This method measures the decrease in optical density as the triglyceride emulsion 31 "clears" during lipase hydrolysis. Manometry (224) is another method of determining lipase activity. No emulsifier is added in the manometer technique. Kramer and Guilbault (123) used a fluorometric determin- ation of lipase activity. The method is supposed to determine lipase activity in the presence of other esterases. Dibutyrl- fluorescein ii; used as the substrate for lipase. The equa- tion for the reaction is shown below: Lipasep_ pH 8.07, H FLUORESCEIN -CH2-CH2-CH3 + 2 CHs-CHg-CHg-COOH DIBUTYRLFLUORESCEIN BUTYRIC ACID The authors reported that only lipase, acylase, and chymo- trypsin, of all hydrolases tested, catalyzed the hydrolysis of the substrate. If tributyrin is used as a substrate, a stalagometric method may be employed to determine the rate of its disap— pearance. The surface tension of a tributyrin emulsion approaches that of water as the tributyrin is hydrolyzed. The dr0p rate of the reaction mixture is counted and compared 32 with that of a control without lipase (126) or the drOps may be weighed (127). A method for determining small amounts of lipase activ— ity by Florisil column chromatography and liquid scintil— lation Spectrometry was employed by Chino and Gilbert (45). The authors added triolein, uniformly labeled in the carboxyl positions with C14, to a pure unlabeled triolein carrier. The two triglycerides were sonicated in 5% polyvinyl alcohol and subjected to lipase hydrolysis. After extracting the products formed by the reaction, the mixture was placed on a Florisil column and eluted with the appropriate solvents to separate the unreacted triglycerides from the diglycerides, monoglycerides and free fatty acids. The various fractions were then subjected to radioactivity assay in a liquid scin- tillation spectrometer. Phospholipases The nomenclature followed in this review will be that of the Commission on Enzymes of the International Union of Biochemistry. It should be pointed out however, that there is disagreement by many workers with this nomenclature. At present there is total agreement that phOSpholipase A removed the 8 fatty acid of a phospholipid. However, there is confusion about phospholipases B, C, D, and lysophosPho- lipase. The enzyme commission calls lysopholipase, phospho- lipase B. The latter removes the fatty acid from a 33 lysophosPholipid. However, other workers say that these are two distinct enzymes. PhOSpholipase B removes both fatty acids on the diacyl phOSpholipid, while lysophOSpho- lipase removes the fatty acid of either the d' or 8 lyso- phospholipid. The disagreement between phospholipase C and D is not quite as severe as with phOSpholipase B and lyso- phOSpholipase. As stated by the enzyme commission, phos— pholipase C releases the diglyceride while phOSpholipase D releases choline. Contardi and Ercoli (52) have this order reversed, i.e., phospholipase C would be the enzyme releas- ing choline, and phOSpholipase D the one releasing the diglyceride. B d' \\\i ,qHEOOCR R'CO TH B O + a A CH20-g~0CH2~CH2N(CH3)3 C ..) D Bonds on L~ClphOSphatidlehOline attacked by phOSpholipaseS A, B, c, and D. (according to the E.C.I.U.B.) PhOSpholipase A (Phosphatide Agyl-Hydrolase) E.C. 3.1. PhOSpholipase A hydrolyzes only one of the fatty acyl 1.4. ester linkages in diacyl phOSphatides. It is found in snake, bee and wasp venoms, pancreas, kidney, bacteria, and in a few plant tissues (6). The naturally occurring phosPhatides that serve as substrates for phospholipase A are phOSpha- tidylwcholine, userine, and -ethanolamine, phosphatidal 34 choline (and probably phOSphatidal—Serine and -ethanolamine) phosphatidic acid and cardiolipin. Hydrolysis of phOSpha- tidyl inositol has also been reported. Diphosphoinositides and sphingomyelin are not attacked (151). The enzyme has steric Specificity, attacking L-a forms and not D—a forms (61,130). It is now clear that in a—phos- phatides the B—ester linkage is specifically attacked by pancreatic phOSpholipase A and by all snake venoms (64,105, 144). Both saturated and unsaturated fatty acids are released from the 8 position (143). It was at first thought that phOSpholipase A released only unsaturated fatty acids, but this was due to the preponderance of unsaturated fatty acids in the 8 positions of naturally occurring phosPhatides (182). The optimal pH for phOSpholipase A function is 7.2 and the enzyme is relatively stable to heat, especially at pH 4—6 (6). Most researchers use an emulsifying agent such as sodium deoxycholate or an "activator“ such as diethyl ether in their reaction media to increase the hydrolysis rate (58,137,138). Ether probably owes its "activating" power to its ability to disperse lecithin particles into smaller units and to prevent the orientation of fatty acids at the lipid water interface (151). According to Dawson (59), ultrasonic diSpersion of a lecithin sol permitted more rapid hydrolysis by phOSpholipase A. 35 Phospholipase B (Lysolecithin Acyl—Hydrolase) E.C. 3.1.1.5. This enzyme, removes the fatty acid from lyso- lecithin and occurs in pancreas, liver, and other animal tissues, Penicillium notatum, Aspergillus opyzae and Serratia plymuthicum (6). Phospholipase B attacks saturated and unsaturated lyso- lecithins and also lySOphOSphatidyl ethanolamine when it is mixed with lysolecithin (6). Dawson (57) found that phOSpha- tidyl ethanolamine competitively inhibits hydrolysis of lyso- lecithin by liver phOSpholipase B. The enzyme is relatively stable to heat at pH 4, but not at pH 7 or above. At present it is unclear if the enzyme requires certain activators (6). For those who distinguish between phOSpholipase B and lysophOSpholipase, future work may Show that systems which remove both fatty acids from a phospholipid really consist of two separate enzymes. In 1957 Hanahan (103) concluded that there was no true phOSpholipase B, and that the removal of two fatty acids resulted from action by a mixture of lecithinase A and lysolecithinase. Kates (121) however, disagrees with Hanahan‘s explanation, because (1) phOSphO- lipase A has a much higher pH optimum than phOSpholipase B and (2) phosPholipase A is activated by ether and by calcium ions, neither of which activates phOSpholipase B. Since most "phOSpholipase B" preparations are relatively crude extracts, the issue will remain unclear until a pure enzyme which removes both fatty acids can be prepared. 36 Phospholipase C (Phosphatidylcholine choline phoSpho- hydrolase) E.C. 3.1.4.3. Phospholipase C was first demon~ strated in 1941 by Macfarlane and Knight (135) in the a toxin of Clostridium perfrinqens (now called El, welchii). The enzyme is also present in other bacteria, plants, and certain animal tissues such as brain, kidney, liver, and Spleen (121). Hanahan and Vercamer (109) showed that the enzyme attacked both saturated and unsaturated lecithins in ether solution yielding the diglyceride. Sphingomyelin can be hydrolyzed in aqueous media and is not activated by ether as shown by Sribney and Kennedy (207). Some preparations of phOSpholipase C attack thSphatidyl ethanolamine and phosphatidyl serine, but more slowly than lecithins: however, glycerothSphoryl choline is not hydrolyzed (6). PhOSpholipase C will attack synthetic saturated lecithins containing C10 acids, since these are readily emulsified in water (216). The longer chain homologues were attacked more slowly. Phosphatidyl ethanolamines are not attacked by thSpholipase C unless lecithin is also present. L-3- (diacetyl) lecithin and L-3~(dibutyryl) lecithin are not attacked by the enzyme. The pH Optimum for phOSpholipase C is near 7 and Ca++ ions activate the enzyme. 37 Phogpholipase D (PhoSphatidylcholine_phosphatido- hydrolase) E.C.3.1.4.4. PhOSpholipase D hydrolyzes satur- ated and unsaturated L—3~lecithins and to a lesser extent, phosphatidyl ethanolamine and phosphatidyl serine. Glyceryl- phoSphorylcholine, phosphoryl choline and lysolecithin are not attacked (6). Hack and Ferrans (101) showed that choline plasmalogen was also hydrolyzed. The enzyme has usually been isolated from plant tissue, but Dils and Hubscher (75) stated that the Ca++ activated incorporation of labeled choline into rat-liver microsomal phospholipids might be due to the reversal of phOSpholipase D action by excess choline. The enzyme was first discovered by Hanahan and Chaikoff (106—108), in carrot root and cabbage leaves. As Shown by Kates (120), the enzyme was found only in the plastin and not in the cytoplasmic fractions of plant cells. However, the preparation Davidson and Long (56) made from a soluble form of Savoy cabbage enzyme was believed to come from the cytoplasmic fraction. Recently, Dawson (60) and Yang §£_§l, (232) independ— ently discovered that phoSpholipase D can catalyze the transfer of a 'phoSphatidyl' unit from lecithin to various aliphatic alcohols such as glycerol, ethanolamine, methanol, and ethylene glycol with the formation of the equivalent phospholipid. EXPERIMENTAL Materials Sample Prpparation Fresh, grade A, large eggs were used as a source of phoSpholipids. Hog pancreas, the source for lipase, was obtained from the M.S.U. meats laboratory. The pancreas was removed from the hog approximately 10 minutes after slaughter and defatted as much as possible by trimming with a knife. Approximately 100 gm. portions were wrapped in 2 layers of aluminum foil and stored in a -200F freezer until needed. Phospholipase D was extracted from Savoy cabbage purchased from a local retail market. Chemicals and Reagents Solvents. All solvents were A.C.S. reagent grade and were used directly from the can unless redistillation was specified in the procedure. Lipids. Egg phosphatidic acid (calcium salt) and egg lysophosphatidyl choline were purchased from Pierce Chemical C0., Rockford, Ill. Methyl esters for GLC standards and 38 39 arachidic cthride for synthesizing lecithin were obtained from the Hormel Institute, Austin, Minn. Tripalmitin was made by Fisher Scientific Co., N. Y. Olive oil (Sultana brand) used for the standard lipase assay was purchased at a local retail store. Methyl myristate was obtained from Sigma Chemical Co., St. Louis, Mo. Synthetic d-L Lecithin was obtained from Nutritional Biochemicals Corp., Cleveland, Ohio. Proteins and Enzymes. Lysozyme, grade I from egg white: albumin from bovine serum, crystallized and lyophi- lized; and phospholipase D from cabbage were obtained from Sigma Chemical Co., St. Louis, Mo. Phospholipase A from Crotalus adamanteus was purchased from Ross Allen's Reptile Institute, Silver Springs, Florida. Crude hog pancreas was prepared by Mann Research Laboratories, New York, N. Y. Column Materials. Sephadex G-200, used for lipase purification was supplied by Pharmacia Fine Chemicals Inc., Piscataway, N. J. DEAE cellulose came from W and R Balston, Ltd., supplied by Reeve Angel, Clifton, N. J., or Eastman Organic Chemicals, Rochester, N. Y. Rexyn 102 (H) resin and Rexyn RG (OH) resin, both research grade, were from Fisher Scientific, Fair Lawn, N. J. Silicic acid used for column chromatography of lipids came from Mallinckrodt Chemical Works, St. Louis, Mo. 40 Thin-Layer Chromatography (TLC). Silica Gel—G, sup~ plied by Brinkman Instruments, Westbury, L. I., N. Y., was used for all TLC work. Gas-Liquid Chromatography (GLC). Diethylene glycol succinate (DEGS), high temperature stabilized was made by Analabs Inc., Hamden, Conn. Chromasorb W, 80/100 mesh Size, acid washed, was obtained from Applied Science Laboratories, State College, Penn. Gases used for the chromatograph came from Liquid Carbonic Division of General Dynamics and were supplied through M.S.U. scientific stores. Bile Salts. Sodium taurocholate and Sodium deoxycho— late were purchased from Nutritional Biochemicals Corp., Cleveland, Ohio. Other Chemicals. NwNitrosomethyl urea used for gener- ating diazomethane was supplied by K & K Laboratories, Plainview, N. Y. All other chemicals used and not mentioned here were of analytical reagent grade. 41 Methods Extraction of Egg Phospholipids The procedure used for extracting egg phoSpholipids was that of Ansell and Hawthorne (6). The yolks were separated from 24 eggs and homogenized with 1 liter of acetone. .The extract was filtered and the solid reextracted in the same way. The residue was next shaken with 600 ml chloroform-methanol (1:1 v/v) and filtered on a Buchner funnel with the aid of vacuum. This extraction was repeated with fresh chloroform-methanol and the extracts were combined and stored in a —200C freezer until further purified by either column chromatography or preparative TLC. Hen egg yolks were chosen as the source of phospholipids because they contained a high abundance of phosphatidyl choline; 73% of the total lipid phoSphorus (6). In addition, during the preparation of egg phOSphatidyl choline by column or thin-layer chromatography, a quantity (15% of the total lipid phosphorus) of phosphatidyl ethanolamine was isolated and used for certain experiments. §ilicic Acid Column Chromatography Silicic acid (Mallinckrodt) was washed with deionized water until no turbidity was observed in the supernatant after the coarse particles settled. After washing once with methanol, it was dried for 24 hours at 120°C, and stored in 42 a tightly stoppered bottle. Two types of columns were used for chromatography: (1) a 2.5 cm o.d. x 30 cm column con- taining a 300 ml reservoir at the top of the column and a coarse sintered glass disc and Teflon stopcock, at the bottom of the column, and (2) a 5 step multibore column (88), constructed of fused glass sections having the follow- ing dimensions: o.d., cm. length of section, cm. 2.80 13.5 2.20 13.5 1.70 13.0 1.50 12.5 1.37 13.5 The 5 step column was designed with a 250 ml reservoir containing a Side arm with a Teflon stopcock to facilitate draining. Glass wool served to keep the Silicic acid from passing through the Teflon stopcock at the bottom of the column. A column of activated Silicic acid was prepared by pouring either 50 (2.5 x 30 cm column) or 62 gm (5 step column) of Silicic acid, slurried in an excess of chloroform, into a beaker with a magnetic stirrer. After Stirring until the mixture was homogeneous and translucent, it was poured either into the 2.5 x 30 cm column or the 5 step column in such a way that no air bubbles were trapped. Since nitrogen was used for speeding up the flow rate during chromatography, the column was conditioned in chloroform under nitrogen prior 43 to the induction of the sample. This procedure allowed the Silicic acid to pack in the column and also removed some of the small air pockets if present. A 1-2 cm layer of powdered anhydrous sodium sulfate was then placed on the top of the packed column to absorb any residual moisture in the sample. Extracts of egg phOSpholipids redissolved in 2-3 ml of chloroform, were carefully applied to the top of the column in a ratio of 0.02 gm lipids/gm Silicic acid. Elution was accomplished by various solvent systems and successive 15 ml (2.5 x 30 cm column) or 27 ml fractions (5 step column) were collected. Fractions were collected by hand from the 2.5 x 30 cm column and by an LKB RadiRac fraction collector, distributor type 3402B, from the 5 step column. The siphon opening at the tip was made smaller to allow the solvents to fill the siphon. Otherwise the solvents would drop in and out without ever filling. Nitrogen was used to adjust the flow rate to approximately 3 ml per minute. Neutral lipids and pigments were eluted by chloroform and monitored by the Salkowski test (124) until a negative reaction was achieved. This effluent was discarded after Spotting on TLC showed the absence of phOSpholipids. The second fraction contained phOSphatidyl ethanolamine (and associated phosphatidyl ethanolamine plasmalogens) which were eluted with 15% methanol in chloroform. This fraction 44 was followed during the course of elution with a rapid nin- hydrin test, consisting of equal volumes of eluate, ninhydrin (4 mg of ninhydrin/ml of 100% butanol) and 2,4, lutidine (20% 2,4, lutidine by volume in 100% butanol). The test was positive if the solution turned blue after heating in a sand bath for 2-3 minutes. When all of the phosphatidyl ethanolamine was eluted from the column as indicated by a negative ninhydrin test, the solvent was changed to 25% methanol in chloroform to elute phosphatidyl choline (and associated phoSphatidyl choline plasmalogens). Elution of the latter fraction was followed by the molybdate test. This consisted of heating a few drops of the sample in a test tube to dryness, adding a few drops of concentrated sulfuric acid and heating, cool— ing, and finally adding molybdate reagent. A blue color indicated a positive molybdate test. The molybdate reagent was made up as follows: 5 ml of 60% w/v perchloric acid, 10 ml of 1N hydrochloric acid, and 25 ml of 4% w/v of ammon— ium molybdate (164). The volumes of solvent used to elute the various fractions depended upon the particular batch of Silicic acid, packing of the column, moisture in the solvents, and the amount of sample applied to the column. Since the objective of column chromatography was to collect phosphatidyl ethanolamine and phOSphatidyl choline for further experimentation, the elution was stopped after all of the phoSphatidyl choline was eluted. Fractions were 45 evaporated under nitrogen to 5-10 ml and Spotted on TLC plates to test for purity. Overlapping of some of the peaks generally occurred and it was difficult to prevent this from happening. Usually phosphatidyl ethanolamine was eluted pure; there was some overlapping of the lysophOSphatidyl ethanolamine and phos- phatidyl choline fractions and of the phoSphatidyl choline and Sphingomyelin fractions. In some cases, the spinogomyelin peak overlapped with the lysophoSphatidyl choline peak. The overlapping usually occurred in only 2 or 3 fractions on either side of the peak and these fractions were discarded. The use of coupled columns with diminishing bores to improve resolution of components eluted from a column was suggested by Hagdahl (102) in 1948. Due to difficulties in packing, their use has not been widespread. Fisher and Kabara (88) reported on the use of multibore columns con- structed by joining glass tubing of various diameters directly, without capillary intermediates. They successfully separated neutral lipids on a Florisil multibore column. In the present study, a 5 step multibore column was used to improve the resolution of egg phospholipids on Silicic acid. Although this column did resolve some of the peaks better than the single bore column, there was still some overlapping. The comparison between the resolutions of the two columns used was made on the basis of TLC of the eluted fractions. Another advantage of the multibore column 46 was its increased sample capacity over that of the conven- tional column. In a single run using a 1.2 gm sample of egg phoSpholipids applied to the column, approximately 700 mg of phosphatidyl choline and 150 mg of phosphatidyl ethanol- amine were obtained. Silicic acid column chromatography was also used to separate unreacted L—a phoSphatidyl choline (NBCo) from phOSphatidic acid after hydrolysis of the former by phoSpho- lipase D. This was accomplished by dissolving the mixture of phOSpholipidS in 1-2 ml of chloroform and placing it on a 2.5 x 30 cm column as previously described. A chromogen, possibly coming from the cabbage cells which were used as a source of phOSpholipase D, was eluted first with chloroform. The phoSphatidic acid was eluted next with increasing concen- trations of 15—35% methanol in chloroform, followed by the unreacted lecithin which was eluted with 50% methanol in chloroform (1). Elution of the chromogen was monitored visually while the other components were monitored by the molybdate test. Since both phosphatidic acid and lecithin gave a positive molybdate test, the change in solvents from 35% to 50% methanol in chloroform was somewhat arbitrary. Usually, the solvent was changed when the molybdate test for phoSphatidic acid gave only a slight blue color. This meant that almost all of the phoSphatidic acid peak was eluted. 47 The third experiment employing Silicic acid column chromatography was the separation of unreacted egg phoSpha- tidyl choline from lysophOSphatidyl choline after hydrolysis of the former by phoSpholipase A. The sample, in 1-2 ml of chloroform, was applied to the column. Free fatty acids were eluted first, with chloroform, followed by the elution of lecithin with 25% methanol in chloroform. Lysolecithin was eluted with 100% methanol. Elution was monitored by the Salkowski and molybdate tests. The final experiment using Silicic acid column chroma- tography was the purification of synthesized lecithin con- taining arachidic acid in the 8 ester position. This purifi— cation involved separating the unreacted arachidic chloride and lysolecithin from the synthesized lecithin. The pro- cedure followed was exactly the same as for the third experi- ment. Diethylaminoethyl (DEAE) Cellulose Column Chromatography The DEAE was prepared according to Rouser §£_§l, (179) and packed into a 2.5 x 30 cm column similar to the one used for Silicic acid chromatography. Sample applications of 0.01 gm lipids/gm DEAE were used. Egg phospholipids were chromatographed on this column to purify phOSphatidyl choline from phosphatidyl ethanolamine. Elution of neutral lipids began by washing the column with chloroform until a negative Salkowski test was obtained. PhoSphatidyl choline 48 (and associated phoSphatidyl choline plasmalogens) were eluted with 9:1 chloroform:methanol (v/v) and monitored by both the ninhydrin and molybdate tests. The complete elu- tion of phosphatidyl choline was indicated when both the molybdate and ninhydrin tests were negative. At this point, the solvent was changed to 7:3 chloroform:methanol (v/v) to elute phOSphatidyl ethanolamine (and associated phosphatidyl ethanolamine plasmalogens). During some runs, the last few fractions of the phoSphatidyl choline peak were contaminated with phOSphatidyl ethanolamine which started eluting with 9:1 chloroform:methanol (v/v). This was indicated by both a positive ninhydrin and molybdate test. Although the phosphatidyl choline obtained from this column contained some Sphingomyelin, it was still used for phospholipase A hydrolysis, since Sphingomyelin did not interfere with the hydrolysis and was not itself hydrolyzed by the enzyme (151). Purification of lysophosphatidyl choline from the unreacted phosphatidyl choline, Sphin909 myelin, and free fatty acids, after phospholipase A hydroly~ sis, was accomplished by Silicic acid column chromatography. In those cases where the fractions contained other impurities besides sphingomyelin, the fractions were pooled, concen- trated under nitrogen and Spotted on preparative TLC plates. The eluted pure lecithin was then subjected to phOSpholipase A hydrolysis. 49 DEAE column chromatography was usually accomplished more rapidly than Silicic acid chromatography, but the lecithin fractions were not as pure as those obtained from a Silicic acid column. However, in cases where the phOSpha— tidyl ethanolamine was collected from DEAE columns, the fractions were of excellent purity. Thin—Layer Chromatography (TLC) Thin—layer adsorption chromatography on silica gel—G was employed both for checking identities of phoSpholipid fractions eluted from columns and for preparative work. RSCo equipment consisting of a plastic mounting board, desiccator, and Spotting template were used. A Desaga vari— able thickness (0-2 mm) applicator distributed by Brinkmann Instruments, Westbury, L. I., New York, was used. The TLC plates used for identification purposes were Spread 0.25 mm thick on 20 x 20 cm clean glass plates, rinsed with methanol. Twentwaive g of silica gel-G contain- ing approximately 13% calcium sulfate binder was shaken with 50 ml of deionized water in a 300 ml glass stoppered Erlen— meyer flask until the mixture was homogeneous. For prepara- tive plates, 0.50 mm thick, 50 gm of silica gel-G and 100 ml of deionized water were used. After spreading, the plates were air dried at room temperature for 15-30 minutes and stored in laboratory drawers. Before use, the plates were activated at 1100C 50 for 1 hour, cooled to room temperature in a desiccator and spotted immediately. Better separation was usually obtained when plates were activated just before use rather than storing activated plates in a desiccator. For preparative TLC work, the plates were spotted in a plastic freezer box, 13.5 x 3.5 x 10.5 inches containing a turned over metal tray, 12 x 2 x 7.5 inches. After placing the TLC plate on the metal tray, chunks of dry ice were placed all around the plate. The plastic cover was posi- tioned on the freezer box in such a manner as to allow for a 2 inch opening at one end of the box for spotting. An atmosphere of C02 was thus maintained and oxidation of the sample was minimized during the 4-7 minute Spotting operation. Micro—TLC plates were prepared by coating clean micro- scopic Slides 2.5 x 7.5 cm with silica gel—G prepared in a 1:2 ratio of silica gel-G to deionized water. Two slides, back to back, were dipped into a tall narrow beaker containing the silica gel-water mixture. The slides were then separated and allowed to air dry. Storage and activation conditions of the micro-TLC plates were the same as for the large plates. Micro-TLC plates offered a rapid method for monitor— ing the purity of lipid samples. Although 4-5 components could be separated on this plate, better results were obtained when the samples contained only 1-3 components. Large TLC plates were used if the samples were composed of more than 3 components. 51 TLC plates Spotted with either L—d lecithin or egg phospholipids were developed in a solvent system consisting of chloroform:methanol:water, 65:25:4 by volume. Samples of lipids methylated for GLC analysis were tested for com- pleteness of methylation by spotting on either micro or regular TLC plates and chromatographing in petroleum ether: ethyl ether:acetic acid, 90:10:1 by volume. Lipid component Spots were detected by spraying with various indicators, e.g., ninhydrin solution for amino phos— phatides (128), molybdic acid for phosphatides (76) and Dragendorf reagent for choline (205). Aqueous sulfuric acid (50%) was sprayed on the plate after first Spraying with the other indicators. After 1—2 hours in an oven at 1100C, any previously undetected lipids or other impurities were visible as a charred spot on the plate. For preparative TLC plates, only 2 edges of the plate were sprayed with molybdate reagent while the center of the plate was protected with another glass plate. After visualization of the phOSpholipid band(s) of interest, the remainder of the band(s) were scraped off into 40 ml centrifuge tube containing approximately 10 ml of chloroform: methanol:water, 65:25:4 by volume. The material in the tubes was stirred with a glass rod and centrifuged for 3-4 minutes in a clinical centrifuge. After decanting the solvent into another container, the procedure was repeated 2 more times. For preparative plates containing phOSphatidic acid, 100% 52 methanol was used for the eluting solvent. To those tubes containing 65:25:4 solvent, 2-3 ml of benzene were added to help drive off the water as an azeotropic mixture during evaporation under nitrogen. Documentation of the chromatography was accomplished by placing the plate on a square cardboard box with its top cut off. A piece of Saran Wrap was placed over the plate and then a piece of thin tracing paper over the Saran Wrap. A light bulb in the box illuminated the plate to facilitate tracing the spots with a fine tipped felt pen. Another method used in documentation involved photo- graphing the plates using a Polaroid copy camera. Either 4 x 5 Pola Pan Type 52 positive film or 4 x 5 positive/ negative, Type 55 P/N was used. Phosphorusggetermination The phoSphorus content in phOSpholipid samples was determined by the method of Rouser §£_al. (181). It was used both for samples in solvents and those taken directly from TLC plates. A standard phOSphorus curve was constructed by using a monobasic potassium phOSphate standard (Hartman Leddon Co., Philadelphia, Pa.). The factor required to multiply absorbance readings in order to obtain ug of P in the sample was calculated from the standard curve to be 11.3 (Appendix B). Rhee and Dugan (172) showed that Rouser's §£_al, method 53 may be extended to give a linear curve up to 17.44 ug of phoSphorus. However, most of the phOSpholipid samples ana— lyzed were usually diluted to give readings within the 2-8 ugm range (0.170—0.708 absorbance). Preparation of Methyl Esters for Gas-Liquid Chromatography The original low temperature procedure of preparing methyl esters according to McGinnis and Dugan (133) was modified by deletion of the 2 ml of sulfuric acid. This modification was based on the work of Zook (235) who found that almost complete methylation of soy and egg phoSpholipids can be effected by using no sulfuric acid and 2—4 gm KOH/15 ml methanol. Evidence for her modification was based on TLC results when samples were chromatographed in petroleum ether: diethyl ether:acetic acid, 90:10:1 by volume. Omission of the sulfuric acid had another desirable effect in that no emulsion was produced during the extraction procedure with petroleum ether. Methyl esters were prepared either from the phoSpholipid fractions obtained from Silicic acid or DEAE columns, or directly from the contents of scrapings off TLC plates. Samples from TLC plates were scraped directly into 125 Erlen- meyer flasks, and 20 ml of peroxide free diethyl ether was added. If the samples were eluted from columns, the materi- al was quantitatively transferred to 125 ml Erlenmeyer flasks, 54 evaporated to dryness under nitrogen, and redissolved in 20 ml of diethyl ether. The flasks were placed in a dry ice—acetone bath and stirred by means of a magnetic stirrer. When the flasks had cooled to -600C, 15 ml of absolute methanol was added and the mixture was allowed to continue stirring at -6OOC for 10 minutes. Methanolic-KOH (2 grams of KOH dissolved in 15 ml absolute methanol) was then added at ~600C. After stirring the mixture for 15 minutes at ~600C, it was allowed to reach room temperature while stirring was continued. The mixture was quantitatively transferred to a 500 ml separatory funnel by rinsing the flask with 150 ml of water. Methyl esters were extracted once with 30 ml of petroleum ether (300—600C B.P.) and twice with 15 ml of petroleum ether. All samples were dried over anhydrous sodium sulfate before they were concentrated to a volume of 0.2 ml in graduated 12 ml centrifuge tubes. Gas—Liquid Chromatography (GLC) Gas-liquid partition chromatography was carried out with a Beckman GC—5 dual column, temperature programmed gas chromatograph. It was equipped with thermal conductivity and flame ionization detectors, and a recorder. All samples were chromatographed using the flame ionization detector. Two coiled stainless steel columns (1/8 inch o.d. x 6 ft.) were used for methyl ester separation. Both columns 55 were packed with 20% by weight DEGS and 1% by weight phos- phoric acid on acid washed chromosorb W 80/100 mesh, as a support phase. Approximately 2.5 gm of column material were packed into each of the columns. The columns were condi- tioned at 2200C for 24-48 hours before being used. Operat- ing conditions used in this study were: column temperature, 1730C; inlet temperature, 2300C; flow rates for air, helium and hydrogen, 250, 30, and 32 ml/min. respectively; tank pressures for air, helium, and hydrogen, 16, 70, and 30 psi reSpectively. The attenuator setting was usually 5 x 102 (2.5 x 10"10 amperes, fullscale deflection) but for samples containing minor amounts of methyl esters it was lowered (higher sensitivity) to 1 x 102 or 5 x 10 (0.5 x 10-10 or 25 x 10""12 amperes, fullscale deflection). Identification of fatty acid methyl esters on the chromatogram was made by direct comparison of major peaks with those of chromatographic standards (99+% pure) passed through the same column under identical conditions. Peak areas were calculated by the triangulation method of multi— plying the peak height by fi-the base (40). gpeparation of.PhOSphatidic Acid and Methylated Phosphatidic Acid Hydrolysis of L- d—Lecithin by PhoSpholipase D. Phospho- lipase D was prepared from fresh Savoy cabbage leaves and lyophilized as described by Tookey and Balls (215). For some 56 preparations, a commercial source of phoSpholipase D was used (Sigma Chemical Co.). Synthetic L—a—Lecithin was hydrolyzed according to the procedure of Davidson and Long (56). The reaction time ranged from 2—3 hours at room temperature with gentle shak- ing on a Burrell wrist action shaker (setting 2). Any un- reacted lecithin, and a chromogen that was present, was separated from the phoSphatidic acid by Silicic acid column chromatography. Dialysis of Phosphatidic Acid. The enzymatic hydrolysis of phosphatidic acid with phospholipase D requires the presence of excess Ca++, which remains as calcium phOSpha— tidate in the final product. Therefore, the phosphatidate salt must be dialyzed before reacting it with diazomethane to produce methylated phoSphatidic acid. The procedure of Abramson §£_al, (1) was followed for the dialysis. The same dialysis treatment was given to the commercial preparation of phoSphatidic acid (Pierce Chemical Co.) Since it was purchased in the form of the calcium salt. Preparation of_Qiazomethane. Diazomethane was prepared by the method of Arndt (8). For small quantities, however, it was not distilled but prepared as follows: To a 50% solution of KOH (cooled) in a test tube, an equal volume of diethyl ether was added. Small quantities of N—nitrosomethyl 57 urea were added to the solution until it appeared deep yellow in color. The ether layer was siphoned off, filtered over dry KOH pellets, and used immediately for the methyla- tion of phosphatidic acid. Diazomethane should not be placed in containers with ground glass stoppers. Any diazo- methane caught between the stopper and the container may explode when the stopper is twisted. Diazomethane may be destroyed by the addition of glacial acetic acid. -Methylation of Phosphatidic Acid with Diazomethane. The diazometholysis for both the prepared phoSphatidic acid and the commercial egg phOSphatidic acid was carried out according to the procedure of Baer and Maurukas (12). After the second methylation (12), the sample was evaporated to dryness under nitrogen and redissolved in carbon tetra— chloride for the IR and NMR analysis. Infrared (IR) and Nuclear Magnetic Resonance (NMR) Analysis of PhoSphatidic Acid and Methylated Phogphatidic AEEQ- The IR Spectra were obtained both before and after methylation of phoSphatidic acid, using a Beckman Model IR-5 spectrophotometer. Samples derived from both synthetic L-a-lecithin and egg lecithin were analyzed. All samples were run in Spectral grade carbon tetrachloride against a carbon tetrachloride reference. After obtaining the IR spectra, the same Samples were used for the NMR analysis. A few drops of tetramethyl 58 silane were added to the phosphatidic acid and methylated phoSphatidic acid samples as an internal standard. The choice of CCl4 as a solvent lies in the fact that it contains no protons to interfere with any of the protons on the phos- phatidic acid. A Varian Model A—60 spectrometer was used for all NMR Spectra. Sonication Experiments Using Crude HoggPancreatic Lipase Apparatus and Materials. A Blue M, Magni Whirl Visi- bility water bath, model number MW 1162, was used to cool the reaction vessel during all sonication eXperiments. An additional motor driven stirrer was used to increase the mixing rate of the water in the bath. A 32 ml aluminum beaker containing 10 ml of crude lipase, 20 mg dried pork pancreas/ml. (Mann Research Laboratories) was positioned in the water bath so that the level of enzyme in the beaker was completely submerged below the water. The probe of a Branson model S~75 “Sonifer” was mounted on a stand and positioned in the center of the beaker containing the enzyme (Figures 1 and 2). The height of the probe from the bottom of the beaker was always kept between 0.2 and 0.3 cm. This fact is important since more heat will be generated when the probe is nearer to the beaker bottom. Positioning the probe too high in the beaker will result in foaming instead of cavitation. All sonication was performed in air. 'afi2r4SApggg . Figure 1. Arrangement of sonifier probe, water bath, stirring motor and metal beaker. Control panel for the sonifier is shown to the left of the water bath. Figure 2. Closeup view of the sonifier probe and metal beaker. 60 The frequency and intensity used during the sonication experiments were kept constant at 20,000 cps and 23 watts/ cmg. The intensity dial on the Branson control box was set to number 5 and the instrument was tuned to 8 amperes. The approximate intensity of sonication the lipase solution received was calculated by sonicating 20 gm of deionized water in a 50 ml glass beaker and noting the rise in tempera- ture per minute with the aid of a thermocouple and recording potentiometer. InsuIation of the beaker was important to avoid heat loss to the sides of the beaker. After calculat- ing the heat produced/minute (Q) and multiplying by 17.58 watt minute, the number of watts produced may be found. The intensity of sonication may then be calculated by dividing the area of the probe tip into the number of watts produced. An example is given below: .Q = 20 cm H20 1 BTU 55.55% = . 454 gm/lb x 1 lb/OF x minute 1.61 BTU/minute _ 1.61 BTU 17.58 watts = Watts _ minute x BTU/minute 28.3 watts . 28.3 watts 2 I = = ntenSity 1.25 sq cm 23 watts/cm In order for the above calculations to be valid two assumptions were made: 1. There was no heat loss to the sides or to the top of the beaker during sonication and 2. All of the energy was emitted from the probe tip and very little was lost to the sides of the probe. These assump— tions were not entirely true, and therefore 23 watts/cm2 can only be considered an approximation of the true intensity. 61 Lipase. In all experiments, a stock solution of 4 gm dried pork pancreas/200 ml deionized water was prepared and kept in a container filled with ice until needed. The enzyme solution was found to be stable for the time re- quired to perform the experiments for a particular day. A fresh stock solution of enzyme was prepared each day to insure that the activity remained constant during the entire experiment. Emulsion. An olive oil gum arabic emulsion prepared according to Marchis—Mouren §£_al, (141) was used as the substrate for all experiments except those with tripalmitin. This emulsion was prepared by adding 30 gm of gum arabic, USP powder (Matheson Coleman and Bell, Cincinnati, Ohio), to a Waring Blender containing 270 ml of deionized water. The mixture was blended for 3 minutes and filtered through a funnel containing glass wool to remove the foam. Then 165 ml of the above 10% gum arabic solution was added to a 250 ml Vir Tis homogenizing cup, along with 20 ml of olive oil, and 15 gm of crushed ice (from deionized water). The mixture was emulsified using a Vir Tis "45“ homogenizer for 4 minutes with cooling by crushed ice contained in the outer plastic cup. Emulsion Sygtem. A 10 ml aliquot of the above emulsion was added to a 200 ml glass beaker, followed by 0.1 ml of 62 1M CaClg, 0.3 ml of 20% sodium taurocholate, and 19 ml of deionized water. The pH of the mixture was adjusted to 9.1-9.2 using a 1% solution of NaOH. The final volume of solution contained in the beaker was 30 ml. Hydrolysis Procedure. The emulsion system was pre- heated for 10 minutes at 380C, before introducing the enzyme. With the concentration of enzyme used, the rate of hydrolysis was slow enough to be titrated manually using a Corning model 12 pH meter fitted with a Sargent combination electrode. An electric motor (600 rpm) employing a glass stirring rod was used to stir the emulsion system during hydrolysis. The system was continuously titrated with 0.1N NaOH to pH 9.0 for 12 minutes. A blank using 10 ml of boiled enzyme was also sonicated and run through the subsequent hydrolysis procedures. The rate of hydrolysis was then calculated by plotting the meq. of 0.1N NaOH used vs. time and determining the slope of the straight line portion of the curve. Experiment_1yr Heat Treatment - No Sonication. A 10 ml aliquot from the stock solution of enzyme at 40C was pipeted into the aluminum beaker. The solution was brought to the desired heating temperature in the shortest possible time using a water bath and was then kept at this temperature for 1 minute. Temperatures of heating were 300, 350, 400, 450 I 500, and 600C. The enzyme solution was then cooled to 250C 63 in an ice bath, and a 0.15 ml aliquot was used for the subsequent hydrolysis. Heating of the enzyme for 1 minute at the above mentioned temperatures served as the zero point for the sonication eXperiments. Experiment 2 - Sonication at Constant Temperatures. A 10 ml enzyme aliquot from the stock solution at 40C was pipeted into the aluminum beaker containing a glass cover slip, 18 sq mm. The glass cover slip protected the beaker bottom from wearing out due to the intensive radiation. Two copper-constantan thermocoUple wires were con- nected to a Minneapolis Honeywell temperature recorder, range 00-1500C, and wired so that thermocouple number 1 printed numbers 1, 3, 5, 7, 9, and 11 and thermocouple number 2 printed numbers 2, 4, 6, 8, 10, and 12. This allow- ed the water bath and enzyme temperatures to be read alter- nately every 5 seconds during the sonication. Positioning the sonicator probe tip 0.2-0.3 cm from the bottom of the aluminum beaker allowed the final sonication temperature to be reached within 15—20 seconds. Sonication was carried 0, 40° and 500 for 1, 5, 5, 10, 20, and 50 out at 100, 30 minutes at each of the temperatures. The subsequent treat- ment of the enzyme and hydrolysis were the same as for experiment 1. 64 Experiment 3 - Sonication of Lipase and Substrate. The emulsion system used for this experiment was the same as in experiment 1 with the exception of the 19 ml of deionized water which was deleted. The pH of the emulsion was adjusted to 8.6 with 1% NaOH. The emulsion contained in an aluminum beaker was allowed to reach bath temperature (210C) and at 0 time the sonicator was turned on. After 0.5 minutes the temperature of the emulsion-enzyme mixture reached 380C (120C), and 0.25 ml of lipase solution (20 mg/ ml), previously equilibrated to 250C, was added to the beaker. The hydrolysis was allowed to proceed for 4.5 minutes after which it was stopped by pouring the reaction mixture into a 50 ml Erlenmeyer flask containing 20 ml of an acetone-ethanol mixture (50:50 v/v), and 4 drops of 1% phenolphthalein in ethanol solution. The beaker was rinsed with an additional 10 ml of the acetone-ethanol mixture and added to the Erlenmeyer flask. The mixture was titrated to a pink end point while being stirred with a magnetic stir- ring bar. Since the white globs of the gum arabic obscured the pink end point, the magnetic stirrer was turned off and the titration stopped so that these globs could settle and the color of the clear solution noted. A blank run using a boiled enzyme solution was also performed. 65 Experiment 4 - Stirring of Lipase and Substrates at 600 rpm. The procedure was exactly the same as in experiment 3 with the exception that the enzyme-substrate mixture re— ceived no sonication treatment. Instead, the mixture was stirred at 600 rpm with a motor fitted with a glass rod stirrer. The aluminum beaker containing the emulsion was allowed to equilibrate at 380C for 5 minutes in a thermo— statically controlled water bath. At 0 time the stirring motor was turned on and allowed to stir the emulsion for 0.5 minutes after which 0.25 ml of lipase solution (20 mg/ml), previously equilibrated to 250C was added to the can. The hydrolysis was allowed to proceed for 4.5 minutes. The re— action was stOpped and the mixture titrated as outlined in experiment 3. A blank run using boiled enzyme was also per- formed. Experiment 5 - Sonication of Lipase and Tripalmitin. Approximately 11 mg of methyl myristate (1 drop) were added to 30 mg of tripalmitin contained in an aluminum beaker. Methyl myristate acted as a carrier for the tripalmitin to help solubilize it. The beaker was heated in an 80°C water bath for 1 minute to melt the tripalmitin. Ten ml of 0.1M Tris-HCl buffer, pH 8.6, 0.1 ml of 1M CaCla, and 0.1 ml of 20% sodium taurocholate were added to the above mixture. The beaker was again heated in an 80°C water bath for 1 minute to bring the tripalmitin and methyl myristate to the 66 top of the mixture. At this point, and until actual soni- cation, there was no emulsion of the mixture in the beaker. The mixture in the beaker was allowed to cool and then placed in a 39°C water bath for 5 minutes. At 0 time, the sonicator was turned on and 0.5 minutes later 0.5 ml of a lipase solution (20 mg/ml), previously equilibrated to 250C, was added to the beaker. The sonication was allowed to proceed for 9.5 minutes after which it was stOpped and the mixture titrated as in eXperiment 3. The enzyme-tripalmitin- methyl myristate mixture reached 450C in approximately 15 seconds after the sonicator was turned on and it remained at this temperature (120C) for the duration of the sonica— tion. Three blanks were run as follows: (1) sonication of a boiled enzyme blank using the tripalmitin-methyl myristate mixture, (2) sonication of a boiled enzyme blank using only the methyl myristate mixture without tripalmitin, and (3) sonication of a non-boiled enzyme blank which would hydrolyze the methylmyristate mixture. These three blanks allowed corrections to be made due to any lipase hydrolysis of the methyl myristate. Experiment 6 - Stirring of Lipase and Tripalmitin at 600 rpm. These experiments were carried out in a thermo— statically controlled water bath at 45°C. The mixture of tripalmitin-methyl myristate used was the same as in experi- ment 5. A 5 minute preheating period at 45°C was given to 67 the aluminum beakers containing the tripalmitinmethyl myristate mixture. At 0 time the stirrer was started (600 rpm) and 0.5 ml of a lipase solution (20 mg/ml) was added. The subsequent inhibition of the hydrolysis and titration were the same as for experiment 3. Three blanks were also performed in this experiment with the exception that they were not sonified. Preparation of Hog_Pancreatic Lipase Fractions with Increased Activity Three different purification procedures were used to obtain lipase for the hydrolysis of egg phOSpholipids. The first procedure was outlined by Melius and Simmons (152) who utilized butanol for obtaining a lipase with a high activity. The second procedure was that of Sarda £5.31, (190) and involved purification of lipase by chromatograph— ing on a series of Sephadex G—200 DEAE columns. However, the latter purification scheme was successfully followed only until the step involving precipitation by ethanol. The third procedure involved purification by electrophoresis (92) of the Sephadex G—200 lipase fraction obtained from the second procedure. 68 Activity Measurement of Lipase Fractions Emulsion. Olive oil emulsions were used for routine assays. The emulsions were prepared in a slightly different manner than previously mentioned for the sonication experi— ments. Olive oil (20 ml) and 180 ml of a 10% gum arabic solution were added to a Waring Blender and emulsified for 4 minutes at high Speed. A typical assay utilized 5 ml of olive oil emulsion, 10 ml of deionized water, and 0.15 ml of a 20% sodium taurocholate solution. Assay System. Lipase was assayed by the titrimetric procedure described by Marchis-Mouren §£_al, (141) using a Sargent recording pH stat connected to a Corning Model 12 pH meter. Operating conditions for the pH stat were as follows: buret size, 2.5 ml; recorder chart Speed, 1 inch/ min.; magnetic stirring Speed, setting of 10; reaction temperature, 37°C; and reaction beaker, 15 ml. Each beaker containing the emulsion (pH 4.7-4.8) was adjusted to pH 9.0 prior to the addition of the enzyme. The free fatty acids liberated by the lipase were automati- cally titrated by the pH stat with 0.05N NaOH to pH 9.0. A unit of enzyme activity is defined as that quantity of enzyme which, acting on a glyceride emulsion under the conditions of the test, liberates 1 microequivalent of acid per minute. Specific activity is expressed as units per mg. protein. 69 Lanearity of Reaction Rate. Linearity of reaction rate was determined for each lipase preparation by taking a series of increasing enzyme aliquots (0.04-0.3 ml) at constant sub- strate concentration and determining,from the straight line portion of the curves, the ml of 0.05N NaOH used per unit of time. Units of activity were obtained as follows: (Ml of Base used/Min) (Normality of Base) x 1000. A second curve was then constructed by plotting units of activity versus ml of enzyme used. An enzyme concentra- tion falling within the linear portion of the curve was used for the subsequent lipase assays. Protein Measurement of Lipase Fractions. The protein content of lipase fractions obtained by various purification methods was determined by the technique of Lowry gp_al. (131). A standard protein curve ranging from 0—80 7 (10007 = 1 mg) of lysozyme was constructed. A factor of 136.36 was obtained from the standard curve. Thus, the factor x absorbance reading=gamma of protein in tube. All readings were made at 650 mu in a pH Coleman Uni- versal Spectrophotometer, Model 14, using a number 14-214 blue filter. Synthesis of_8 Arachidoyl Lecithin from Egg_Lysolecithin Hydrolysis of Egg Lecithin by Phospholipase A. Egg lecithin was hydrolyzed by phospholipase A venom (Crotalus 70 adamanteus) using the procedure of Tattrie (211). Usually the reaction was not complete and the lysolecithin was subsequently separated from the free fatty acids and un- reacted lecithin by silicic acid column chromatography. Esterification of Lysolecithin with Arachidic Acid. Two lysolecithin preparations were used as the starting material for lecithin synthesis. The first preparation was made by hydrolyzing egg lecithin with phOSpholipase A, while the second preparation, also derived from egg lecithin, was purchased from the Pierce Chemical Co. The procedure involves a technique originally described by Baer and Buchnea (11) and used by Hanahan pp 3;. (105), De Haas §£_al, (63), and others. Except for scaling down the amount of reactants in the synthesis, the procedure was exactly the same as described by Hanahan §£_al, (105). All solvents in this procedure were redistilled except for the absolute ethanol. It is extremely important to exclude any trace of moisture when reacting the lysolecithin- CdClg complex with arachidic chloride in chloroform contain— ing pyridine. (1) A solution of 290 mg of CdC12°2.5H20 dissolved in 0.2 ml of deionized water was added to 3.2 ml of 99% ethanol. (2) Lysolecithin (200 mg) dissolved in 3.8 ml of warm 99% ethanol was then added to (1). After two hours at 0°C, the precipitate was collected, and washed with ethanol and 71 ethyl ether and dried in a vacuum desiccator containing calcium chloride. (5) To 662 mg of arachidic chloride in 2 ml of chloro- form (free of EtOH and dry), 200 mg of the lyso-CdClg complex were added. This was followed by the addition of 0.3 ml of dry pyridine in 3 ml of dry chloroform while stirring the whole mixture with a magnetic stirring bar. The reaction was carried out in a 25 ml Erlenmeyer flask fitted with a CaClg drying tube. Within a short time after the addition of the pyridine- chloroform solvent, the mixture became clear and subsequently the CdClg-pyridine complex precipitated. Stirring was continued at room temperature for 2 hours. At this stage, the original procedure called for wash- ing the precipitate with chloroform, however, this step was omitted because much of the synthesized lecithin would have been lost in the washings. This was shown to be true during two preliminary trial runs. The material in the flask was evaporated to dryness under nitrogen and low heat (350C) and then redissolved in chloroform:methanol:water, 5:4:1 by volume. The excess cadmium chloride and pyridine hydrochloride were removed from the reaction mixture by passing the solution through a 1.5 cm x 30 cm column containing equal mixtures of Amberlite IR—45 and IRC-50 (Rohm and Haas Co.) or Rexyn 102 (H) and Rexyn RG (OH) resins (Fisher Scientific). The advantage 72 of using the latter resins was that it was not necessary to regenerate to their respective H or OH forms while the Amberlites had to be regenerated by washing the IR-45 with 0.5N NaOH and the IR-50 with 1N HCl. The column was washed with approximately 100 ml of chloroform:methanol:water, 5:4:1 by volume. If the eluant contained any cadmium (157), the solution was passed through another column containing the same resins as the first one. Since the flow rate through the mixed resin column was very rapid, the stopcock was partially closed so that only 2 ml/ min was collected. This allowed more time for the cadmium chloride and pyridine hydrochloride to come into contact with the resin. The effluent was evaporated to dryness under vacuum by a Rinco rotatory high vacuum-type evaporator at 40°C. After redissolving the material in 2-3 ml of chloroform, it was chromatographed on a Silicic acid column to separate the synthesized lecithin from arachidic chloride and lysolecithin. Egg PhoSpholipid Hydrolysis by Various Lipase Preparations Units of Lipase Used for Phospholipid Hydrolysis. All lipase preparations were adjusted to contain 800 Inter- national Enzyme Units (I.E.U.). When hydrolyzing phoSpho- lipids, a ratio of 800 I.E.U. per mg of phOSpholipid was used for all experiments. 73 Experiment 7 - Reaction of Crude Hog Pancreatic Lipase with Egg Phosphatidyinholine. PhOSpholipidS were hydro— lyzed by pancreatic hog lipase using the procedure of De Haas pp_ai, (62). The following materials were added to each of 2 test tubes (13 x 185 mm): 7 mg of sodium deoxy- cholate, 4.5 mg of serum albumin, 1 ml of 5 x 10‘3M borate buffer, pH 8.0, containing 5 x 10‘3M CaClg, 1 mg of pure egg phosphatidyl choline, and 1 ml of crude hog pancreas (Mann Research Laboratories) containing 20 mg/ml deionized water. The test tubes were stoppered with corks wrapped in Saran Wrap and shaken until homogeneous. Incubation was conducted for 3 hours at 300C without shaking. The reactions were stopped by the addition of 3 ml of chloroform:methanol 2:1 (v/v) followed by 2 ml of benzene. After complete evaporation of the total mixture under nitro- gen (no extraction of the phOSpholipids were made), it was taken up in 0.5 ml of chloroform:methanol (1:1 v/v) Spotted on preparative TLC plates, and chromatographed in chloroform: methanol:water, 65:25:4 by volume. By spraying the edges of the plates with molybdate reagent, the presence of both lecithin and lysolecithin was demonstrated. The remainder of each band was scraped off into 125 ml Erlenmeyer flasks, methylated, and GLC analysis performed. Experiment 8 - Reaction of The Butanol Extracted Lipase with Egg PhoSphatidyl Choline. The procedure followed in 74 this experiment was identical to experiment 7 with the ex— ception of the lipase source. For this experiment, 1 ml of butanol extracted lipase (Step V) was used. Experiment 9 - Reaction of The Sephadex G-200 Lipase Fraction with Egg Phosphatidyl Choline and Phogphatidyl Ethanolamine. (Duplicate 1 mg samples of either phosphatidyl choline or phoSphatidyl ethanolamine were added to separate test tubes and the solvent evaporated off under nitrogen. Borate buffer, deoxycholate, and albumin were added, followed by sonication of the mixture for 1 minute at an intensity of 23 watts/cma. Cooling of the material during sonication was accomplished by placing the test tubes in an ice-water bath. After equilibrating the solution at 300C for 5 minutes, 0.5 ml of the Sephadex G-200 lipase fraction was added. The mixture was shaken at 300C for an additional 15 minutes and the reaction was stopped and analyzed as described in experiment 7. Experiment 10 - Reaction of The ElectrOphoretic Lipase Exaction withiggg PhOSphatidyl Choline. Duplicate 1 mg samples of phosphatidyl choline were added to separate test tubes and the solvent evaporated off under nitrogen. The reaction mixture mentioned in experiment 7 was added, followed by the addition of 1 ml of the electrophoretic lipase fraction. After shaking the tubes until the solutions 75 were homogeneous, they were incubated at 300C for 4 hours without shaking. The subsequent treatment of the reaction mixture was described in experiment 7. Experiment 11 - Reaction of The Sephadex G—200 Lipase Fraction with Egg Lysophosphatidyl Choline. Four 10 mg samples of egg lysophOSphatidyl choline (Pierce Chemical Co.) were weighed out into test tubes. Two of the samples con- tained the complete-reaction mixture mentioned in experi- ment 7, while in the other 2 samples, sodium deoxycholate was deleted. One ml of the Sephadex G—200 lipase fraction was added and the mixture was incubated at room temperature (230C) for 4.5 hours with shaking. The subsequent treatment of the reaction mixture was described in experiment 7. Experiment 12 - Reaction of The Sephadex G-200 Lipase Fraction with EggpPhQSphatidic Acid. The powdered phoSpha— tidic acid obtained from the Pierce Chemical Co. was dis— solved in 25 ml of a chloroform:hexane solution 9:1 (v/v) and stored in the freezer until needed. Two 5 ml aliquots were taken from the above stock solution, evaporated under nitrogen to:¥ 0.5 ml and streaked on separate preparative TLC plates. After chromatographing in chloroform:methanol: ammonium hydroxide, 75:25:4 by volume, the plates were Sprayed at the edges and 2 phosphorus positive spots were revealed. The R values were 6.8 and 0.29 respectively. f 76 Only the upper bands Rf = 6.8 were used for the lipase re— actions. They were scraped off the plate, eluted with 100% methanol, and evaporated to dryness under nitrogen. Approxi— mately 1 mg of phosphatidic acid was eluted from each of the bands. The reaction mixture mentioned in experiment 7 was added to the test tubes, and they were sonified for 1 minute as described in experiment 9. A 0.5 ml of the Sephadex G-200 lipase fraction was added to each tube and they were incubated at room temperature (230C) without shaking, for 0.5 and 4.5 hours respectively. The subsequent treatment of the reaction mixture was described in experiment 7. Experiment 13 - Reaction of Sephadex G-200 Lipase Frac— tion with Synthetic_8 Arachidoyl Phosphatidyl Choline. Two samples containing 0.54 mg of synthetic phoSphatidyl choline were prepared and sonicated as described in experiments 7 and 9 respectively. A 0.5 ml aliquot of the Sephadex G-200 lipase fraction were added to each tube and they were incu- bated at 300C for 20 minutes without shaking. The subsequent treatment of the reaction mixture was described in experi-‘ ment 7. Sonication of EggpPhoSpholipids and The Sephadex G-200 Lipase_§raction Experiment 14p- Sonication of EggiPhosphatidyl Choline and Phosphatidyl Ethanolamine for 15 Minutes. Duplicate 1 mg 77 samples of either phoSphatidyl choline or phoSphatidyl ethanolamine were evaporated to dryness in small metal beakers 1.75 cm cud. x 3.85 cm in height. The reaction mix- ture mentioned in experiment 7, under the previous set of experiments, was added to each of the cups, followed by 2 for 1 minute at sonication at an intensity of 23 watts/cm 300C. The sonication setup was previously described under the section entitled Sonication Experiments Using Crude HogPancreaticLipase. After the addition of 0.5 ml of the Sephadex G—200 lipase fraction, the reaction mixture was sonicated for an additional 14 minutes. Phosphatidyl choline and phosphatidyl ethanolamine blanks were run in the same manner as described above, except for the absence of the enzyme. The mixtures were analyzed as described in experiment 7 of the preceding section. PhoSphorus tests were made on samples obtained directly off TLC plates to determine the percentage of lysophophatidyl choline and -ethanolamine formed from the original phosphatidyl choline and -ethanol- amine. Experiment 15 - Sonication pi Egg PhoSphatigyl Choline §pg_PhoSphatidyl Ethanolamine for 1, 3y,5, and 10 Minutes. The same sample size and reaction mixture as used in eXperi-- ment 14 were used for this experiment with the exception that the material was sonicated for 10 seconds (23 watts/cma) prior to the addition of 0.5 ml of the Sephadex G-200 lipase 78 fraction. EXperimental conditions used were as follows: Additional Reaction Time of Sonication Time without Sonication Sample No. with Lipase at 300C at 300C, No Shaking Control 0 30 minutes 1 1 minute 29 minutes 2 3 minutes 27 minutes 3 5 minutes 25 minutes 4 10 minutes 20 minutes After the predetermined reaction time, the samples were treated in exactly the same manner as described in experi— ment 7. Experiment 16 — Sonication of The Sephadex G-200 Lipase Fraction for 1y 5L_5, and 10 Minutes. Five samples (2 ml) of the Sephadex G—200 lipase fraction were placed in small metal beakers without the addition of deoxycholate, albumin, buffer, or phospholipids. They were sonicated (23 watts/cmg) for 1, 3, 5, and 10 minutes at 300C. A sample which was not sonicated served as the control. After sonication, the samples were tested for lipase activity on an olive oil-gum arabic emulsion. RESULTS AND DISCUSSION Preliminary Experiments with Lipase and Its Reaction with LecithianPhosppatidic Acid and Methylated PhoSphatidic Acid The Specificity of hog pancreatic lipase for the a, 0' positions of triglycerides and other primary ester groups is well established. Therefore, it is reasonable to assume that lipase would react with the diacylglycerophoSphatides to produce their corresponding 2-acyl—lysocompounds. To test this assumption, experiments were designed to hydrolyze syn- thetic dipalmitoyl lecithin or soybean lecithin with a semi- purified preparation of hog pancreatic lipase. An emulsion system similar to that used for testing lipase activity (141) was employed. The main criteria used for determining whether lipase had actually hydrolyzed any fatty acids from the lecithin molecule was a decrease in pH of the reaction mixture. In addition, the lipids were extracted from the reaction mixture and Spotted on TLC plates to test for any liberated free fatty acids. Results of these experiments indicated that there was a small decrease in pH, however, the TLC results were inconclusive. It was also recognized that any decrease in pH could have been the result of the 79 80 liberation of fatty acids by phospholipase A activity present in the lipase preparation. In conjunction with the above experiments, the problem was approached from the stereochemistry of the lecithin molecule. It was conceivable that lipase did not hydrolyze L-d lecithin very rapidly due to the presence of the choline moiety on the lecithin molecule. The choline moiety may have acted as a steric hindrance to the enzyme, thus prevent— ing the formation of an enzyme-substrate complex. Keeping this in mind, synthetic L-d lecithin was hydrolyzed by phos- pholipase D to produce phosphatidic acid. Upon reacting phoSphatidic acid with lipase for 1 hour, again a small decrease in pH was noted. PhoSphatidic acid proved to be more difficult to work with than lecithin. Due to the acidic nature of this phos- pholipid, it was difficult to maintain the pH of the emulsion system at pH 8.5 or 9.0 without the use of a buffer. The latter pH range was desirable because the optimum activity of the lipase enzyme fell between these pH values (92). In addition, phosphatidic acid is very unstable unless it is converted to its salt, usually the sodium form (104). Olley (195) showed that phOSphatidic acid underwent hydrolytic decomposition by an intramolecular attack. The phosphoric acid group attacked the two fatty acyl esters with the sub— sequent formation of free fatty acids and equal amounts of a and 8 glycerophoSphoric acid. 81 Since there were difficulties in working with phoSpha— tidic acid, it was reasoned that methylation of the free phosphatidic acid (acid form) would solve the problem of unstability and would still allow lipase to hydrolyze the a fatty acid ester of the dimethylphoSphatidate without steric hindrance. It had already been established by Baer and Maurukas (12) that diazomethane could produce phoSphatidic acid directly from synthetic phoSphatidyl serine or phoSpha- tidyl ethanolamine without first hydrolyzing off their re- spective bases by phoSpholipase D. They could not, however, produce phoSphatidic acid from phOSphatidyl choline. >Crone (53) subsequently showed that phosphatidyl choline also may be converted directly to phOSphatidic acid, but that the reaction was much slower than with either phoSphatidyl serine or phOSphatidyl ethanolamine. In this experiment, synthetic L-d-lecithin was first hydrolyzed by phOSpholipase D to produce phoSphatidic acid according to the procedure of Davidson and Long (56). The phOSphatidic acid was then methylated with diazomethane to yield the dimethylphOSphatidate. This procedure was used later by Wurster and Copenhaver (229) in their work. ‘ Methylation of both phosphate ester positions was con— firmed by both I.R. and NMR spectroscopy. Infrared spectra of both the free phoSphatidic acid (acid form) and the methylated phoSphatidic acid are shown in Figure 3. The infrared spectra of free phosphatidic acid agreed very well 82 w chHUHZ CH zumcmdm>mz e we .3 me. ma 3 ca m m a m m. a w No _ - 1 _ _ _ W _ d _ _ _ _ _ 1 .l .1 on T: IJ .1 .1 ca Fl IL. T: .4 Op 1' I .I ooe T 504 0334585 I. T. . IL I l c as l .1 ON T I. oa 1| L .Ilow 304 83.2585 1 l anagram: A .. A _ 1 a. 7 C/ l ooe T h L L L L L L r L P L _ L r .L T» 4» T» .L T. T» 70 70 c. .V n: 9 .L 8 6 0 TV 2 Ca .7 C.. L O 9 O O O C._ O O O O O O O O 0 9 O O O O O O O O O O O O O O O O O O O O O HIE0 HmQEscw>m3 .saoo .u:0>aom .UHUM UHUHumnmmOSQ UmumH%£umE UGO Odom UHUHumzmmOSQ m0 muuommm UOHMHMCH .m whsmflh 83 t pi, (2). These with the Spectra published by Abramson authors stated that the acid form of phosphatidic acid had a broad Shoulder in the 1020 cm"‘1 region and a maximum at 1055 cm71. .As the cation content increased, the absorption at 1020 cm’1 decreased, and the absorption at 1100 cm‘1 became relatively stronger than at 1055 cm'l. The spectra of the methylated sample agreed with the one published by Crone (53) for the dimethylphOSphatidate derived from phOSphatidyl ethanolamine. Although the dimethylphoSphatidate for this eXperiment was derived from L-a—lecithin, the correlations of the phOSphate ester group still held true. Crone stated that the untreated phoSpha— tidic acid had a strong absorption band between 1200 and 1250 cm“1 which, in the methylated compound, appeared between 1250 and 1300 cm‘l. The former condition was characteristic of the P=O stretching vibration in compounds with two electronegative substituents or in which there was hydrogen bonding of the P=O group. The condition found in the methyl- ated compound was reported for compounds with three substi— tuents on the phoSphorus (19,54). The second main effect of the diazomethane treatment on phOSphatidic acid was the appearance of a strong absorption band at 1045 cm‘l, which is characteristic of aliphatic substitution on phosphates. The NMR spectra of phOSphatidic acid (acid form) and methylated phoSphatidic acid are shown in Figure 4. The ) co ’elations used here are mainly from Ramirez et pl, (169). 84 _. ‘." 1M5 «(mom snows S _ J -‘ 6 o 3 O ' (H J=ll.5 cps , g1. \ j \ 1‘ x - . W '! )Vi ) Ii. . ,1". 2:: UA‘R‘lvufii'i‘n‘ : , .. ‘k L (H “Maw“! )“ MYNYIAYED | h if M) )‘n. h l. ,‘ "I'M ,_ 1, , ~ \ ‘10 'VI, H" mosvmmmc ACID 1 ) x t | .. , . F p U) ()4 7’ n mosrmnmc A .u . u ‘ '. 5,.) a W nv 0° . I I.,! u‘J 1"; ‘ _ ’,I-‘ '1 .. . ‘1' l1 1 I l 1 l a“ 6 7 8 9 10 Nuclear magnetic resonance Spectra of phosphatidic acid and methylated phosphatidic acid. Solvent: CCl4. Figure 4. 85 Methylated phosphatidic acid was easily distinguished from that of the unmethylated sample by the presence of a doublet at 6.30 Tau with JHP=11.5 cps. Chapman and Morrison (44) have published NMR spectra of phOSpholipids. However, they did not Show Spectra for either phOSphatidic acid or methyl- ated phOSphatidic acid. They assigned Tau values of 6.0-6.2 for the —CH2NMe3 and —CH20CO groups of lecithins. The -CH20CO group is also present in phosphatidic acid, but, at the concentration of phosphatidic acid used in this experiment, there was no recognizable response even at higher magnifica- tion. The low concentrations used may account for the absence of the Spectra characteristic of olefinic groups in the un- saturated fatty acid moieties in the molecule. After methyl- ation of phOSphatidic acid, however, the presence of the two methoxyl groups was readily apparent even at a 2% concentra— tion in CC14. When the methylated phosphatidic acid was hydrolyzed by lipase in the same manner as previously described, the pH decreased after a 1 hour reaction period and free fatty acids were noted on TLC plates. The experiments cited above gave evidence that some reaction was taking place between the phOSpholipid substrate and lipase. However, it was also quite obvious that the criteria used for determining whether phOSpholipid hydrolysis had actually occurred, i.e., a de- crease in pH and establishment of the presence of liberated free fatty acids by TLC, needed modification so that conclu- sive evidence could be presented. 86 During the course of these studies, De Haas pp_ai, (62) showed unequivocally that a pure lipase preparation devoid of phoSpholipase A activity will hydrolyze synthetic 1—oleoyl-2-Stearoyl—glycero—3-phoSphorylcholine at the primary acyl ester position. Evidence presented by De Haas p£_ai, as proof of the specificity of hog pancreatic lipase included a TLC diagram of the lysolecithin formed and GLC data for the fatty acid composition of this lysolecithin. The GLC data showed that only stearic acid, which was in the secondary acyl ester position, remained on the lyso- lecithin molecule. Recently, Renkonen (171) elegantly demonstrated that a pure lipase preparation will hydrolyze methylated phoSphatidic acid to produce the dimethyl lyso- phosphatidate. The proof advanced for this reaction was similar to that given by De Haas p£_ai, (62) i.e., it was based on TLC and GLC data. In addition Renkonen confirmed the NMR data on methylated phoSphatidic acid presented in this experiment. In retrOSpect, it should be stated that although the basic assumptions that lipase would hydrolyze a phoSpholipid was first tested in the experiments performed in this labora- tory, it was De Haas and his colleagues who actually advanced unchallenged proof that lipase hydrolyzed the primary fatty acid position of a phoSpholipid. With the establishment by De Haas pp.ai. (62) that hog pancreatic lipase acted on lecithin, it was of interest to 87 determine whether or not lipase would hydrolyze other phos- pholipids such as phoSphatidyl ethanolamine, phoSphatidic acid, and lysophosphatidyl choline. In conjunction with the above, investigations were performed to determine the effects of sonication on the lipase-phospholipid reaction. To establish the effect of sonication on enzyme activity under various conditions sonication experiments were first performed on the lipase enzyme without the addition of a substrate. These experiments were followed by sonication of lipase in the presence of triglycerides as a substrate. Finally, purified egg phoSphatidyl choline or phosphatidyl ethanolamine were reacted with lipase during sonication. Sonication Experiments Using_Crude Hog Pancreatic Lipase (Experiments ij6)— Heating the enzyme or sonicating it for 1 minute at various temperatures produced the same type of inactivation curve (Figure 5) however, the lipase was more rapidly inac- tivated when sonicated. The latter was attributed to the intense cavitation produced during the sonication treatment. There was no activation to the enzyme during the first 5 o o , or 50 , as was minutes (Table 1) of sonication at 100, 30 indicated by the sonication experiments of other workers (57-59, 42, 117, 174, 175, 214, 254). At 40° there was a slight activation of the enzyme during the first 5 minutes of sonication (Figure 6 and Table 1). In subsequent m..;- .n, U liv,V in“: FHA .- 8 “UFNIW hrs-h 88 00 .mmDBauom summed co .nmusumummfimu msoaum> um .muDCHE H How UCHUMOHCOw paw mcflummn MO uomwmm 0:8 .m musmflm °NIW/GIOV 'AIHOHOHDIW - ALIAILDV HSVdIT ..a. .vh-JJIJ ,-.vau.:.m,v d 89 mMBDZHZ .MEHB ZOHBdUHZOm on mm om me oe m _ 1 J _ _ _ Q 000m B) coca coon MWfll UOOH nlJf\ {MWWI F _ L r l r so muswmummfimu pct mEHu coauMUHCOm m0 uummmm One .wufi>fluom mmmmaa .m madman oa ‘NIW/GIDV"AIHOSOHDIW - ALIAILDV ESVdIT 90 experiments with a semi-purified lipase preparation, an in— crease in activity was again noted after a 3 minute soni- cation treatment (Figure 16). This phenomenon was reported by many workers and is discussed in the section entitled, Sonication of Egg,Phospholipidpiand The Sephadex G—200 Lipase Fractionl Experiment 16. The literature contains numerous reports on both the activation and inactivation of enzymes for various sonication treatments (Appendix A). It is difficult to compare these reports since the conditions for each experiment varied with the frequency, intensity, duration, temperature of sonica- tion, atmosphere used, and with the particular enzyme em- ployed. There is some indication that in cases where increas- ed enzyme activity was reported during the first few minutes of sonication, the enzymes were not pure. This has been shown with milk xanthine oxidase (175) where the enzyme is attached to the surface of fat globules as lipoprotein cenapses. Upon sonication, these cenapses break and the enzyme is intensely activated before loss of activity finally occurs. In impure pepsin preparations, it was postulated (42,156) that there may be a conversion of inactive pep- sinogen to the active pepsin during the first 3 minutes of sonication, thus accounting for a rise in activity. The increase in activation noticed in these experiments during the first 5 minutes of sonication may be similar to the case of milk xanthine oxidase (175), since lipase is also 91 known to exist as a lipoprotein complex before its purifi- cation to a pure protein (186). The latter condition would imply that there was no actual increase but just an apparent increase in lipase activity, i.e., the enzyme was now becoming available to the substrate after breakdown of the lipoprotein complex. Since a large amount of heat is produced during sonica- tion, the initial bath temperature at which the enzyme was equilibrated before sonication was usually 90-13OC below the final desired temperature (Table 2). The final desired temperature was usually reached and maintained within 15-20 seconds after sonication was started (Figure 7). It should be pointed out that the temperatures given for the enzyme during sonication, were average temperatures of the entire material in the beaker. It is well known that the particles of material undergoing sonication may be at higher tempera- tures at a given moment than the average values (221). Although many workers have reported that N2, H2 or other gases decrease enzyme inactivation during sonication, in the case of lipase similar results were obtained by lowering the sonicating temperatures. Nearly all of the original lipase activity was retained after a 30 minute sonication treatment in air at 10°C. It was evident (Figure 6) that any temperature between 100 and 300C gave nearly the same rate of lipase activity for a given sonication treatment. As the temperature was increased above 92 Percent of original lipase activity after sonica- Table 1. tion treatment at constant temperature (12 C). Sonication Time (min) 10°C 50°C 40°C 50°C 0 100 100 100 100 1 101 101 107 65 3 100 98 104 58 5 98 100 105 62 10 98 101 93 52 20 96 95 79 38 30 98 95 79 28 Table 2. Starting and final temperatures of lipase systems undergoing sonication at 1, 3, 5, 10, 20, and 30 minutes. Starting Temp. of Temp. of Enzyme Run No. Bath and Enzyme, OC dpiing R n 12°C 1 0 10 2 17 30 3 31 40 4 40 50 93 (C W m El; 1.33-3.5“. 0 H11.“ J.,-.c 211.11.03.11ugv62ivudlbbi , A m c . ., . a .. 27...,1.-- on 8 5L 3 77.03.01], 1.! we? ,. l I )Inl )(J i ) . . ..7 .. NJ,.n1csw.nceun.n55-n./adan vac , .. .. GIL 1|; 352:2 £2: TEMP” °c Chart recording temperature of water bath and Figure 7. °c, _-t2°C. enzyme during a sonication run at 30 94 300C there was a progressive inactivation of the enzyme. Therefore, 30°C was considered to be the approximate critical temperature for sonication of the enzyme. This meant that there was practically no alteration in lipase activity during sonication at any temperature below 300C for times up to 30 minutes. The decreased lipase activity at higher temperatures of sonication may be attributed to enzyme oxidation. It was demonstrated that oxidation of lipase with H202 or other oxidizing agents destroyed or reduced lipase activity (114, 202). Since sonication treatment of aqueous solutions is known to produce H202, it is likely that oxidation of the enzyme occurred. Table 3 shows the results of the lipase-olive oil sonication experiment. It was not possible to determine the rates of hydrolysis for this experiment since it would have been necessary to have placed the pH electrode in the same aluminum beaker as the sonifier probe. This in turn might have caused damage to the electrode. Instead, the total amount of fatty acid liberated after 4.5 minutes was deter- mined. The sonicated system liberated 2.7 times as much fatty acid as the stirring system. This increase in rate was directly attributed to two facts: 1) a better emulSion Of olive oil and gum arabic was obtained and 2) there was a greater turnover rate of substrate at the oil-water inter- face for the enzyme to act upon. 95 Since sonication produced a 2.7 fold increase in the amount of fatty acids liberated in the lipase—substrate experiment, it was of interest to see whether sonication would increase the hydrolysis rate of a high melting point triglyceride such as tripalmitin. Preliminary experiments showed that triplamitin would not hydrolyze even with soni— cation unless the temperature of hydrolysis was raised to 64°C, the melting point of the tripalmitin. This temperature of course would soon inactivate the lipase (Figure 5). However, with the addition of methyl myristate which acted as a carrier for the tripalmitin (15), the hydrolysis temperature could be lowered to 450C. Even at 450C the hydrolysis rates were very slow (Table 4) and the duration of the experiment had to be increased to 9.5 minutes instead of the 4.5 minutes used with the olive oil experiment. No fatty acids were liberated during the lipase-tripalmitin stirring experiment. The latter was attributed to the fact that there was no emulsion formed between the tripalmitin, methyl myristate, and buffer. Therefore, it may be stated that sonication by itself will not increase the hydrolysis rates of the higher melting point triglycerides unless a carrier is present to lower the original melting point or maintain the component in solution. Raising the temperature above 38°C may be unnecessary if enough carrier is present to solubilize the high melting point triglycerides during hydrolysis. 96 Table 3. Effect of sonication and stirring of enzyme and olive oil-gum arabic emulsion for 4.5 minutes at 38 C. u Equiv. of total fatty acids liberated Run No. Sonication Stirring 1 144 44 2 134 58 3 141 58 4 134 48 5 129 49 Avg. 138 51 Table 4. Effect of sonication and stirring of enzyme and tripalmitinl for 9.5 minutes at 45°C. u Equiv. of total fatty acids liberateda Run No. Sonication Stirring 1 11 0 2 15 0 5 19 0 lThe tripalmitin contained methyl myristate as a carrier. 2The methyl myristate blanks have been subtracted in the above figures. 97 Preparation of Hog Pancreatic Lipase Fractions with Increased Activity Due to the fact that the crude hog pancreas used for the preceding sonication experiments contained only 11% lipase activity and 89% phOSpholipase A activity (PhoSpho- lipase A:lipase ratio, 8.45:1, Table 7), it was necessary to increase the lipase activity by purification before it could be used for the reaction with phospholipids. Other— wise, most of the lysocompound formed during the lipolysis would be due to phoSpholipase A activity. The crude enzyme was purified by three different methods. The activities of the three lipase preparations are shown in Table 5. The Sephadex G-200 lipase fraction was slightly higher in specific activity than the butanol ex- tracted lipase. Electrophoresis of the Sephadex G-200 fraction increased the specific activity 1.5 fold. .Chromatography of the ammonium sulfate precipitate of lipase on Sephadex G-200 resulted in the elution of the active lipase peak immediately following the void volume. Only the lipase peak is shown in Figure 8, however, two other protein peaks were eluted after the lipase peak, but they did not contain any lipase activity. These results are in good agreement with those obtained by Sarda 23.2;- (190) . When testing for the linearity of reaction, a straight ' ion line relationship between enzyme concentration and react 98 wnu How poms mmB %uH>Huum mmmmaa u .coauumum mmmmfla OONIO xmpmnmmm map mo mans .omfi mucmummmu mom .mucmEAHOQXm pamaaonmmonmtmmmmmw nonman 0:» pmcamucoo £0flg3 a: 095» H a Monmouuomamh .coflumuamausm xopmnmmm umuahm .de mocmummmn mom .> moum coaumoflmaucma )l'l ooa.m aaa.d oao.m mm a.nmammuonmouuooam was.e oce.e meo.m ome moomuo xdtcsamm cam.e new. mse.e mme acoeeossuxm Hocscam WNH>HHM¢ HE\chuoum m2 HE\muacD mocmummmm COApmowmqum .mwvw m m0 UOSumz .mmuspooonm usmnmmmflp wows» ma summed oaumouosmm mos mo coeuwoflmausm .m magma ,. l. a. u .. Tani m a n a _ u.— 1 a... IJQA nun-J ‘ III ran-llllw v Im/UISQOId EN 99 m.. Hogan: 0939 o---o .Aomd .wmmv 00m: mmB :SDHOU xmpmzmmm So we x am.m a .mnsu\as m .msoeuomnm .maomo ammo.o ca Homz Smh.0 .Gowufiaom mcflusdm «macauflpcoo .mmmmHH Oflummuocmm m0: m0 OUMDHQHUOHQ Ugandsm ESHCOEEM 0:» m0 >£QMHmOumEOH£U OONIO XwUmnmmm .m Guzman \-| N N) C) BOT X Im/sqtun Kaintioe esedtq 100 rate was obtained for enzyme concentration greater than 0.15 ml. After that concentration, the rates were no longer proportional to the amount of enzyme added (Figure 9). Although the linearity of reaction was tested for all three of the preparations, the enzyme concentration range (0.02- 0.15 ml) tested with the Sephadex G-200 lipase fraction was applicable to the other preparations. The lipase from the Sephadex G-200 column was usually eluted in 8-9 fractions containing a combined volume of 24-27 ml. Before application of this fraction to the electrophoretic bed, the lipase solution was dialyzed against deionized water overnight at 20C and concentrated to a volume of 7-10 ml by the use of Sephadex G-25 (5). The electrophoretic pattern of the Sephadex G-200 lipase showed that at least 3 peaks were present (Figure 10). However, almost all of the lipase activity was recovered in tubes 0, -1, -2, +1, and +2. The electrophoretic pattern of the Sephadex G-200 lipase was always reproducible. The standard protein curve used in determining the protein in each lipase fraction is shown in Appendix C. Upon converting y to mg of protein (10007 =1 mg), the specific activity of each lipase fraction may be calculated by dividing the number of units activity/ml by the number of mg protein/ml. 101 coauomum mmmmaq OONIO xmpmcmmm mo .H2 mm. em. ON. 3.. we. _ _ z _ e a _ _ _ z d l a: N) BOT X Situn KQIAIqoe esedtq l at -mcmz.zmo.o,uo,geauattm70aumecuso 0nd Nb Umdflmugflma mm3 O.m m0 mm ucmumcoo d .OOSM um mflmhaomfla mCflHDU Umuspoum huw>fluum MO mudcs Hmuou Cam coaumuucmocoo mmmmfld :mm3umn AHE mH.O Ou may QHSmCOflumamu HMOCHA .m mhzmflm fired fl QHUHA VIL,v-)ulJ-A &.viuifl .J... .- Fa. I I‘lll‘lil 102 nm 093 is soueqxosqv 'utsiOJd .—- 0 m0. 0 :4 L0 \--l O O N LO N V7 DO 0 mponuumam e HOQEDZ mass mpouuomam m +ma +ca +ae +me +oe +8 +8 +4 +N 0 N: ,ww c- m. can me: as- can me: o ,m _ 4,4 mWIL .4. q _ _7_ a L _ ,— _ _ Aw \L \~MVIf:4 _ — _ 7— q.~ ll (((OIIO’ . Ir IO_IAIO\\O I1 .InVAu, . 1' / . Ii . 17§4 . 1. L fin A/. HM , a M. . TI .1 5 1| In 1. II I. coaumoaamm¢\\(\vw :1 mo usaom I, .1 mm. J noumum may Eoum Congas mp3 dawned one .0 N .wusumummfimu cam muHo> oom “0.5 mm .mumnmmozm 2mm .0 .ummmsm emcoauflpcoo .Amm .mmmv unmammm EU H Hem m.m mm .uommsn mans Zfi.o no He OH £ua3 own “mason we now ME ON .mmwuocmm mos Bonn coauumnw mmmmfla OONIO memnmmm mnu MO coauwummwm oaumuonmouuomam .Od musmflm SOT x Imysiiun AqrArqoe 982511 -CD CD. 103 Synthesis of 8 Arachidoyl Lecithin from EggLysolecithin To ideally determine the actualeumnnnzof phoSpholipase A activity present in a lipase preparation a known synthetic lecithin is necessary as a substrate. The fatty acid composition of the a“ and 8 positions should be different so that phospholipase A and lipase activity may be distinguished by analyzing the remaining lysolecithins after reaction. It is also possible to make this distinction without having to synthesize the complete lecithin molecule starting from glycerol. In this study lysolecithin was prepared from natural egg lecithin and reacted with arachidoyl cholride to form a new lecithin. Since natural egg lecithin contains only 0.1% arachidic acid in the 0' position (125), essentially all of the arachidic acid on the newly formed lecithin was in the 8 position. Therefore, any lipase preparation react- ing with the synthesized lecithin and yielding a lysolecithin with little or no arachidic acid, contained a high amount of phospholipase A activity. Although some of the egg lecithin-phOSpholipase A re— actions were hydrolyzed for periods of up to 24 hours, complete hydrolysis of the lecithin was not always achieved. Thin-layer chromatography of the concentratedcether phase after centrifugation and removal of the lysolecithin showed the presence of lecithin. Many workers consider the absence of phoSphorus in the ether phase, after removal of the 104 lysolecithin precipitate, as evidence of complete reaction. However, this may not be a truly indicative test for com— plete conversion to lysolecithin. If the phosphorus test used on the aliquot of ether sample was not sensitive enough to pick up traces of phOSphorus, it would appear as though the reaction were complete. Conversely, Rouser _p _i (180) pointed out that phOSphatides which contain a great deal of unsaturated fatty acids may not give a precipitate, as the lysocompound containing unsaturated fatty acid is relatively soluble in ether. In the latter case, the re- action may have gone to completion and yet a phoSphorus test without TLC would lead to the conclusion that lecithin was still present. .Nutter and Privett (158) state that quantitative conversion of lecithin by phoSpholipase A to fatty acids and lysolecithin is not generally attained and there is some evidence that different fatty acids may be hydrolyzed at different rates. Reactions of Lipase and Egg_Phospholipids Thin-Layer Chromatography. During some of the prelimi- nary experiments with phospholipid hydrolysis by lipase, it was demonstrated that extraction of the phoSpholipids from the reaction mixture by the use of chloroform:methanol 2:1 (v/v) resulted in a small portion of the phoSpholipids remaining in the aqueous buffer of the reaction mixture. 105 Since methylation was to be performed directly from the samples taken off the TLC plates, it was of importance to chromatograph the complete reaction mixture if ratios of phospholipase A:lipase activities were to be calculated. If however, the assumption was made that the portion of phoSpholipids remaining in the aqueous phase was identical in molecular composition to that portion being extracted, then the phoSpholipids remaining in the aqueous phase may be disregarded. Since various phoSpholipids have a greater tendency to dissolve in water than others, the amount of phospholipid remaining in the aqueous phase varied with the nature of the phospholipid. It was therefore decided to concentrate the entire reaction mixture and spot it on TLC plates. Figures 11-13 show the results of chromatographing the reaction mixtures of phosphatidyl ethanolamine, phoSphatidyl choline, and phoSphatidic acid. The R values in Figure f 11, 12 and 14 where the solvent system was chloroform: methanol:water, 65:25:4 by volume was 0.53—0.60 for sodium deoxycholate, 0.47 for phosphatidyl ethanolamine, 0.32 for phOSphatidyl choline, 0.25 for lysophosphatidyl ethanolamine, and 0.16 for lysophoSphatidyl choline. The R values in Figure 13 where the solvent system f was chloroform:methanol:ammonium hydroxide, 75:25:4 by volume were 0.66 for phOSphatidic acid, 0.41 for lysophos- phatidic acid and 0.23 for sodium deoxycholate. Figure 14 106 Figure 11 Figure 12 Figure 11. Preparative TLC plate of the phosphatidyl ethanolamine mixture after reaction with 0.5 ml of the Sephadex G-200 lipase fraction, spots 1-6. Adsorbent: Silica Gel-G; solvent system: chloroform:methanol:water, 65:25:4 by volume; indicators: molybdate reagent followed by 50% sulfuric acid and charring. Dotted outline indi— cates phoSphorus positive spots. SF: solvent front; U: unknown; DOC: sodium deoxycholate; PE: phosphatidyl ethanol— amine; LPE: lysophosphatidyl ethanolamine; O: origin. Figure 12. Preparative TLC plate of the phosphatidyl choline mixture after reaction with 0.5 ml of the Sephadex G—200 lipase fraction, spots 1-5. Adsorbent: Silica Gel-G; solvent system: chloroform:methanol:water, 65:25:4 by volume; indicators: molybdate reagent followed by 50% sul— furic acid and charring. SF: solvent front; U: unknown; DOC: sodium deoxycholate; PC: phosphatidyl choline; LPC: lysophosphatidyl choline; O: origin. Figure 13 Figure 14 Figure 13. Preparative TLC plate of the phosphatidic acid mixture after reaction with 0.5 ml of the Sephadex G-200 lipase fraction, spots 1-6. Spots 7 & 8: phOSphatidic acid as purchased from the supplier. Adsorbent: Silica Gel—G; solvent system: chloroform: methanol:ammonium hydroxide, 75:25:4 by volume; indicators: molybdate reagent followed by 50% sulfuric acid and charring. SF: solvent front; PA: phosphatidic acid; LPA: lysophoSphatidic acid; DOC: sodium deoxycholate; O: origin. Figure 14. Preparative TLC plate of sodium deoxycholate and serum albumin used in the reactions of phOSpholipids (Figures 11-13) with the Sephadex G-200 lipase fraction. Spot 1: sodium deoxycholate; 2: sodium deoxycholate plus serum albumin; 3: serum albumin. Adsorbent: Silica Gel—G; solvent system: chloroform:methanol:water, 65:25:4 by volume; indicator: 50% sulfuric acid followed by charring. 108 shows the TLC pattern of sodium deoxycholate both in the absence and presence of serum albumin. It is evident from the TLC plate that the sodium deoxycholate contained at least two unknown minor components. When serum albumin was Spotted together with sodium deoxycholate, its presence caused increased tailing of the sodium deoxycholate Spot. Serum albumin itself did not chromatograph in the solvent system used for these experiments. Phosphatidyl ethanolamine overlapped with the sodium deoxycholate spot (Figure 11). The dotted outline represents the phosphorus positive spot after spraying with molybdate reagent. The remainder of the spots were made visible by Spraying with 50% sulfuric acid and charring. Analysis of Synthetic 8 Arachidoyl Lecithin and Egg Phquholipids by Gas Liquid Chromatography. After synthesiz- ing 8 arachidoyl lecithin from egg lysolecithin it was re— acted with phospholipase A and the residual lysolecithin was analyzed for its fatty acid composition. No arachidic acid was found in the d' position. Analysis of the complete syn? thesized lecithin is shown in Table 6. A 1.03:1 ratio of 0' t0 6 fatty acids was obtained. These results were in very good agreement with the theoretical ratio of 1:1. The synthesis of lecithin from lysolecithin involves a lengthy procedure in which the lecithin formed may become oxidized easily if due care if not taken. This is eSpeCially 109 a..5 seems. 4e.mo.e . . IIIII . u D" oapmm H0.aud d.m0 a IIIII WW.Mmfi mmmum 00.0 ea.0m mm we IHDSMmcc Hmuoe o o o 00.00a mm.ma 0H.Hm mm.www mw.mwa mmumusumwpmww a O 0 O I“ . . 00 00a mm mm H0 00 maumm aspoa IIIII >0.H mm.md IIIII 0H.m muom N0.m¢ momuu IIIIIIIIII momuu ouom lllll QUMHU. QUMHH lllll @UMHU mum.“ QUMHH Nmofifi Hm..mfi OUMHU Omofifi Nam.“ moon» mm.mm mo.>H momuu Ow.mN Humd s¢.ma mm.¢a mm.dm m.mm 0m.mH onma 00mm» m¢.a 0m.m woman mm.fi auoa Hm.mm 0N.mm mm.md w.¢m 00.Hm oumfi OOMH# OUMHH OUMHU OUMHU OUMHH Onflfi mcaaono pave OGHEMHOSMSum mcaaonu mcwaozu pave > MI. Hmpaumnmmosm oapwumsmmonm Hmpflu8£mmonm HmpaumsmmOSQOmzq Hmpflumnmmosm . up m Hmopwnomud a oeuiscmm HGQUHOQ MORAN I) 110 true during the evaporation procedure to remove excess pyridine, which is accomplished at 30-350C, preferably under vacuum. In addition, the lecithin must be chromatographed on two columns (resin and Silicic acid) followed by prepara- tive TLC and elution. An advantage in this particular syn- thesis was that almost all of the fatty acids on the newly synthesized lecithin were saturated, thus the chances of the material becoming oxidized were minimized. To determine the ratios of phOSpholipase A:lipase activity for the various lipase preparations, it is necessary to know the composition of the phoSpholipid undergoing hydrolysis. Since it is difficult to synthesize lecithins, a natural source is desirable for routine lipase assays. Egg lecithin may be used because the majority of fatty acids on the 0' position are saturated while those on the 8 position are almost entirely unsaturated. Inasmuch as lipase attacks the a' position of the phOSpholipid while phOSpholipase A attacks the 8 position of a phospholipid, the ratios of phospholipase A:lipase activity may be determined by knowing the ratios of 8:0' fatty acids produced in the resulting two lysolecithins. A small correction factor must be made for phoSphatidyl choline and phosphatidic acid to take into account the fact that there are some unsaturated fatty acids in the a' position and some saturated fatty acids in the 8 position (125,167). The correction factor was calculated as follows: 111 1. The average composition of egg lecithin as reported by two different groups was taken (125,167). 2. The d' osition contains 90% saturated fatty acids and 10 unsaturated fatty acids, while the 8 position contains 98.7% unsaturated fatty acids and 1.3% saturated fatty acids. 3. Total saturates on molecule: 100 x 2%8é = 45.65% 1 O 08.7 _ Total unsaturates on molecule: 100 x 200 — 54.35% or Saturates:Unsaturates = 1:1.19 For example when crude hog lipase acts on phOSphatidyl choline, a ratio of saturates:unsaturates of 7.1:1 is obtained in the lysolecithins formed. The correction would be made as follows: 1 (s) 1 (Phoppholipase A) 1.19_iu) 1 (Lipase) 7.1 (s) x (PhOSpholipase A) 1 (u) 1 (Lipase) x = 8.45 5. Therefore the ratio of phospholipase A:lipase activity is 8.45:1. The same results may Simply be obtained by multiplying 7.1 x 1.19 = 8.56. 6. It should be emphasized that all of the analyses were performed on the remaining lysolecithins. 7. For the above correction to work, the total amount of unsaturated fatty acids in an experimental analy- sis must always be set to 1. 8. Using egg lecithin as a substrate, the highest lipase activity almost free of phOSpholipase activity would be attained by an experimental ratio of saturates:unsaturates, 1:75.9. In correcting the ratios to determine the actual amount of phospholipaSe A:lipase activity both numbers are divided by 75.9 to give a ratio of 0.01321. Then 0.013 x 1.19 = 0.015, or a ratio of saturates:unsaturates, 0.015:1. Upon dividing both numbers by 0.015, the corrected value obtained would be 1:66.66. 112 Corrections could not be made for the egg phoSphatidyl ethanolamine samples since there were no reliable published reports on the fatty acid composition for the a' and 8 positions. Although Hawke (110) reported on the fatty acid distribution of phoSphatidyl ethanolamine on the d' and 8 positions, his results were not in good agreement with the total fatty acid composition of phoSphatidyl ethanolamine as analyzed in this laboratory. It is very likely that some of the procedures Hawke used to isolate his phospholipid samples contributed to their oxidation, e.g., freeze drying, refluxing, and dialysis. The fatty acid composition of the egg phOSpholipidS used in the following experiments are shown in Table 6. Hydrolysis of Egg Phosphatidyl Choline by Various Lipase Preparations. The fatty acid composition of the lyso— phOSphatidyl choline produced by the action of various lipase preparations (experiments 7-10) on phosphatidyl choline are presented in Table 7. The ratios of saturatedzunsaturated fatty acids along with the corrected ratios for phospholipase A:lipase activity are also shown in Table 7. From the data presented, it was established that crude pancreatin (Mann Research Laboratories) contained 8.45 times more phospholipase A activity than lipase activity. The butanol extracted lipase contained only 1.92 times more phospholipase A activ— ity than lipase activity. Both the Sephadex G—200 lipase 113 e.mm.e dum¢.m mmmmfland mmmmflaonmmonm . anom. u . fi.dm.fi fiuam.w wumm.w dufi.> bum MO Ofiumm mm.m¢ 0¢.m¢ mm.mm mm.ma mmumusummcs Hmuoe mm.wm fi>.0m mh.am m0.>m mmumugumm Hmuoa em.mm ea.ooe Ne.ooe mm.mm meant Suede etude 00mph mo.m momma momma snow momma mm.m mumuu momma mums mm.m me.m mm.m mm.e m.me mm.am mm.me me.ce mm.e e.me cm.m em.me om.ae om.cm o.me om.a mm.me oa.me mm.m e.ce No.44 om.mm ma.ee mm.om o.ce momuu mommy momnu woman ouea one“ .mmmmaq cowuomum unseen madman mmmnocmm paod munch oeumuosmouuowam 00NIO xmpmnmmm pmawausm Hocmbsm mom mpsuo LDH ungflummxmv Am unwefiuomxmv Aw ucmEfluwmxmv as ucwawummxmL pcmuumm mmu< .mcflaono Hmpaumnmwozm 000 so mcoauumum ommmfla m50aum> >9 UOUDUOHQ OCHHOQU prflumnmmonmomma mnu mo GOASHmOQEOO peom hunch .m mHQMB 114 fraction and the electrophoretic lipase gave similar ratios of phoSpholipase A:lipase activity, 1.56:1. Although the - specific activity for the Sephadex G—200 isolated lipase was 1,795 and that of the electrophoretic lipase 2,700, the ratios of phospholipase A:lipase remained the same. This may be explained by the fact that electrophoresis probably removed other protein impurities from the lipase without removing any of the phospholipase A protein. Thus there was an increase in the specific activity of the lipase, but the . ratio of phospholipase A:lipase remained the same. .As ad- ditional evidence for this argument, Sarda pp pl. (190) found a second protein peak when the Sephadex G-200 lipase fraction was treated with ethyl ether and deoxycholate and rechromatographed on Sephadex G-200. Furthermore, when the latter lipase was purified by DEAE cellulose chromatography, aside from the lipase peak, two additional peaks were found. AS evidenced from the ratios of phospholipase A:lipase activity presented in Table 7, none of the lipase prepara- tions were free of phospholipase A activity. De Haas pp_ai, (62) succeeded in obtaining a lipase free of phOSpholipase A activity by using the Sarda ep_ai, (190) method of lipase purification. However, when the latter method of purifica— tion was tried in this laboratory, it was not successful. The main problem arose after dialysis of the first Sephadex G-200 lipase fraction. The lipase from that fraction is isolated as a lipoprotein complex of which more than 65% of 115 the protein moiety consists of lipase (186). The lipid must then be dissociated from the lipase, phOSpholipase A, and other proteins present by adding ethyl ether and sodium deoxycholate, after which both the lipids and proteins are precipitated (186). It was the difficulty encountered in obtaining a precipitate that caused the deletion, from the original procedure (190), of the second Sephadex G-200 chromatography step and the DEAE chromatography. In some preliminary experiments where the first Sephadex G—200 lipase fraction was chromatographed directly on DEAE, no lipase activity was recovered. This was due to the fact that when lipase is bound to lipids, it does not chromatograph on ion exchange resins. Therefore, delipidation is a compulsory step prior to purification because lipase tends to adsorb on the lipids which are present in the extract, even if only a very small amount of lipids is present (186). Experiments 9 and 10 demonstrated that the Sephadex G-200 and electrophoretic lipase preparations gave identical ratios of phospholipase A:lipase activities (Table 7). Since it was less difficult to prepare lipase by chroma- tography on Sephadex G-200 than by electrOphoresis, the former method was chosen as the purification procedure for obtaining lipase. Therefore, in all subsequent experiments, the Sephadex G-200 lipase preparation was used for the reaction with various phospholipids. 116 The fatty acid composition of phOSphatidyl choline and phosphatidyl ethanolamine hydrolysis products (experi- ment 9) after the reaction with lipase are Shown in Table 8. The fatty acid composition of the unreacted phosphatidyl choline was quite similar to that of the original phospha- tidyl choline (Table 6). However, with phosphatidyl ethanol- amine there were slight differences in the fatty acid compo- sition of the unreacted and original materials. An apparent increase in stearic and linolenic acids was observed. When Renkonen (171) reacted methylated phOSphatidic acid with pancreatic lipase he also noted that the unreacted dimethyl- phoSphatidate differed in fatty acid composition from that of the original starting material. Moore and Williams (153) observed that during the first stages of hydrolysis of natural lecithins by phospholipase A, different molecular Species are attacked at different rates. A similar situation exists when lipase attacks natural triglycerides (33,115, 233). Attwood pp_ai, (10) found definite differences in the observed rates of hydrolysis by phospholipase A on synthetic lecithins containing different fatty acids. These differ- ences appeared to be related to the ability of phospholipids to form micelles in aqueous dispersions (similar to the conditions in the present experiment) as well as the size and shape of the micelles formed. The above explanation by Attwood e£_ai, may account for the observed differences in fatty acid composition between the starting material and the unreacted phosphatidyl ethanolamine. rnvnu Idlznpi a. five; ‘14-; & r1- - \/ Tu F 0 C Fill—fa.- H,I.I flilfift U,\f\llu.II. 1,! ll 117 e.m..e . . . . . . 5.. seems 55.0m ww.mma Wemmea mw.wma mmumusuMmcs Hmuoa HNomm mmomuv abomm mmcfiw WQUMHSUQW HMUOH. mmomm HOoOOH fiHoOOH mmcmm mUHUM WHUQM HMUOB m>.m em.ma m0.m Hm.e snow co.c em.m mm.m em.m mmme mm.m sm.me ms.m mo.me «.me mm.me em.ae mm.me mm.mm e.me am.cm ce.cm am.me ma.me o.me on.m ac.m mm.me mm.m e.me em.cm em.om om.mm aa.mm o.ee mommy momma momnu momma ouwa maesmaosmrum maesmeosmsbm mceaoro mceeoro thud muumm Hmpflumzmmozmommq Hapwumnmmonm prflumnmmonmommq ahpflumnmmosm pmuomwncD Umpummunb ucounmm mend .Am unweflummxmv coauumum summed OONIO Kmpmnmmm one QuHB coeuommh umbmm muuspoum mflmmaoupxn mcaEmHocmnum H%pflum£mmocm pcm OCHHOQU ahpaumzmmosm mo coauflmomfioo UHOM wubmm .0 dance 118 Both the lysophosPhatidyl choline and lysophosphatidyl ethanolamine formed by the lipase hydrolysis contained a high amount of saturated fatty acids. As previously men- tioned, this was due to the high proportion of phospholipase A activity present in addition to the lipase activity. The amount of lysophosphatidyl ethanolamine and lyso- phosphatidyl choline produced after a 15‘minute reaction period was 33.23% and 18.75% reSpectively. Phosphatidyl ethanolamine therefore, reacted 1.77 times as fast as phos— phatidyl choline. This increase in reaction rate for phos- phatidyl ethanolamine may be attributed to two factors: 1. The fatty acid composition of the phOSphatidyl ethanol- amine differs from phosphatidyl choline in the amount of total polyunsaturates present, especially in arachidonic acid (Table 6). The differences in the observed rates may be due to the difference in fatty acid composition between the two phospholipids. Since both phospholipase A and lipase activities are present in the Sephadex G-200 lipase fraction, one or both of the enzymes may react more readily with those fatty acids distributed on a particular molecular Species of phOSphatidyl ethanolamine rather than those on phOSpha- tidyl choline. If only lipase were present, the reaction rates may have been quite different from those observed for this experiment. 2. The nature of the ethanolamine or choline base must certainly play an important role in deter- mining the reaction rates of different phospholipids with 119 various lipolytic enzymes. As will be shown in experiment 12, phOSphatidic acid reacted more rapidly than either phoSphatidyl ethanolamine or phoSphatidyl choline. In this case, the fatty acid composition of phoSphatidic acid was identical to that of phoSphatidyl choline and yet the re- action rate was more rapid for phoSphatidic acid. These results would tend to give more emphasis to the theory that the nature of the phospholipid base is important. Experiment 11 - Reaction of The Sephadex G-200_iipase Fraction with Egg LysophoSphatidyl Choline. To establish whether the Sephadex G—200 lipase fraction contained any lysophospholipase activity, the enzyme was reacted with lysophoSphatidyl choline both in the presence and absence of sodium deoxycholate. Since deoxycholate is a known in- hibitor of lysophoSpholipase (137), it was omitted from one of the reaction mixtures. No glycerophOSphoryl choline was produced either in the presence or absence of sodium deoxycholate from the reaction mixture. Lysolecithin was recovered from the reaction mixture unchanged. These experi- ments established that (1) no lysolecithinase was present in the Sephadex G-200 lipase fraction and (2) lipase did not react with the lysophoSphatidyl choline even though the fatty acid was on the d' ester position. Since these experiments were carried out for 4.5 hours, there was sufficient time for the enzyme to react with the lysolecithin substrate. 120 Experiment 12 - Reaction of The Sephadex G-200 LipeSe Fraction with EggpPhosphatidic Acid. Thin-layer chroma— tography of the purchased phosphatidic acid (calcium‘salt) revealed two phosphorus positive spots after Spraying with molybdate reagent (Figure 13, spots 7 and 8). A phOSphorus analysis of the upper (Rf = 6.8) and lower (Rf = 0.29) spots revealed that the upper spot contained 29.27% of the total phosphorus while the lower Spot contained 70.72%. The upper spot was considered to be the free phosphatidic acid, while the lower spot was actually the dicalcium salt of phoSpha- tidic acid. Gas-Liquid Chromatography of the fatty acid methyl esters derived from the two Spots showed that they both con- tained similar fatty acid compositions. Only the upper band or free phoSphatidic acid was used in the following experi- ments. After 4.5 hours of reaction time, all of the phos- phatidic acid was converted to lysophoSphatidic acid. This was indicated by only one phosphorus positive band (Rf=0.4) on the TLC plate. When the reaction time was decreased to 0.5 hours, 20.63% of the total phoSphorus appeared in the phosphatidic acid band while 79.37% was found in the lyso- phOSphatidic acid band. Results of the fatty acid analysis of the unreacted phosphatidic acid and the lysophosphatidic acid formed are given in Table 9. 121 OQB mza co mHQHmme Uaom oflpaamnmmonm 0c mm3 mmmna mocam mamHmEoo mmB coaaom .mmamam mm mamas Huwa.m Humo.m IIIII mmmmaaud mmmmmaozmmonm a.m.a mume.a a.mo.e S.m oaamm >>.mm Hm.0m Hm.>m mmammDammcs HmaOB am.a0 mm.m0 m0.mm mmammsamm Hmaoa mm.mm am.mm mm.mm mmaom maamm acaos aa.o am.o em.o 4.0m momma momma momma mama ma.m oa.a aa.m mums we.mm wa.am mm.mm fiumd mm.ma mm.mfi 0N.MH Game 00.0 m0.> we.> Hume mm.>a mm.ma mm.mm ouma momma momma momma Quad cave cave omtaacaamOSd Sada seams oapaamgmmOSQOmmq oapmamnmmoamommq UmaommmcD Ammson m.av AmHDOS m .Ov AwHDOE. m oOv acmommm mmm< .Amfi acmEHmmmxmv mmsos m.w Ucm m.0 mom coaaommm mwmmfla 00NIO xmpmcmmm mza £aa3 coaaommm mmamm memom paom oapaamflmmoam0mha 0cm Uaom oapaamtmmonm mom mo coaawmomfioo Uaom Maamh .a manta 122 The unreacted phosphatidic acid differed in composition from the original starting material (Table 6). Largest of the differences noted was in the linoleic acid content. The original phOSphatidic acid contained 11.62% linoleic acid while the unreacted phosphatidic acid contained 6.11%. As previously mentioned, the explanation of Moore and Williams (153) may be applied to clarify this occurrence. Both of the lysophOSphatidic acids formed after reacting the phos— phatidic acid for 0.5 and 4.5 hours, contained almost identi- cal fatty acid compositions and phospholipase A:lipase ratios. These results indicate that at least after 30 minutes of reaction time, there was no Specificity of phospholipase A or lipase to react with certain molecular Species of phos- phatidic acid rather than others. If this were not the case, then different fatty acid compositions of the lysophOSphatidic acids should have been obtained for the 0.5 and 4.5 hour experiments. Experiment 13 - Reaction of The Sephadex G-200 Lipase Fraction with Synthetic 8—Arachidqyl Phosphatidyl Choline. After 30 minutes of incubating the synthetic lecithin with lipase, only 13.18% of the total phoSphorus appeared in the lysolecithin band. The remainder of the phoSphorus, 88.82%, was found in the unreacted lecithin band. Evidently the re- action of the Sephadex G-200 lipase fraction with synthetic B-arachidoyl lecithin was much slower than with phosphatidyl 123 choline, phOSphatidyl ethanolamine, or phosphatidic acid (Table 10). Although the conditions for some of the experi— ments varied in time of reaction and temperature, the order in which the various phospholipids ranked with reSpect to the amount of lysocompound formed could still be determined by inspection. The fastest reaction occurred with phospha- tidic acid followed by phOSphatidyl ethanolamine, phosphatidyl choline, and synthetic phoSphatidyl choline. The fatty acid analysis of the unreacted synthetic lecithin and the lysolecithin formed are shown in Table 11. The composition of the unreacted synthetic lecithin was almost similar to the lysolecithin. However, when comparing the fatty acid composition of the unreacted lecithin to the original starting material (Table 6), a 20% increase in the oleic acid content was noted for the unreacted lecithin. There was also a 19% decrease in the arachidic acid content for the unreacted lecithin when compared to the original starting material. Upon analyzing the lysolecithin formed by the reaction, the ratio of total fatty acids liberated from the 0' position to those liberated on the 8 position containing the arachidic acid was found to be 2.83:1. No correction factor was necessary for determining the phOSpholipase A:lipase ratio Since both the saturated and unsaturated fatty acids aside from the arachidic acid were esterified to the 0' position of the molecule. Therefore, by simply taking the d':8 ratio of fatty acids the enzyme ratios could also be determined. 124 .mmsammmmama BoomQ .mammamcm memonmmonm mmm mcmaono Hmpaamnmmonm ma.ma on on ma IamopaaommmIm omamnacwm >m.m> 9mm 0m Na Umom oapaamnmmonm mm.mm on ma m mcaamaocmnam praamnmmosm ms.me on me a acamoso Haemamsmmora m memom oo.mmsammmmama A.cmav mama acmEammmxm pamaaonmmosm pcsomaoo comaommm coaaommm ommq mo R .comaommm mmmmaa OONEO xmpmnmmm mna Sam? mommaaosmmOSm mmm msoamm> mo comaommm mmamm UmEmOM pcsomEooomha acmommm .0fi magma dumm.m IIIII mmmmmaud mmmmmaommmonm mu.o oaamm 125 acmommm mmmd H«W®.N flamm.N oe.mm mo.om coaaamom a so made umcasommm mmaos mm.m> mm.mm comaamom .o co mpaom aaama Hmaoe $0.0m No.00H mpaom waamm Hmaoe 0a.mm 00.0m . ouom IIIII IIIII mama O¢.fi OUMHU Numfi mm.m m0.m auma mm.>m Hm.mm Oumd mm.m momma dame m0.mm mm.mm ouma momma momma owed mcamono mammoao mammamsmmoam Odom haamm praamzmmozmommq Hmopanommmrm UmaommmcD . .Ama acmEammmxmv coaaommm mmmmaa OONIO xmpmndmm mna naa3 coaaommm mmamm memom mcaaoao ampaam . . . . smmosmomma psm mcaaono mwpwamammonm ahopaaommmlu oaamaachm mo coaaamomfioo Uaom Maamm .afi mHQmB ac 51“? O ti 126 There appeared to be appreciable phOSpholipase A activity present in the Sephadex G-200 lipase fraction when synthetic lecithin was used as the substrate for the reac- tion. The use of the same Sephadex G-200 lipase fraction on phOSphatidyl choline, phOSphatidic acid, and synthetic phosphatidyl choline, gave increasing ratios of phoSpholipase A:lipase activity; 1.56:1, 2.11:1, and 2.83:1 respectively. It was evident that the phoSpholipase A activity was changing with respect to the nature of the substrate. ,One question still not answered is why there should be any difference in phOSpholipase A activity between phOSphatidyl choline and phosphatidic acid since both contain identical fatty acid compositions. However, Rimon and Shapiro (173) also noted Similar results when using a purified ox pancreatic phos— pholipase A on various phOSpholipid fractions. The authors unfortunately, did not give any explanation for this occur- rence. Perhaps the rate of phOSpholipase A activity is related to the presence or absence of the choline base. It appears that the reaction is more rapid when the base is not present as in the case of phosphatidic acid. The increase in phoSpholipase A activity for the syn- thetic lecithin, where essentially almost all of the fatty acids on the molecule were saturated may be explained by the observations of Nutter and Privett (158). These workers found that when 47.8% hydrogenated egg lecithin in soybean lecithin was hydrolyzed by phoSpholipase A snake venom, 127 the disaturated classes of lecithins hydrolyzed more rapidly than the other classes. Sonication of EggpPhOSpholipids and The Sephadex G-200 Lipase Fraction In the previous section, it was established that a semi— purified lipase preparation acted on phosphatidyl choline, phoSphatidyl ethanolamine, and phOSphatidic acid to produce their respective lysocompounds. It was also established that the enzyme did not hydrolyze lysophoSphatidyl choline to produce glycerothSphoryl choline. The final objective of this study was to determine the effect of sonication on the lipase-phospholipid reaction. Since an increase in the rate of lipolysis was obtained with the simultaneous sonication of lipase and its olive oil sub- strate (Experiment 3 under the section entitled, Sonication Experiments Using Crude Hog Pancreatic Lipase), it was of interest to test this procedure with the Sephadex G-200 lipase fraction using phoSpholipids as substrates. The in- tensity of sonication in the following experiments was 23 watts/cma, the same as for the lipase—olive oil experiments. Experiment 14 - Sonication of Egg Phosphatidyl Choline apd PhOSphatidyl Ethanolamine for 15 Minutes. Sonication of phosphatidyl choline or phoSphatidyl ethanolamine with lipase produced interesting results. It was originally expected 128 that sonication would either increase the rate of phospho- lipid hydrolysis by lipase or maintain the same rate of hydrolysis as the unsonicated samples. However, upon analyz— ing the results, there was actually a decrease in the total amount of hydrolysis. No lysophOSphatidyl Choline was formed during the 15 minute reaction period, while only 17.85% lysophOSphatidyl enthanolamine was formed. Under identical reaction conditions with no sonication (Experiment 9, under the section entitled Reaction of The Sephadex G—200 Lipase Fraction with Egg Phosphatidyl Choline and Phosphatidyl Ethanolamine), 18.75% lysophOSphatidyl choline and 33.23% lysoPhosphatidyl ethanolamine were formed. Obviously, soni— cation radically decreased the amount of lysocompound formed. At least three possibilities exist for this occur- rence: 1. Oxidation — If oxidation of the phoSpholipid occurred because of the high intensity of sonication, then it may be possible that the orientation of the fatty acids on the phospholipid molecule was changed in such a way as to prevent the enzyme from attacking it. 2. Enzyme Inactiva- tion - If oxidation of the phospholipids or of other chemicals in the reaction mixture occurred, then free radicals, perox- ides, or other ion Species formed during the sonication may have inactivated the enzyme. Although experiments with crude pancreatic lipase showed that the enzyme did not lose any activity after sonication periods of up to 15 minutes at 30°C (Table 1), a semi-purified enzyme may be more rapidly 129 inactivated. Inasmuch as phosphatidyl ethanolamine reacted twice as rapidly as phosphatidyl choline, it was conceiv- able that there was sufficient time for the phoSphatidyl ethanolamine to react before the enzyme became inactivated, while the phosphatidyl choline did not receive sufficient time to react.’ Another possibility was that sonication preferentially inactivated the phospholipase A enzyme while leaving the lipase enzyme untouched. This possibility will be discussed later in experiment 16. 3. Phospholipid Micelletgprmation - As demonstrated by Papahadjopoulos and Miller (161), when various phOSpholipids were hydrated in water or salt solutions, they formed different types of liquid crystals. The degree of swelling and the size, Shape, and general configuration of the resulting liquid crystals depended on the particular phospholipid used as well as the ionic strength, the valency of the ionic Species and the pH of the aqueous medium. ,Mechanical agitation promoted frag- mentation of the liquid crystals, producing Spheroidal particles of varying sizes generally within the range of 5-50 u. Ultrasonication of the phospholipid suspension produced further fragmentation of the particles, but the lamellar arrangement of the individual molecules was pre— served. The authors found that in a 145mM KCl or NaCl solu- tion, phOSphatidyl ethanolamine formed large irregular liquid crystals with rough external surfaces. Phosphatidyl choline on the other hand, usually formed a prolate spheroid _‘ 130 type of liquid crystal, while phosphatidic acid produced a Sphericalparticle with remarkable homogeneity in particle size (3-5 uein diameter). Photographs taken by optical and election microscopes clearly revealed the different types of liquid crystals for the various phospholipids. An attempt will be made to explain the results of this experiment using the findings of Papahadjopoulos and Miller (161). It should be recognized however, that in this experi- ment the reaction mixture consisted of sodium deoxycholate, albumin, borate buffer containing Ca++, lipase, and a phos- pholipid, while in the experiments of Papahadjopoulos and Miller, the phospholipid was suspended in only a salt solu— tion. Therefore, the type of phospholipid liquid crystal aggregation for this eXperiment may be different than for the experiments performed by the above named authors. It is postulated that the rate of lipase reaction is related to the size and shape of the phospholipid micelles in solution. In other words, phosphatidyl ethanolamine is hydrolyzed faster than phoSphatidyl choline because phos- phatidyl ethanolamine forms larger liquid crystals in solu- tion and the enzyme can evidently orient itself to react with the substrate. When the liquid crystal size is dis- rupted by sonication, the enzyme for some reason cannot react with the smaller sized liquid crystals as rapidly as with the larger ones. Apparently sonication decreased the liquid crystal Size of phosphatidyl choline to such an extent that 131 no hydrolysis occurred. This reasoning is supported by the unpublished work of Horne and Watkins as cited by Papahad- jopoulos and Miller (161). The former authors Showed that dispersions of phoSphatidyl choline and phOSphatidyl choline— phosphatidyl ethanolamine mixtures made in the presence of cytochrome C and, to a lesser extent bovine plasma albumin contained a large number of small particles in the range of 100-1000 A°. The GLC traces obtained for the methyl esters of the sonicated phospholipids, i.e., unreacted phoSphatidyl ethanolamine, lysophOSphatidyl ethanolamine, and unreacted phosphatidyl choline, could nOt be used for calculations because many unknown peaks appeared on the chromatographs. Also, the retention times for the fatty acid methyl esters in the sonicated samples differed from those of the unsoni- cated samples, and the methyl ester standards of known fatty acids did not match those peaks of the sonicated samples. It is proposed that some of the unknown peaks caused by the sonication treatment were due to oxidized fatty acids (peaks appearing right after the solvent peak) and to hydroxy fatty acids which may have formed at or adjacent to the double bonds of unsaturated fatty acids. The reaction of linoleic acid with water to yield a saturated hydroxy acid or acids was demonstrated by Mabrouk and Dugan (134). These reactions occurred under a saturated nitrogen atmosphere, when 0.5 g of linoleic acid was shaken 132 with 1 liter of distilled deionized water for 3 hours at 6.7OC. Under the conditions for this experiment (300C; sonication time, 15 minutes) it was very likely that the formation of hydroxy compounds had taken place at a faster rate than was observed in the experiments of Mabrouk and Dugan. This can be attributed to the higher temperature used, and to the sonication treatment itself. Experiments 15 and 16 were designed to test some of the obvious possibilities for the causes of the decreased rate of phospholipid hydrolysis by lipase during the sonication treatment. EXperiment 15 - Sonication of Egg Phgpphatidyl Choline and Phosphatidyl Ethanolamine for 1, 3, 5, and 10 Minutes. If sonication was indeed breaking up the phoSpholipid micelles in solution, then sonicating the phoSpholipid and enzyme for increasing periods of time, followed by a period of no soni- cation, should show a continuous decrease in the amount of lysocompound formed. This assumption was made on the basis that there was no re—aggregation of the phOSpholipid micelles after they were broken down into smaller subunits. If the size of the phospholipid micelles were continuously being modified due to the breakdown, reformation, and breakdown etc. of the micelles, then decreases, increases, and de- creases etc. of the lysocompounds formed should also be noted. In fact Figure 15 does show such a curve for the formation of Fvfldn.\.l!.uxv vfifiuJaJnhfilnmvm ®-.« NIAA an». a rah-i. _ 5...?- nnwf. u... — uvnl u 3 a f. .I... .u . . I- >q€ v Iv Amer—A—shnvu-An :— Ayv and u..~\fi.*thHfii\f-I- ~ '1 u :- n. 14:49:... A. u ..,u.~ v-5-JAvPfiEfervs uei.\fl 1 (a p-flu... - . vnIN I . I A,“ ~ .1. m ~43 H ..~ 133 mmBDZHZ .MEHB ZOHBfiUHZOm 0a . m w . a N as msaaozo ampmamammoamom>q U mcaEmHocmnam / / I \O :1 praamammonm0qu \\ / // \\.\\ / la/ .Amfi acmEHmmmxmv :Oaaomma mmmmma OONIO xmpmammm msa ma mcafimaocmaam Hapaamzmmoam pcm mcadono Hmflaamsmmosm m0 mammaompws mmamm UmEmOm upcDOQEOOOmmd acmommm .mfi mmsmam Ofl ,ON on Ca aswu0d sannoawooosxq J0 6 g 134 lysophosphatidyl ethanolamine from phoSphatidyl ethanolamine. However, in the case of phosphatidyl choline, after 5 minutes of sonication there was a continuous decrease in the amount of lysophosphatidyl choline formed. AS shown in the first experiment of this section, no lysophoSphatidyl choline was formed when phoSphatidyl choline and lipase were sonicated for 15 minutes. Conceivably in the case of phosphatidyl choline there was no reformation of the micelles into larger aggregates, hence, there was no increase again in the amount of lysophoSphatidyl choline formed. The only explanation offered for the continuous increase in the formation of lyso- phOSphatidyl choline during the first three minutes of soni- cation is that the lipase was activated during that period of time. (See discussion for experiment 16.) Experiment 16 — Sonication of the Sephadex G-200 Lipase Fraction for 1y 3, 5, and 10 Minutes. As mentioned previously, enzyme inactivation was one of the possibilities for the de- crease in the amount of lysocompounds formed in experiment 14. To determine whether the Sephadex G-200 lipase fraction was becoming inactivated by the sonication treatment, the enzyme was subjected to sonication for time periods of Up to 10 minutes and then tested in an olive oil-gum arabic emul— sion. Following the first three minutes of sonication, the lipase activity increased 4.4%. After 5 minutes of sonication the lipase activity slightly decreased and then remained con- stant until the termination of the experiment after 10 minutes (Figure 16). The loss in lipase activity after 5 minutes of 135 mmBDZHZ .mSHB ZOHBdUHZOm .Amfl acmfiammmxmv mmascHE 0H 0cm .0 .m .fi mom coaamoacom mmamm coaaommw mmmmaa OONIO memammm mSa mo >aa>aao¢ .mfi mmsmam 907 X TW/SLIND iiIAIiov ’SSVJIT 136 sonication represented only 6.6% of the original activity. An increase of plasma lipase activity during the first 3 minutes of sonication was also noticed by Buonsanto ep_ai, (39). This increase was followed by a continuous decrease until the termination of the experiment after 15 minutes. Workers using enzymes other than lipase have also reported an increase in activity during the first 5 minutes of soni- cation (37-39,42,117,174,175,214,234). When examining the results of experiment 15, Figure 15 shows an increase in the amount of lysophoSphatidyl ethanol— amine and lysophosphatidyl choline formed after three minutes of sonication. This coincides with the increase in lipase activity after 3 minutes of sonication and can account for the increase in the amount of lysocompounds formed in experiment 15. rSince the Sephadex G—200 lipase fraction contained 61% phOSpholipase A activity as determined by the ratio of saturated:unsaturated fatty acids formed from egg lecithin hydrolysis, it was conceivable that the decrease of the lyso- lecithins formed in experiments 14 and 15 were caused by the preferential inactivation of phoSpholipase A during the sonication treatment. If this occurred and lipase was left as the only active enzyme, then it represented only 39% of the total starting activity. In addition, the decrease in lysolecithins formed could also be a result of the fact that lipase acts more readily on a triglyceride than a phOSpholipid (219). 137 Waite and Van Deenen (219) showed that rat liver mitochondria acted on both the 1 and 2 acyl positions of phosphatidyl ethanolamine to produce the two lysocompounds. However, when the mitochondria were sonicated prior to incu- bation with phoSphatidyl ethanolamine, there was an increase in the residual amount of 1-acyl lySOphoSphatidyl ethanol- amine, indicating an increase in phoSpholipase A activity by 50%. The author did not mention the duration, frequency, intensity, or temperature used during their sonication experiments. In this experiment, the answer to the question of whether the phOSpholipase A was inactivated or not could have been provided by the GLC traces of experiments 14 and 15. Any decrease of the saturated fatty acids originating from the lysocompounds formed would indicate a decrease in phoSpholipase A activity. Unfortunately, these traces could not be calculated due to the numerous unknown peaks and differences in retention times between the fatty acids of the sonicated and unsonicated samples. SUMMARY AND CONCLUSIONS The results of this study showed that sonication of lipase or phoSpholipids at 20,000 cps, intensity 23 watts/ cm2 can have either an advantageous or deleterious effect, depending upon the conditions used during the treatment. In general terms however, sonication usually produces unde- sirable effects, i.e., lipase is inactivated and phOSpho- lipids are oxidized or in some manner altered. Lipase may be protected from inactivation during periods of sonication up to 30 minutes by simply lowering the temperature during treatment. It is doubtful however, if the latter method would be effective for phospholipids, especially if the duration of sonication was for 15 minutes or longer. Since sonication has become a routine means of dis- persing lipids in aqueous solutions, it is recommended that a minimum treatment be given to disperse the sample, preferably 1 minute or less at 00C. If longer periods of sonication are necessary, a portion of the sample should be methylated and analyzed by GLC to establish that no alterations of the lipid occurred. The conclusions reached during this study are summarized below: 1. Lipase may be sonicated under certain conditions up to 30 minutes without becoming inactivated. 138 139 There is a slight but definite increase in lipase activity during the first 5 minutes of sonication at certain temperatures. Sonication of triglycerides during hydrolysis by lipase increases the liberation rate of free fatty acids. Sonication of the high melting point triglycerides during hydrolysis with lipase does not increase the liberation rate of free fatty acids unless a carrier is present to lower the melting point of the mixture or to maintain the components in solution. Lipase apparently cannot act on solid triglycerides. Lipase preparations may be tested for their phos— pholipase A activity by reacting the enzyme preparation with egg lecithin and determining the ratio of saturated to unsaturated fatty acids in the lysolecithins formed. A high level of saturated fatty acids in the lysolecithins is indicative of high phOSpholipase A activity in the enzyme prep- aration. Lipase reacts with phosphatidyl choline, phosphae tidyl ethanolamine, and phosphatidic acid to produce their reSpective lysocompounds. It does not react with lysophosphatidyl choline to produce glycero- phosphoryl choline. The variations in reactivities among the phOSpho- lipids hydrolyzed by lipase was attributed to their abilities to form different sizes of micelles in solution. It was postulated that those forming large sized micelles were more reactive. The nature and presence or absence of the phoSpholipid base is important because it in part determines the shape and size of the micelle in solution. Sonication of phOSphatidyl choline or phoSphatidyl ethanolamine during hydrolysis with lipase decreased the amount of lysocompound formed during a specified time period. It was demonstrated that this phenomenon was not attributed to inactivation of the lipase activity. Sonication of phospholipids produced unknown GLC peaks. Those peaks appearing immediately after the solvent front were oxidation products. Other unknown peaks were attributed to hydroxy compounds formed at the site of the double bonds of the unsaturated fatty acids during the sonication treatment. PROPOSALS FOR FURTHER RESEARCH Differentiation Between the 1 and 2 Aeyl Lysocompounds At present there is no published method for the separa- tion of the two lysocompounds produced when glycerophos- pholipids are hydrolyzed by both phOSpholipase A and lipase simultaneously. Once they are separated, the two compounds may be distinguished from each other by NMR Spectroscopy (44). However, the latter method is limited to lysocompounds containing saturated fatty acids. When unsaturated fatty acids are present, the identification becomes more difficult because of the overlying interference arising from the ole— finic protons (44). It is desirable to have a TLC system which can separate the two compounds without causing acyl migration during the separation. Lipase Purification The methods used for lipase purification are long and tedious. In addition it is very difficult to obtain lipase preparations free of phospholipase A activity. Studies should be undertaken either to improve the present methods 140 141 of purification or selectively inhibit phoSpholipase A activity'without having to remove it from the preparation. Unfortunately most of the known inhibitors for phospho- lipase A activity also inhibit lipase activity. Sonication Studies 1. Since sonication is frequently used by many lipid chemists to disperse triglycerides or phOSpholipids in aqueous buffer systems, it would be useful to have a detailed study of the frequency, intensity, duration, and temperature, that various lipids could withstand without undergoing decomposition. Attention should especially be devoted to the GLC analysis of the sonicated samples. In many cases, workers have used sonication to diSperse radioactive phOSpho- lipids in buffer systems before hydrolyzing them by various enzymes. They separated the products by TLC and then pro- ceeded to take the radioactivity counts in the various iso- lated fractions. Since no GLC was ever performed, these workers had no idea if any alteration or oxidation of the sample had taken place. It was shown during the proceeding experiments that the TLC Spots of the sonicated samples appeared to be unoxidized, as noted by the absence of streak— ing. In effect however, this was not a reliable indication for establishing that no alterations of the lipids had taken .place. 142 Many researchers have used ice baths to cool their samples when dispersing them by sonication treatment. The present studies showed that although the temperature of the phOSpholipid-buffer system might be 0°C at the beginning of the sonication, the temperature will rise to 100C within 15-20 seconds, depending upon the frequency and intensity of sonication. It is speculated that sonication for periods of even 2-3 minutes at 100C may cause the formation of com- pounds which produce unknown peaks in the GLC traces. 2. In conjunction with the study suggested above, it would be of interest to identify the oxidation or hydroxy products formed during the sonication of a lipid. This type of study would probably require the use of sophisticated instruments such as a rapid scanning IR or mass Spectrometer connected to a GLC. The unknown products might also be separated by TLC and identified by conventional chemical methods such as functional group analysis. 3. Studies should be undertaken to determine the Size of the phospholipid micelles undergoing sonication treat- ment. Samples should be taken at various time intervals during the treatment and analyzed by optical or electron microscopy. It was postulated in this study that the size of the phoSpholipid micelles may continuously be changing by first breaking down, reforming, and breaking down again. This was given as a possible reason for the variation in the amount of lysophatidyl ethanolamine found during lipolysis 143 while undergoing sonication treatment. In the case of phos- phatidyl choline, it appeared that the micelles were not reaggregating after breakdown because there was a continuous decrease in activity after the first three minutes of soni- cation. The latter two statements implied that in order for lipase to attack the phOSpholipid, a certain size micelle was required. 4. Due to the limitations of the sonicating instrument used in this study, all experiments with lipase were per- formed at a frequency of 20,000 cps. Since the brute force effect of cavitation below 100,000 cps often results in enzyme inactivation, it would be of interest to use frequencies above this range and note the effect on lipase activity under various experimental conditions. Preparation of Intact Plasmalogens Many natural sources of phosphatidyl ethanolamine and phosphatidyl choline contain fairly large amounts of plasmalogens. These compounds are difficult to separate intact because their properties are very similar to those of phosphatidyl ethanolamine and phoSphatidyl choline. Therefore, they are usually isolated together with the latter compounds. If phosphatidyl ethanolamine or phosphatidyl choline contained plasmalogens and were reacted with a pure lipase (free of phospholipase A activity), the hydrolysis products would contain a lysocompound, free fatty acids, 144 and the unreacted plasmalogen. These products may be separated easily from one another by TLC. It is important however, that the reaction go to completion. Otherwise the so-called pure plasmalogen would still be contaminated with the diacyl phOSphatidyl ethanolamine or phosphatidyl choline. Initial experiments in this laboratory have shown that plasmalogens may be separated intact from phOSphatidyl ethanolamine even with crude lipase preparations containing as much as 60-65% phOSpholipase A activity. It appeared that there was a preferential attack by the phospholipase A on the phosphatidyl ethanolamine rather than on the plasma- logen. Further studies will determine the extent of plasma- logan hydrolysis by the phospholipase A enzyme. 10. 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