,. V-.. \ ‘3'! ,J' u: mm a, a?» ' DJ s i”: .‘ Mr {A Q»: A ‘ :5 ”wit ? .q 3‘. “A; a.“ 431%5“ QT.;3'{'.-I 5““ May-m .uV WWE. 3! F. ‘-.,“-3 .1‘4' (“$3505. ”I A ‘ , i?- - r ‘u ' ‘ ' ,g./{:\ y ' , ' w: 9‘ 5’?! u 4.— M Ir p. § gm. 55? é' . V- .J'." ' I 'r' z: “9?? .' K: ‘A ’33sz ‘ .n‘ r c-A'HIA‘~ ’39? .‘B‘fi-V’ .‘ 3' . s V . 4 .T.‘ _ \2) I v \ .> ;;4 I v - . Iii“. . . ‘ " . ' .. - ‘7 - p—_. t ‘ . . L ‘5’ f ‘ '.‘ ‘ z ‘1 - _ - > I . , n . ,,- . . H. - ~ . l , ' ' ‘ ‘ ' > . \ .' ',.' 3 l 1 ‘ . x ‘v , p , I. n l . . f ' ‘ I .. l ‘ 1 1» , . . u t . “ 3 . h , > ' 1 m . ' '5“ .' 5 ~ ._.;‘.~ J I ' V r‘ ‘ I LIBRARY V r\ A ru PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 45‘}; R1 Q IAN-d Z 1 IOCD 2/05 plClRC/DateDueJndd-pd STUDIES ON THE MECHANISM OF THE CLOTTING OF CASEIN BY THE ACTION OF RENNIN By Clarence A. Broomfield A THESIS :iubmitted to the School of Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1958 r ’quW ar .1 " o IQ'QVU ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. Hans A. Lillevik for his patience, encouragement, interest and advice during the course of this investigation. Acknowledgment is also due to certain other members of the chemistry department, particularly those in the physical section, for their help and advice. The author is also grateful to the National Science Foundation, and to the American Dairy Association, who provided financial support during part of the course of his study, v Finally, special acknowledgment is due to Mrs. Jane Smith Post for technical assistance in the ultracentrifugal studies, and to the author's wife for her help in the typing of this manuscript. ii VITA The author was born September 18, 1930 in Mt. Morris, Michigan, and his primary and secondary education was obtained at Mt. Morris Consolidated Schools. He entered the University of Michigan in 19kg, and graduated in 1953 with a Bachelor of Science in Chemistry. In 1953 he entered the School of Graduate Studies, Michigan State University where he has majored in biochemistry and minored in physical and organic chemistry. He has served as an undergraduate laboratory assist- Eant in the Department of Chemistry,-University of Michigan, during the school year 1952-1953: a laboratory assistant in the Engineering Experiment Station, Michigan State University during the summer of 195h; a Graduate Teaching Assistant in the Department of Chemistry, Michigan State University from 1953.1957; a Fellow of the National Science Foundation during the school year 1957‘1958; and a Special Graduate Research Assistant during the summer of 1958. Upon graduation, he shall assume a postédoctoral research position in the Department of Chemistry, Cornell University, Ithaca, New York. He is a member of the American Chemical Society, and the Society of the Sigma Xi. iii TABLE OF CONTENTS Page I. IZ‘ITRODUCTIONOOO;OOOOOOOOOOOOOOOOOOOO0.00.0000... II. HISTORICALOOOOOOO00.0.00....0OIOOOOOOOOOOOOOO... A. CaseinOOOOOOOOOOOOOOO.OOOOOIOOOOOOOOOO... B. BanninOOOI...00......OOOOOOOOOOOOOOOOOOOO \O 0‘ u: x» I4 C. The Clotting Reaction.................... III. mERIMENTALCOOIOOOO0......0......OOOOOOOOOOOOO. 1'6 A. EquipmentOOOOOO.OOOOOOOOOOOOOOOOOOOOOOOOOO 16 BO Reagents and MethOdsoooeeeeeeeeeoeeeeeoeo 21 C. Experimental Procedures.................. 33 D Data...........l0...0..................... ” Iv. RESULTS AND CONCLUSIONS......................... 65 The Effect of Ageing and of Heating.......... 65 Comparison of Electrophoretic and Sedimentation Behavior.................... 72 N.P.N. Liberation and Clotting Rate.......... 86 Changes of Turbidity and Viscosity........... 112 Effect of Different Buffers on Clotting...... 126 Optical Rotatory StUdieSOOOOOOOOOOOOOOCOOCOO. 1” v. SWRYOOOOOOOOOOOOOOODOOOOOOOOOOOOOO0......O... 135 BIBLIOGRAPHYOOOOOOOOOOOOOOOOOOOOOOOO...OOOO...O....... 138 TABLE II III IV VI VII VIII IX XI XII LIST OF TABLES Physical Properties of Crystalline Rennin...... Effect of Age on Clotting Rate and N.P.N. Before and After Rennin Clotting............... Rates of Liberation of N.P.N. by Rennin from Solutions of Heated and Unheated Calcium Caseinate at 10° and 30°....................... The Rate of N.P.N. Liberation by Rennin from Citrate Buffered Sodium Caseinate Solution, pH 5.20, at 00 and 300.000.000.0000000000O...O. The Rate of N.P.N. Liberation by Rennin from Phosphate Buffered Calcium Caseinate SOlution, pH 6.20 at 00 and 300.00.000.00000000 The Rate of N.P.N. Liberation by Rennin from Phosphate Buffered Sodium Caseinate Solution, pH 6.uo at 0° and 309.... ........... The Rate of N.P.N. Liberation by Rennin from Phosphate Buffered Sodium Caseinate Solution, pH 6.h0 at Various Temperatures...... The Rate of N.P.N. Liberation by Rennin from Phosphate Buffered Sodium Caseinate Solution, pH 6.h0 at Various Temperatures...... The Rate of N.P.N. Liberation by Rennin from Citrate Buffered Sodium Caseinate Solution at Various Temperatures............... The Rate of N.P.N. Liberation by Rennin from Citrate Buffered Sodium Caseinate Solution, pH 5.25 at Various Temperatures...... The Effect of Temperature on the Clotting Rate of a Solution of Calcium Caseinate at pH 02.0.0000000000000OOOOCOCOOOOOOOO.00.... The Effect of Temperature on the Clotting Rate of a Solution of Citrate Buffered SOdium caseinate at PH SOZSeeeeeeeeeeeeeeeeeee. V Page 8 ho A1 ha h3 as #7 1+9 51 Sir LIST OF TABLES ’ Continued Table XIII XIV XV XVI XVII )GHII XIX XXII The Effect of Temperature on the Clotting Rate of a Solution of Citrate Buffered Sodium Caseinate at pH 5. h5.................... The Effect of pH on the Rate of N.P.N. Liberation from Citrate Buffered Solutions ofo Sodium Caseinate by Rennin at 300 centigrado OOOOOOOOOOOOOOOOOOOOOOOO00...... The Effect of Enzyme Concentration on Clotting Rate in a Citrate Buffered Solution of Sodium Caseinate at pH 5.25 at 0° Centigrade... The Effect of Enzyme Concentration on Clotting » Rate in a Citrate Buffered Solution of Sodium Caseinate at pH 5.25 at 12.50 Centigrade The Effect of Enzyme Concentration on Clotting Rate in a Citrate Buffered Solution of Sodium Caseinate at pH 5.25 at 30° Centigrade.. Rate of the Primary Reaction in a Solution of Calcium Caseinate as Measured b N. P. N. Liberation and Flash Heating to 30 C.......... Rate of the Primary Reaction in a Solution of Citrate Buffered Sodium Caseinate, pH 5. 20 as Measured by N. P. N. Liberation and Flash Heating to 30° C............................... N.P.N. Liberation and Turbidity Development in a Citrate Buffered Solution of Sodium Caseinate with the Action of Rennin at 30° C... Changes of Turbidity and Viscosity During Rennin Digestion of a Solution of Citrate Buffered Sodium Caseinate at 300 c............. Change of Viscosity During Digestion of a Solution of Citrate Buffered Sodium casej‘nate at 00 0.0.0.000...OOOOOOOOOOOOOOOOOOO Change of Viscosity During Rennin Digestion of a Solution of Citrate Buffered Sodium Caseinate in h.6 M Urea and pH 5.20 at 30° C... vi Page 55 56 57 58 58 ‘59 60 61 62 63 63 LIST OF Table XXIV XXV XXVI XXVII XXVIII TABLES * Continued Changes of Turbidity, Viscosity and N.P.N. in a Citrate Buffered Solution of 'Enriched a‘Casein During Rennin Digestion... Effect of pH, Ionic Strength, and Specific Ion on Clotting.................... Optical Rotation Measurements During Rennin Digestion of Phosphate Buffered casein SalutionOOOOOOOOO00000000000000.0000. Optical Rotation Measurements During Rennin Digestion of thuffered Casein Solution..... Optical Rotation Measurements of Casein and Paracasein in the Presence and Absence or ureaOOOOOOOOIOOOOOOO0.0.0.0...IOOOOOOO... vii Page 128 131 131 132 III. IV. VI. VII. VIII. IX. XI. LIST OF FIGURES Electrophoretic Patterns of Three Casein Preparations....................... Electrophoretic Patterns of Crystalline Rennineeeeeeeeeeeeeeeeoeeeeeeeeeeeeeeeeeee N.P.N. Content of Unbeated Sodium Caseinate Solution, Before and After Rennin ClettingOOOOOOOOOOOOOOOO0.... N.P.N. Content of a Heated Sodium Caseinate Solution, Before and After Rennin C10tting....OOOOOOCOOOOOOOOOOOIOOC. Plot of a/(arx) for N.P.N. Liberation from an Unheated Casein Solution, as a Function of Digestion Time........... Plot of a/(a.x) for N.P.N. Liberation from Heated Casein Solution, as a Function of Digestion Time........... N.P.N. Liberation from a Citrate Buffered Sodium Caseinate Solution, pH 5.20, as a Function of Digestion Time.. Electrophoretic Patterns of Citrate Buffered Sodium Caseinate Solution, After Various Periods of Rennin Digestion. N.P.N. Liberation from a Phosphate Buffered Calcium Caseinate Solution, pH 6.20, as a Function of Digestion Time.. Electrophoretic Patterns of Phosphate Buffered Calcium Caseinate Solution, After Various Periods of Rennin Digestion. N.P.N. Liberation from a Phosphate Buffered Sodium Caseinate Solution, pH 6.h0, as a Function of Digestion Time.. viii Page 31 66 O\ O\ 70 75 76 77 78 79 LIST OF FIGURES - Continued FIGURE XII. XIII. XIV. XV. XVI. XVII. XVIII. XIX. XXII. Electrophoretic Patterns of Phosphate Buffered Sodium Caseinate Solution, After Various Periods of Rennin Digestion.. Plot of a/(a-x) as a Function of Digestion Time for N.P.N. Liberation from a Citrate Buffered Solution of Sodium Caseinate, pH 5.20......................... Plot of a/(a-x) as a Function of Digestion Time for N.P.N. Liberation from a Phosphate Buffered Calcium Caseinate SOIUtion, pH éeeoeeeeeeeeeeeeeeeeeeeeeeeeee Plot of a/(a-x) as a Function of Digestion Time for N.P.N. Liberation from a Phosphate Buffered Sodium Caseinate SOIution’ pH éouoOIOOOOOOOOOOOOOOOOOOeee... Arrhenius Plots of Rate Constants for N.P.i. Liberation from Three Different Substrates Under Identical Conditions...... Sedimentation Patterns of Casein SOIUDIOH, 2.6% in vemnaIQQQOOOOOOOOOOOOOO Sedimentation Patterns of Paracasein SOIUDIOH, 2.6 f; in veronal .ooeeeoeoeeeeeoo 'Sedimentation Patterns of Casein Solution, 1.3 % in Veronal................. Sedimentation Patterns of Paracasein Solution, 1.3 % in Veronal................. Semirlog Plot of a/(arx) for N.P.N. Liberation from Citrate Buffered Casein Solution for Rate Constant Determination 0° to 16.50 C................ Semi-log Plot of a/(a-x) for N.P.N. Liberation from Citrate Buffered Casein Solution for Rate Constant Determination, 22.50 to 37.20 o............ Arrhenius Plots of Clotting Rate and N.P.N. Liberation from Calcium Caseinate in Phosphate Buffer at pH 6.20................ ix Page 80 81 82 89 9O 91 LIST OF FIGURES ‘ Continued FIGURE XXIV. XXV. XXVI. XXVIII. XXIX. XXX. XXXI. XXXII. XXXIII. XXXIV. XXXVI. Arrhenius Plots of Clotting Rate and N.P.N. Liberation from Sodium Caseinate in Citrate Buffer at pH 5.20.0.0...OOOOOOOOOOOOOOOOOOO00....0O. Arrhenius Plot of Clotting Rate of Sodium Caseinate in Citrate Burfer at pH SOb‘SO0.0000000000000000000000 Comparison of Arrhenius Curves for Clotting Under Three Different Conditions. Effect of pH on Clotting Rate of Citrate Buffered Sodium Caseinate Solution at 30 C 0.000.000.0000...OOOOOOCOOOOOO... Electrophoretic Patterns of Citrate Buffered Sodium Caseinate Solutions at pH 5.20 and 5.h5 After Rennin Digestion Effect of Enzyme Concentration on Clotting Rate of Citrate Buffered Casein SOluti-On at 30°C Oeeeeeeeeeeeeeeeoeeoeeee Effect of Enzyme Concentration on Clotting Rate of Citrate Buffered Sodium Caseinate Solution at 12.50 0...... Effect of Enzyme Concentration on Clotting Rate of Citrate Buffered Casein SOlution at 00 COOOOOIOOOOOOOOOOOOO Effect of Enzyme Concentration on Clotting Rate of Citrate Buffered Casein Solution at 30°, log-log Plot...... Effect of Enzyme Concentration on Clotting Rate of Citrate Buffered Casein Solution at 12.50 C., logrlog Plot. Effect of Enzyme Concentration on Clotting Rate of Citrate’Buffered Casein Solution at 0° C., log‘log Plot.... Change of N.P.N. and Turbidity in a Citrate Buffered Casein Solution During Rennin Digestion. 30° C............ X Page 92 93 9h 99 100 101 102 103 ion 105 106 LIST OF FIGURES - Continued FIGURE XXXVII. XXXVIII. XXXIX. XL. Change of Viscosity and Turbidity During Rennin Digestion 30° C Change of Viscosity During Rennin DiECStion at 00 Cece-eeeeeeeeeeeeeeeeeeeo N.P.N., Turbidity and Viscosity Changes in a Solution of Citrate Buffered Enriched a‘Casein............... Sedimentation and Electrophoresis Patterns of Enriched a-Casein Dllrim; Rennin DigeStionOIOOO0.0.0.0...00. xi Page 115 116 117 118 STUDIES ON THE MECHANISM OF THE CLOTTING 0F CASEIN BY THE ACTION OF RENNIN By Clarence A. Broomfield AN ABSTRACT Submitted to the School of Advanced Graduate Studies of Michigan State university of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1958 Approved ‘ c / ABSTRACT Casein clotting by rennin action was studied in an attempt to draw definite conclusions concerning the mech- anism of the overall process. Most of these studies have utilized crystalline rennin and citrate-buffered sodium caseinate solution at pH 5.20 wnich forms a clot even in the absence of calcium ion. This clot is physically simi‘ 1ar to the calcium paracaseinate clot, and in each experi‘ ment the clotting behavior of the calcium‘free substrate was compared with that of phosphate-buffered calcium caseinate solution. Since many workers have mentioned that ageing increases the clotting rate of casein solutions, this effect was studied to determine whether the increase was due to un- catalyzed hydrolysis of rennin-susceptible bonds. This appears to be the case. The effect of heating a casein stock solution to 90° for ten minutes, as recommended by Nitschmann and Varin (1), was also studied with respect to ageing and clotting behavior. The clotting rate was es- sentially unaffected, though rate of uncatalyzed N.P.N. liberation was decreased. There was also an indication that heating might have changed the temperature dependence of the clotting reaction. The change in the electrophoretic pattern of casein by rennin, as noted by Nitschmann and Lehmann (2), has been related directly and solely to the primary enzymatic reaction, independent of subsequent steps in the clotting process. Changes in the sedimentation patterns were less apparent, but it was not possible to make this study as complete. Plots of clotting rate versus the inverse of absolute temperature indicated that there are at least two separate reactions in the clotting process, and that one of them has a very high activation energy; therefore an extremely high temperature dependence. This was previously noted by Berridge (3), and was attributed by him to a thermal der naturation of the rennin-altered casein. Since the calcium- free and calcium caseinate substrates gave similar curves, here is a great probability that the temperature-sensitive step is an intermediate reaction, between the primary re- action and clot formation. Furthermore, results of studies relating enzyme concentration to clotting rate at various temperatures suggest that there are two separate mechanisms for destabilizing the rennin-altered casein, thermal and enzymatic, and that the former might be reversible. The clotting behavior of the citraterbuffered substrate is very pH sensitive. If the solution is pH S.h5 instead of PH 5.20, a much higher enzyme concentration is required to obtain a clot, and the high temperature coefficient is no longer observed at low temperatures. Turbidity and viscosity changes upon rennin action on both citrate-buffered and calcium caseinate substrates were studied, in the absence and presence of urea. The results of these experiments suggest that the actual clotting step might be a polymerization involving hydrogen-bond crossr linking. That clotting in the calcium-free medium was not a specific effect of the citrate ion, was demonstrated by also preparing sodium caseinate solutions in malate, acetate and phthalate buffers. All solutions below pH S.h behaved essentially the same upon rennin treatment. Optical rotation studies failed to establish a de* naturation reaction; evidence was found that the specific rotation of paracasein might be different from that of casein, thus explaining the absence of a change during the reaction. 1. Nitschmann, Hs., and R. Varin, Helv. Chim. Acta, _3_L£, 114.21 (1951). 2. Nitschmann, Hs., and W. Lehmann, Experientia,‘2, 153 (1951). . Herridge, N. J., in The Enzymes, vol. I, pt. 2, p. 1079 1951). I. INTRODUCTION The phenomenon of milk clotting by the enzyme rennin has been known since ancient times, and through the years numerous investigators have been curious about the changes accomplished by this enzyme, which converts fluid milk into a solid curd. In spite of the numerous investigations of this reaction, however, its precise mechanism has eluded elucidation. As long ago as 1918, Hammersten (l) recog‘ nized that the reaction consisted of at least two stages: a primary enzymatic stage, followed by a non-enzymatic clot formation. Although this idea has been modified and ex- panded, the great volume of work that has been done on this reaction has produced only comparatively minor variations of this original poetulate. Berridge has suggested recently that there is perhaps a third step, between the enzymatic primary reaction and the actual clot formation, which is, or resembles, a denaturation of the rennin'modified protein (6). It was further suggested by Heinicke in 1953 (3) that this "denaturation" could be brought about by either a temp- erature above in degrees Centigrade or else by proteolytic enzymes such as trypsin or bromelain. . One of the princip11_objectives of the present in- Vestigations has been to ascertain, if possible, the precise nature of the reactions involved in the rennin clotting reaction and particularly to either establish or else disprove the postulated denaturation step. In the main, a calciumrfree casein solution, buffered with citrate at a pH near 5.0 was utilized in order to avoid the complex colloidal system of calcium phosphate and calcium casein- ate: a system previously suggested by Lundsteen (h). During the course of this research, two new components of casein have been announced, called respectively kappaf (k-) casein by Waugh (5) and lambda-(X0) casein by Gould, 33 El; (15). The former is purported to be a substrate for rennin and indeed it has been suggested that this may be the primary substrate for this enzyme (7, #3). A1‘ though this component has been unavailable to us in pure form and thus was not studied as such, it and its possible role in the clotting reaction have been kept under consid- eration with respect to design and interpretation of the experiments to be described. II. HISTORICAL A. Casein Casein is a phosphorus-containing protein of milk, which was first isolated in pure form by Mulder (8) in 1838. His method of separation, isoelectric precipi- tation by the gradual addition of dilute acid, is still generally used for the preparation of this.protein, though in 1956 a new method was patented by Waugh (5) which is based on the fact that casein is insoluble at high calcium ion concentrations. These two methods of preparation have been shown by Nielsen (9) to be very similar with respect to molecular weight and electro- phoretic behavior. Casein was considered to be a single protein until Osborn and wakeman (17), in 1918 isolated a small amount of alcohol-soluble protein from isoelectric casein. Other fractionation studies, particularly those of Linder- strdm-Lang in 1925 (10), supported the contention that casein was heterogeneous and then, in 1939, Mellander (11) demonstrated electrophoretically the presence of at least three distinct components, which he designated as alpha- (a-), beta-(pf), and gamma-(7.) in decreasing order of their mobilities. In 19hh, Werner (12) reported the isolation of a- and p- fractions from whole casein, and in 1950 Hipp, h et. a1. (13) successfully separated the 7- fraction. The same year Cherbuliez and Baudet (1h) isolated two sub‘ fractions from a-casein with almost identical phosphorus, tyrosine and tryptophan content, which they named a1 and a2 caseins. While attempting further to purify their calcium precipitated casein, Waugh, gt .gl.(5) in 1956 observed a new component which they designated as kappa casein. This fraction was characterized by its solubility in 0.25 M calcium chloride, a low phosphorus content (less than 0.33 %) and a very high sedimentation constant in pH 6.98 phosphate buffer (13.18 S). The next year McMeekin and his corworkers (16) also reported the isolation of a new com- ponent from acid precipitated casein which they designated a¢2 casein and which is characterized by a low phosphorus content (0.10 to 0.15 %). It is possible that this com‘ ponent is identical to Waugh's k-casein, as has also been suggested by Nielsen (9). Early this year, 1958, Gould, 33 13;, (15) reported still another new component, isolated from a crude prepara- tion of k‘casein, which was designated by them as l-casein. This component was characterized by a high phosphorus content (1.15 %), solubility in 0.25 M calcium chloride and a low sedimentation rate (1.10 S) in phosphate buffer 01‘ pH 6.98. Naugh has very recently (#3) expanded his findings to include a description of a stoichiometric complex between S the caseins. According to this description, whole casein consists of car, k-, p-, and “m” caseins. The most imr portant complex involves three molecules of as linked through calcium and phosphate groups to one molecule of k, to form a very stable complex. At sufficiently high cal- cium ion concentrations, 0 is also complexed. It is pre‘ sumed that "m" includes the fraction previously denoted "7", but this has not been explicity stated. The properties of several casein fractions, except 1. and "m-" caseins are summarized by Hielsen (9). A1‘ though it cannot at present be stated how many separate components constitute casein, it is nevertheless obvious that the system is very complex, and that there are many interactions between the several components. There is evidence that the kappa fraction is largely responsible for the stability of the casein complexes, and that many of the interactions do not take place in the absence of this component. B. RenninI Rennin is defined as the main milk-clotting enzyme secreted by the abomasum of the suckling calf. It is secreted as its zymogen, pro-rennin, but is activated at relatively low hydrogen ion concentration, e.g. at pH 3.6 and 26° C. activation is complete in seven minutes. The clotting activity of rennin preparations has been defined on the basis of time required to clot a standard substrate under an arbitrary specified set of conditions. The substrate recommended for use as a standard in this assay is a 12 % solution of spray-dried, fat-free cow's milk reconstituted with 0.01 M calcium chloride. This tends to average out variations between milks from in- dividual animals because such commercial preparations are usually made from the pooled milk of a large number of animals. Since the clotting rate is not precisely linear with enzyme concentration, the enzyme solution is then diluted in such a way that one m1. of solution will pro- duce a clot in ten ml. of substrate after about five minutes at 30° C. One "rennin unit” (R.u.) is defined by Berridge (2) as that amount of enzyme which will clot ten Inl. of standard substrate in 100 seconds at 30° C., and activity calculations are made using the formula: R.u. = 100 D t iJI which D is the dilution required to achieve a desirable 7 concentration (about 5 min. clotting time) as described above, and t is the clotting time in seconds. This enzyme is usually prepared by extracting dried vells with five to ten per cent sodium chloride solution, decanting the renninrcontaining brine and clarifying it by adsorption on alumina, followed by elution with potassium phosphate. Crude dried rennet preparations may be made by precipitating the protein with sodium chloride (to sat- uration), drying, and powdering the precipitate. The first preparation of crystalline rennin was re- ported by Hankinson (19) in 19h3 who described his com! pound as forming "needle-shaped white crystals." A few months later Berridge (20) succeeded in isolating rennin in the form of flat plates, but this was believed to contain small amounts of impurity, and in l9h5 he reported the preparation of cubic crystals which were claimed to be of very high purity (2). A scheme for the preparation of such crystals is outlined by Berridge in a review article (22). The physical and chemical properties of Crystalline rennin are tabulated below: (Table I). Table I Molecular weight ho,ooo Nitschmann, 9.12. 9.1.(21) Isoelectric point h.5 (21) Sedimentation constant h.0 S (21) Difmsion constant 9.5 cmg/sec. (21) Crystalline form cubic Berridge, N. J. (20) pH optimum, proteolysis 3.8 (20) pH optimum, clotting 5.1; Lundsteen, (h) C. The Clotting Reactiont As was mentioned in the introductory paragraph, a great deal of work has been done in attempts to understand the rennin clotting of milk, and several theories have been advanced to explain various experimental observations. But none of the postulates that have been proposed so far are able adequately to explain all of the phenomena con- nected with the clotting reaction. Since this work has been extensively reviewed by Berridge (22, 23), and more recently by Pyne (2h), all of the details of past research will not be discussed here. Rather, the various theories on the mechanism of the renninrcasein reaction will be pre- sented, and then those observations pertinent to each par. ticular hypothesis will be discussed. The earliest hypothesis advanced to explain the clot‘ ting reaction was that of Hammersten (1), who proposed that casein coagulated because part of the molecule was broken <>ff by the action of rennin, forming a whey proteose (Molkenalbumose) and leaving a residue whose calcium salt is insoluble. Although this idea, per se, has been gener‘ ally shown to be untenable, there is nevertheless much data accumulated which indicates that the proposal was not as erroneous as was once believed. Holter, for example (25), disclaimed the theory on the basis of his measurements of the extent of proteolysis, which indicated that less than one peptide bond per "molecule" of casein was broken under 10 normal conditions. Since it has been subsequently shown by electrophoretio analysis and by studies on pure frac- tions that only a single fraction of the casein,the a- component is affected by rennin, Holter's findings fail to disprove this basic idea. The "Molkenalbumose" that Hammersten believed to be'released by rennin action was later shown by Cherbuliez and Jeannerat (26) to be a normal casein constituent which they called "delta" casein, and which has more recently been generally considered to be identical with v-casein (30). But Nitschmann and Zahler (27) have recently demonstrated that nonrprotein nitrogen (N.?.N.) is indeed released by rennin, and Alais (28) has isolated a glycomacropeptide from this N.P.N. which con- tains approximately ho % carbohydrate and has a molecular weight of 6,000 to 8,000. In addition, susceptibility of a casein solution to clotting is increased upon ageing, which might be explained to be due to uncatalyzed proteo- lysis. The main argument against such a simple scheme as IHammarsten proposed is a finding by Berridge (6) that the taggregation phase of this reaction has an extremely high isemperature coefficient, between 1.3 and 1.6 per degree (or’between 1h and 100m per ten degrees), and thus has a ‘Iery high heat of activation, of the order of130 kilo- calories per mole. Temperature coefficients for most Chemical reactions are of the order of about two per ten 11 degrees (or about 1.07 per degree), and the only pertinent reaction for which such a great temperature dependence has been observed is the denaturation of proteins. For example, the thermal denaturation of egg albumin between 70° and 90° C. shows a temperature coefficient of about 1.9 per degree; (010 = 600); that for the denaturation of hemoglobin in the same temperature range is about 1.3 per degree (Q10 = 1h). On this basis, Berridge (23) has proposed that there is a denaturation step between the initial enzymatic reaction and the visible coagulation. This hypothesis has been supported by Heinicke (3), who asserts further that this denaturation may be accomplished either by a high tempera- ture, above lh° C. or else a proteolytic enzyme, such as trypsin. Such a scheme may be represented: Rean1—n A I! N Casein + H20 limited proteolysis'— modified Casein 0 "modified" Casein oge$$§psin;hetg*%"denatured" Casein 9 0 084+ "denatured" Casein 30a Paracaseinate C T Such a scheme would be consistent with most observations ‘NMich have been made, and none of the reported findings (iirectly contradict this hypothesis. However, aside from ‘the high temperature coefficient observed, and the fact that clotting is essentially inhibited by low temperatures, there has been no direct evidence to substantiate this Proposed denaturation. 12 An older theory, and a slightly more widely accepted one is that proposed by LinderstrdeLang (18) in 1929. His explanation involved the presence of a protective colloid in the casein system which served to stabilize the solution to calcium ions, and which was the primary sub- strate for rennin. When this projected protective colloid was destroyed by the enzyme action, the remaining com? ponents of the system became unstable and formed an in- soluble precipitate. This idea accounts for nearly all of the observed phenomena except for the high temperature coefficient demonstrated by Berridge. Otherwise, none of the data reported so far contradict such a scheme. This postulate gained renewed impetus upon the discovery of kappa casein by Waugh. It has been asserted that perhaps kappa casein is the longOsought protective colloid, and that it is therefore the primary substrate for rennin. The glycomacropeptide described by Nitschmann 23, El; (7) wbuld be split off this component, and since this fragment is very hydrophilic, such a reaction would tend to decrease the solubility of the remaining part of the molecule, causing it to lose its protective properties. The observa- tion by Nitschmann and Lehmann (32) that casein and para- casein are precipitated together when mixed and subjected to the action of calcium chloride is in agreement with this theory, particularly if the a - k complex is assumed to be in dynamic equilibrium with free component molecules in SOlution. 13 Hankinson and Palmer (33) have postulated a completely different action of rennin on casein on the basis of their studies on conductivity changes, electroviscous effects and changes of zeta potential in casein solutions to which have been added various concentrations of rennin and/or calcium ion. They have shown that viscosity of sodium caseinate solutions is much greater than that of casein solutions containing calcium phosphate; further, the viscosity decreases when rennin is added. Digestion times are not cited, however, and change of viscosity seems to be considered only as a function of enzyme concentration, while it has been shown that viscosity of a given casein- rennin system decreases with time. From these data, the authors reached the conclusion that rennin acts mainly as a dehydrating agent, freeing bound water from various sites on the protein and thus making it less soluble. This hypothesis is at odds with the observations of Hammersten and of Nitschmann, gtL‘gl‘ that a limited proteolysis is catalyzed by rennin as a primary step. However, such a Irelease of bound water might conceivably account for the lligh temperature coefficient in lieu of a denaturation. Thais theory has found little support, perhaps because of Inany important factors which were apparently not controlled. Another theory which has not received a great deal of attention is that of Beau (3h), who suggested that rennin acts on the caseinate.phosphate micelle to depolymerize it 1h to some extent so that carboxyl, amino, and phosphoryl groups, and calcium ions become available. Then the casein molecules can combine by linkage of their carboxyl groups through calcium ions, and their amino groups through phosphate radicals to form a network of continually in- creasing size and of indefinite molecular weight. Although this postulate does not account for some of the phenomena on which the other ideas are based, e.g. N.P.N. liberation and high temperature coefficient, it is nevertheless con- sistent with the finding of Hankinson and Palmer that con‘ ductivity of the solution is intreased, and, perhaps more significantly, does explain the-physical nature of the clot: a gel which retracts and becomes more firm with the passage of time. Also, such a picture would help to show why the clotting reaction should be so very sensitive to calcium ion concentration and has a requirement for phos- phate radicals in order to produce a normal clot. The fact that no "modus operandi" is suggested for the depoly- merization step allows one to speculate that perhaps this postulate might fit into one of the other mechanisms {fib- ready described. Although almost all the observations on this reaction Inay fit into one or more of these postulated mechanisms, there are a few that remain unexplained so far. For ex- ample, it has been shown that heating of milk in pasteur‘ ization decreases the sensitivity to rennin, and that 15 sodium nucleate inhibits clotting. Also, as mentioned in the introductory paragraphs, it has been found that clot- ting will occur in the absence of calcium if the pH of the solution is low enough, i.e. near 5.0. In order to be considered valid, any proposed mechanism for the rennin clotting reaction should in some way account for all of the observed phenomena. This is obviously not easily accom- plished because of the complicated system of colloids being dealt with, but eventually such an explanation should be possible. 16 III. EXPERIMENTAL A. Equipment Temperature Control. Most determinations of enzyme activity, clotting times and viscosity were carried out in a modified all-glass water bath manufactured by Fisher Scientific Company, Pittsburg, Pa., consisting of a 12 by 12 inch pyrex battery jar. This bath was equipped with a Cenco Conedriven stirring motor, type NSl * 12 (Central Scientific Company, Chicago, Ill.) with a stainless steel stirrer, a l/h inch outside diameter copper coil through which tap water could be circulated for attaining tempera- tures below room temperature, and a modified Sargent Mer- curial Thermoregulator (E.H. Sargent and Company, Chicago, Ill.)which, in conjunction with a thyratron¢tube electronic :relay unit (constructed in the Kedzie Chemical Laboratory Inachine shop) and a 125 watt knife-blade heater (Cenco) 53erved to maintain the temperature within i 0.020 when set eat 300 C. Tolerances were a little greater at temperatures IPequiring use of the cooling coil because of the difficulty <>f maintaining a constant rate of flow of water from the 13ap. A stainless steel test tube support was used to sue. IDend samples in the bath for clotting time measurements. UDhe sample-containing test tubes were brightly illuminated 'by two h5 watt, four inch show-case bulbs immersed in the bath. 17 For experiments requiring temperatures between five and 150 C. a metal insulated water bath equipped with a refrigeration coil was used. This bath, obtained from the American Instrument Company, was slightly modified so that while it was being used, the compressor was operated con- tinuously and the desired temperature was maintained with a 250 watt Cenco knife-blade heater, regulated by a he- lical bimetallic thermoregulator through a mechanical relay with a six-volt solenoid. Later, because the points of the relay became quite badly burned, a deKhotinsky type thermo- regulator was used, which was not quite as satisfactory but which served its purpose. All work at zero degrees C. was carried out in a six 'by ten inch Dewar flask in which an ice-water mixture was Inaintained. The measured temperature of this mixture was consistently found to be between zero and 0.10 C. A :fairly uniform temperature was assured by constant agita- tion with a Sargent cone-driven stirring motor fitted with a small glass stirrer. For viscosity measurements a hSO- (+50‘900 glass right prism was immersed in the ice-bath in 1ution of powdered rennet was prepared by dissolving 25 grams of R-li Rennin, N.P‘. in 250 ml. of distilled water, 29 and then centrifuging down the small amount of insoluble matter at 10,000 rpm. in the Servall refrigerated centrir fuge. The supernatant liquid, which has a rennin activity of about 32 R.u. per ml., was placed in a sac of Visking tubing and equilibrated with saturated sodium chloride solution by dialyzing overnight on the external rotating liquid dialyzer in the cold-room. The resulting precipr itate was separated by centrifugation at 10,000 rpm., the supernatant, which was practically devoid of rennin activity, was discarded, and the precipitate redissolved in distilled water. Then this solution was admitted into £1 sac of Visking tubing and the equilibration with salt lurine was repeated. This process was repeated until no :further increase of activity was found. After the third Iprecipitation, an activity of 33 R.u. per milligram of Iiitrogen was found, and after the fourth the activity had (dropped to 2h R.u. per mg. N, so it was assumed that op“ ‘timum purification had occurred after three steps. It was Iaoted that this activity compares rather poorly with that <3f crystalline rennin as reported by Berridge (171 R.u. Iper mg. protein, or, assuming 16 % N, 1070 R.u. per mg. of Iiitrogeni). There seems to be some discrepancy in our trespective methods of assay, however, because he obtains a ‘value of 16 R.u. per mg. as the activity of his commercial :rennet, whereas a value of about 0.32 R.u. per mg. was :found fbr the rennet used in these experiments by the assay 30 xnethod used here. It will be noted that in each case the order of magnitude of the degree of purification is app- roximately the same. Crystalline Rengig was obtained from Sigma Chemical Corporation, St. Louis, Missouri; part of Lot number 'R78-052. This was found to contain 1h.15 % nitrogen on a total weight basis, had an activity of 15.7 R.u. per millr igram and was electrophOretically homogeneous at pH 6.h, .having a mobility of h.0 Tiselius units. At pH 8.h it was completely inactive and its electrophoretio pattern dis‘ jplayed two peaks (see Fig. II). Microscopic examination :revealed a mica-like structure; irregularly shaped thin jplates, similar to those described first by Berridge, :rather than the cubic crystals obtained by him in later emxperiments. This enzyme was used for most final studies. lieagents For Non-Protein Nitrogen Analysis. Eighteen per cent {wa1 trichloroacetic acid (T.C.A.) “M13 prepared by dissolving 180 grams of T.C.A. crystals in distilled water and diluting to a total weight of 1000 grams. The microkjeldahl digestion mixture was made by adding SCH) ml. of concentrated sulfuric acid to 500 m1. of dis- ti.11ed water in which had been dissolved two grams of C‘HDric sulfate (hydrated) and 100 grams of sodium sulfate. Forty per cent (w/w) Sodium hydroxide solution for rmicro-kjeldahl distillation was prepared by dissolving 160 31 Figure II J- A Ascending Descending Electrophoretic patterns of crystalline rennin, Sigma, in phosphate buffer, pH 6.38, p c 1.0, current a 20 ma. time a 7200 seconds Ascending ,- Descending 1E1ectrophoretic pattern of crystalline rennin, Sigma, in Veronal buffer, pH 2 8.h, u a 0.05, current = 9 ma. time = 2200 seconds 32 grams of sodium hydroxide pellets in distilled water and diluting to a total of too grams. 3932 pg; _c_e_p_t_:_ M _B_g_1_~_ig_ geld was prepared by disr solving 20 grams of boric acid crystals in distilled water and diluting to a total weight of 500 grams. Methyl Egg ggd Methylene £132 solutions were prepared from the water‘soluble forms of these indicators, and were each made to a concentration 0f 0.02 % by dissolving 20 mg. of indicator in 100 ml. of water. Both of these solu- tions seemed to be quite stable for up to six months. Standard Hydrochloric acid for titration was prepared by diluting 25 ml. of 0.08605 N hydrochloric acid, standard- ized against a simultaneously standardized solution of sodium hydroxide, to 200 ai., providing a 0.01076 N working solution, equivalent to 0.151 mg. of nitrogen per m1. Thirty ESE cent Hydrogen peroxide was Baker's Reagent Egrade, assayed at 30.6 % hydrogen peroxide and containing (3.05 % sodium pyrophosphate as a stabilizing agent. Nessler's Reagent was prepared according to the method (If Fblin and Wu as described by Hawk, User and Summerson 1r1 Practical Physiological Chemistry. twelfth edition (38). Egg Digestion Mixture for determination of nitrogen by tflle Nessler's method was prepared by adding 500 ml. of con- °entrated sulfuric acid to 500 ml. of a one per cent solur tillonof mercuric chloride. This seems to be a little more £Effective than the copperrcontaining digestion mixture. 33 Egggrimental Proceduggg Clotting Tlggg, All clotting times were determined by the same procedure. Five m1. of substrate was pipetted into a 0.5 by h.5 inch culture tube and allowed to equilir brate in a water bath at the desired temperature for at least fifteen minutes. Then 0.5 m1. of enzyme solution was layered carefully on top of the substrate solution. As rapidly as possible, the tube was inverted three times to assure complete mixing and simultaneously a stoprwatch was started. If a period of longer than a few minutes was expected, the actual time of mixing was also noted. At the first sign of heterogeneity in a film of substrate allowed to flow from a flattened stirring rod down the side of the tube, the timer was stopped and the elapsed time noted. In the case of samples requiring more than a :few hours of digestion to clot, the tubes were kept stop- I>ered until it was noticed that they were almost about to Clot. . . I Non-Protein Nitrogen (N.P.N.) Aliquot 5 m1. samples Were taken at intervals from the digestion mixture with a ‘Rblumetric pipette/and transferred as rapidly as possible, arm: with constant swirling into a 50 ml. Erlenmeyer flask containing 10 ml. of 18 9% trichloroacetic acid (T.C.A.). After allowing to stand for ten or fifteen minutes (always tune same for any set of data) the precipitate was filtered off on a fluted 9 cm. Whatman number ’12 ashless filter 311 paper and the filtrate collected in a six inch pyrex test tube. This tube was stoppered until its contents could be analysed for N.P.N. SemimicrorKjeldahl Method: Ten ml. of the T.C.A. fil- trate was pipetted into a 50 ml. kjeldahl flask, two m1. of digestion mixture and two borosilicate beads were added, and a three inch straight drying tube was inverted in the tmouth of the flask to act as a condenser. Then the samples were heated on the digestion racks until all dense fumes had abated and the solutions had become colorless. Then the flasks were removed to a holding rack for about three ndnutes, and two drops of 30 % hydrogen peroxide were dropped directly onto the cooled liquid. The flasks were ‘then replaced on the digestion racks for at least 15 min- lites longer. After'complete digestion, the flasks were sallowed to cool, the condensers were rinsed with distilled “water, and the samples were steamrdistilled in the usual Hninner, the distillate being collected in a 125 m1. Erlen- meyer flask containing three m1. of four per cent boric atkid solution, two drops of 0.02 % Methyl Red indicator and one drop of 0.02 % Methylene Blue. A total of approximately 50 m1. of distillate was collected. The distillate was then titrated with 0.01076 N hydrochloric acid to the first permanent pink color. Analysis of known solutions consis- tOl'ltly showed a recovery of 97 to 98 per cent. 35 The Nessler Method (38) was adapted with modifications 'that follow for nitrogen determinations in early experir lnents. This had the advantage that duplicates could be :run for each sample. The T.C.A. filtrate was prepared as Iareviously described, and five m1. aliquot portions were jpipetted into N.P.N. digestion tubes which were calibrated aat 35 and 50 ml. One ml. of digestion mixture used for Nessler nitrogen analysis and two borosilicate beads were added to each tube, and digested until all the water had evaporated and dense fumes filled the tube. Then a 20 m1. beaker was inverted over the top of the tube While digesr tion continued until all color had disappeared from the liquid, after which the tube was removed from the rack, allowed to cool for 90 seconds, treated with one drop of 30 % hydrogen‘peroxide added directly onto the liquid, and returned to the digestion rack for five more minutes. After decomposition of the hydrogen peroxide the tube was removed, allowed to cool, and then diluted to 35 ml. with distilled water and thoroughly mixed by pouring back and forth into a 125 ml. Erlenmeyer flask. When mixing was complete, the solution remained in the flask and was placed in an ice bath to cool. After about ten minutes of cooling, fifteen m1. of Nessler's reagent was added from a graduated cylinder, as rapidly as possible and with constant swirling, to form quite a stable suspension, which was allowed to stand at room temperature for fifteen minutes before ab- 36 :morbancy was measured in a Klett-Summerson colorimeter with 51 green filter. This method was not dependable enough to alllow one to be confident of the results obtained. Ultraviolet Absorbancy Measurements. The absorbancy sat 280 mp of several T.C.A. filtrates was measured prior to N.P.N. sampling, against a blank of 12 % trichloroacetic acid. Although the findings were very promising, the readings were extremely low and excessively erratic, so that they were considered to be of little quantitative value. In an attempt to obtain more significant results, an adapter was constructed for the Beckman DU spectro- photometer in order to utilize ten cm. capillary polar- imeter tubes as cells. Since there were no quartz windows available for these tubes, further absorbancy measurements were omitted. Turbidity Measurements. Changes of turbidity as a function of digestion time were made in the Beckman model DU spectrophotometer at 360 mu. This wavelength was chosen because no absorption could be noticed which would compli- cate the measurement of turbidity. A11 effects of light scattering and reflection were neglected, and it was as- sumed that the absorbancy measured was a fairly accurate measure of the increase in particle size. A sample of the substrate diluted by ten per cent with distilled water was used as reference. For these measurements, 0.5 m1. of rennin solution was added to five m1. of substrate in the 37 Inanner described for clottingrtime determination. As soon 518 possible after mixing, 0.5 ml. of the reaction mixture vnas transferred to a one cm. silica Beckman Cell and a 9.0 nun. quartz insert was placed in the cell so as to reduce 'the light path length to one millimeter. Readings were :recorded at intervals after the time of mixing. The 'temperature was maintained at 30° C. by a water jacket ‘through which was circulated water from a constant tempera- ture bath. Viscosity_Measurements. The Ostwald viscosimeter con- veniently required a volume of four to six ml. of solution, so that the sample could be admitted in either of two ways. In the first experiments, five ml. of substrate was pip- etted into the instrument and 0.5 m1. of rennin solution was layered on top as in clotting-time measurements. Then these solutions were mixed by blowing air through the capillary arm for about thirty seconds, and flow time measurements could immediately be begun after this. A more satisfactory method was adopted a little later: The enzyme and substrate were mixed in a test tube, and then admitted as rapidly as possible after mixing into the viscosimeter. Flow time measurements could be started almost as soon after mixing as with the other method, and less difficulty with air bubbles was encountered. Measurement of The Primarngeaction (at low tempera- tures by clotting time measurements at thirty degrees). 38 ESince it was noticed that at low temperatures there seems 'to be a considerable lag between the end of the primary :reaction and the observed clotting, and that if the solu- 'tion is warmed to a suitable temperature after this initial :reaction has gone to completion, the solution will clot 21mmediate1y, a procedure was devised to flash-warm samples 'which.had been digested at a low temperature with rennin. IEor this measurement, a small sample, about 0.5 ml. was transferred from the digestion vessel (0.75 by 12 inch jpyrex test tube to a test tube of 11 mm. inside diameter. This was most satisfactorily accomplished by means of a pumping device which was kept in the digestion vessel and thus transferred a minimum of heat to the digesting sample. As rapidly as possible, the small tube was placed in the water bath at 30° C. and simultaneously a glass rod of 10 mm. diameter was admitted and a stop-watch started. Thus was formed a film of approximately 0.5 mm. thickness be‘ tween the rod and the wall of the tube, which warmed to 30° C. By rotating the glass rod back and forth while holding the tube stationary, the first appearance of heterogeneity was readily and reproducibly noted. C. Data The following tables contain all data used in the construction of figures . to Table II The Effects of Ageing on Casein Clotting time N.P.N., before tAge, Days seconds rennin action N.P.N., after rennin action Solution prepared with old casein, unheated 0 3flh 0.165 287 0.212 139 0. 380 12 115 0.636 to 220 1.89 0.313 0. 382 0.517 0.759 2.0h .§2lnii2a_az2aaz2a_zith_£:anh_aesain._anheated. 0 227 ' 0.097 0.311 1 221 0.125 0.325 13h 0.202 0.38u 12 137 0.331 0.511 to I 165 0.79h 0.952 .§alnii2ae2za2azaa_ziia_£raaa_asaaia._haeien_ 0 225 0.088 0.328 1 233 0.121 0.328 5 138 0.181 0.369 12 1113 0.269 0.1162 to 155 0.656 0.850 hl Table III Rates of Liberation of N.P.N. by Rennin from Solutions of Heated and Unheated Calcium Caseinate at 10° and 30° Digestion tine N.P.N. a minutes milligrams a - x a - x unheated Solution, 10° 0. 0 0.190 0.285 1.6; 5 0.205 0.270 1.7 10 0.213 0.262 1.81 20 0.238 0.237 2.00 0.250 0.225 2.11 6 0.280 0.195 2.h3 0 0.320 0.155 3.06 90 0.32h 0.151 3.15 unheated Solution. 30° 0, 0 0.202 0.273 1.7h 5 0.25h 0.221 2.15 10 0.28% 0.191 2.%z 20 0.36 0.109 a. 30 0.381 0.08h 2.65 5 0.h00 0.075 .33 0 0.h20 0.0h5 10.55 90 0.h 8 0.007 .00 Heated Solution, 10° 0. 0 0.202 0.2 3 1.7h 5 0.211 0.2Zh 1.80 10 0.230 0.215 wt 20 0.2g0 0.225 2.11 30 0.2 0 0.215 2.21 5 0.280 0.125 2.h3 2 0.3 0.1 1 2.25 105 003 0.1” 3. 1 #2 Table IV The Rate of N.P.N. Liberation by Rennin from.Citrate Buttered Sodium Caseinate Solution, pH 5.20, at 0° and 30° Digestion time N.P.N. a Electrophoretic minutes _£!Eh. a - x a - x .pattern ngmbeg_ At 30° Centigrade 0 0.156 0.172 1.93 5 0.189 0.1173 2. 3h 10 0.2%2 0.090 3.69 15 0.2 0 0.072 n.61 20 0.287 0.0%3 7.39 2h.1 0.293 0.0 8.52 602 At 0°Centigrade 0 0. 2 0.190 1.75 20 0.1 2 0.170 1.95 0 0.175 0.157 2.12 1 0.196 0.136 2.h% 160 0.236 0.096 3.h 320 0.278 0.05 6.15 386 0.299 0.03 10.05 608 1h70 0.331 0.001 332 610 7500 0.363 616 #3 Table V The Rate of N.P.N. Liberation by Rennin.from.Phosphate Buffered Calcium.Caseinate Solution, pH 6.20 At 0° and 30° Digestion time N.P.N. a Electrophoretic minutes mam. a - x a - 1 pattern number At 30° Centigrade 0.5 0.160 0.200 1.80 5 0.1 9 0.161 2.2h 10 0.2éfi 0.118 3.05 15 0.2 0.036 3.75 20 0.275 0.0 h.23 25 0.296 0.0 5.63 25.7 0.318 .th 8.57 60h At 0° Centigrade 0 0.160 0.200 1.80 6 20 0.178 0.182 1.98 07 0 0.201 0.159 2.26 0 0.230 0.130 2.77 160 0.27 0.08 h.23 320 0. 0.0 10.60 h11 0. 0.01 20.00 606 1h70 0. 0 611 7500 0.375 617 uh Table VI The Rate of N.P.N. Liberation by Rennin‘from Phosphate Buffered Sodium.Caseinate Solution, pH 6.h0 At 0° and 30° Digestion time N.P.N. a Electrophoretic minutes mgm. a - x a -’x pattern number At 30° Centigrade 0.5 0.175 0.185 1.95 5 0.190 0.170 2.12 10.5 0.207 0.153 2.35 20 0.2g% 0.136 2.65 25.7 0.2 0.122 2.95 603 30 0.2%6 0.1 3.16 60 0.2 2 0.07 %.62 120 0.3h1 0.019 1 .95 3600 612 ,At 0° Centigrade 0 0.16 0.132 1.8 20 0.27 0.1 1.9 0 0.17 0.182 1.9 0 0.199 0.161 2.2h 160 0.218 0.1h1 2.55 320 0.2% 0.112 3.21 h11 0.2 g 0.091 3.96 605 1h70 0.35 0.002 180 60 7500 0.398 61 hS 'Table VII The Rate of N.P.N. Liberation by Rennin from Phosphate Buffered Sodium.Caseinate Solution, pH 6.h0 At Various Temperatures Digestion time N.P.N. a minutes milligrams a - x a - At 10° Centi rade 0 0. 0.256 1.56 5 0.i%% 0.23h 1.71 10 0.189 0.211 1.90 15 0.192 0.201 1.99 20 0.20 0.19u 2.07 30 0.2%3 0.157 2.55 5 0.2 3 0.137 2.92 0 0.282 0.118 3.39 30 0.330 0.070 5.72 1 1 0.351;, - 0.0116 8.70 _ At 15° Centigrade 0 0.1h0 0.260 1. 5 0.152 0.2M 1.53 18 “:31. °-i it: 1 0. 0. . 30 0.2%0 0.120 2.50 50 0.2 0 0.120 3.3h 70 0.290 0.110 3.6h 0 0.328 0.072 5.55 1 0 0.363 0.037 10.80 At 20° Centigrade 0 0.217 0.283 1.77 5 0.250 0.250 2.00 10 0.266 0.2! 2.05 15 0.289 0.211 2.37 20 0.300 0.200 2.50 30 0.330 0.170 2.9h 5 0.368 0.132 3.79 0 0.38% 0.116 h.32 0 0.h0 0.092 S.hh 1 o 0.156 0.00:, 11.1; Digestion time minutes 5.5 16 20 5 0 0 126 he Table VII, Continued N.P.N. milligrams At 2 ° Centi rade §§§§ EEE’: -:4=§: 0000.000000 VIN] 0H At ° Cent ra 0.206 0.209 0.18u 0.167 0.175 0.119 0.090 0.05h 0.025 0.020 At 35° Centigrade e e a“) WNN HQQN 000mm 3: as 0000000000 0 a e a a 555 F’NH» 0.222 0.218 0.210 ‘ ‘-§ 0.0.0....O \»~PQ4 Digestion time N.P.N. , a minutes milligrams a - x a r at pH 5.58 0.5 0.1 0.211 1.%g 5 5 0.1g? 0.182 1.3 10. 0.1 1 0.1 2. 17 0.200 0.111 2.8g 20 0.22 . 0.081 3.88 25 0.23 0.072 1. 30 0.272 0.033 9.56 15 0.270 0.035 9.00 at pH 5.51 0.5 0.089 0.226 1.39 5.5 0.139 0.176 1.72 15 0.201 0.111 2.7 20 0.223 0.092 3.113 25 0. 0.071 %.hh 30 0.2 0.052 .05 17 0.293 0.022 11.30 at pH 5.30 0.5 0.076 0.2 1.32 5 0.127 0.18 1.68 10 0.157 0.158 2.00 15 0.18% 0.131 2.10 20 0.20 0.107 2.90 25 0.230 0.085 3.71 30 0.2%fi 0.075 %.20 10 0.2 0.051 .18 at pH 5.20 0.5 0.057 0.258 1.22 2 0.097 0.218 1. 5 5 0.122 0.1 3 1. 3 10 0.166 0. 9 2.11 15 0.186 0.129 2.00. 20 0.2 0.101 3.12 25 0.2 0.079 3.99 30 0.251 0.061 5.17 57 Table XIV, continued IDigestion time N.P.N. minutes milligrams a r x a - x at pH 5.10 0.5 0.091 0. 221 1.%2 5 0.122 0.193 1. 3 10 0.162 0.153 2.06 16 0. 206 0.10 2.82 20 0. 219 0.09 3.2 Table XV The Effect of Enzyme Concentration on Clotting Rate in a Citrate Buffered Solution of Sodium Caseinate at pH 5. 25 at 0° Centigrade Enzyme Enzyme Clotting time Clotting Concentration Concentration seconds Rate m8./m1. R.u./ml. 10 160 6,900 11 0 1 16 72: 000 l a 1121:: 25; 0.2 19 00 l. 0.13 2 3210 30.9 0.06 1 500. £5180 000 19.3 — 12.8 0.03 0.5 0,000 16.h 0.015 0.25 1,130, 000 6 7.0 0.008 0.125 more than 1.5 X 100 a - The ice in the bath melted, causing the temperature to rise to 9°C. for a short time. 58 Table XVI The Effect of Enzyme Concentration on Clotting Rate in a Citrate Buffered Solution of Sodium Caseinate at pH 5.25 at 12.50 Centigrade g Enzyme Enzyme Concentration Concentration Clotting time Clotting mg./m1,_ R.u.Zm1I seconds Rate 1 16 6,900 115 0.5 8 8100 r 10,200 98 - 123 0.25 1 18,000 5 .5 0.13 2 28,800 .7 0.06 1 60,000 1 .7 0.03 0.5 122,100 8.16 0.015 0.25 270,000 3.80 0.008 0.125 189,600 2.01 Table XVII The Effect of Enzyme Concentration on Clotting Rate in a Citrate Buffered Solutionoof Sodium Caseinate at pH 5.25 at 30 Centigrade Enzyme Enzyme Concentration Concentration Clotting time Clotting mg./m1. R.u./ml. seconds Rate 0.25 1 12 83.181 0.125 2 20.9 17. 0.063 1 39.13 25.1 0.032 0.5 70. 11.2 0.015 0.25 1.0 7.10 0.008 0.125 2 2.3 3J13 0.008 0.125 239.5 1.18 59 Table XVIII Rate of the Primary Reaction in a Solution of Calcium Caseinate as Measured by N.P.N. giberation and Flash Heating to 30 0. Flash Digestion time N.P.N. a Clotting time minutes mgm. a r x a r x seconds At 30° 0. 0 0.2 0.226 2.01 5 0.23% 0.192 2.10 10 0.309 0.151 3.05 15 0.332 0.128 3.60 20 0.351 ' _0.106 1.31 25 0.382' 0.0 8 5.90 30 0.398 0.0 2 7.13 38.5 0.108 0.052 8. 5 At 9.0° c, 0 0.226 0.2 1.97 2280 5 0.237 0.22 2.06 10 0.272 0.18 2.15 20 0.275 0.185 2.18 30 0.288 0.172 2. 50 0.332 0.128 3.60 110 0.370 0.090 5.12 150 0.12% 0.036 12.80 255 0.12 0.031 13.53 37.5 300 0.120 0.010 11.50 39.2 385 28.7 2 3° 165 3%‘1 0 0. 3000 0.168 62 1200 0.188 66.7 10100 0.180 23 11200 spontaneous clot 60 Table XIX Rate of the Primary Reaction in a Solution of Citrate Buffered Sodium Caseinate, pH 5.20, as Measured by N.P.N. Liberation and Flash Heating to 30 -; -_ Flash Digestion time N.P.N. a Clotting time minutes mgm, a - x a - 1 seconds 0 0.166 0.17 1.95 5 0.172 0.16 2.02 10 0.172 0.168 2.02 380 15 383 20 0.202 0.138 2.16 22 227 30 0.212 0.128 2.66 £2 2%? 0 1 15 63.1 50 25.0 52 31.3 56 25.6 60 0.257 0.083 1.05 12.1 70 6.8 80 %.9 90 0.282 0.058 5.87 .9 105 3.5 120 0.302 0.038 8.95 2.8 750 ' 3.5 1080 0.322 0.018 18.89 1 1110 0.338 0.002 170 0.1 2100 ' clot 61 Table XX N.P.N. Liberation and Turbidity Development in a Citrate Buffered Solution of Sodium Caseinate with the Action of Rennin at 30° C. IDigestion time Optical Density N.P.N. minutes 360 mu milligrams 0 0.16 3 0.18 6 0.160 7 0.208 8 0.185 9 0.207 10 0.227 12 0.30 13 0. 312 15 0.100 0.210 16 0.158 17 0.191 18 0.510 19 0.591 20 0.269 21 0.635 21 0.8 8 25 0.281 29 1.18 30 0.293 35 1.50 10 1. 70 15 . 1.88 SO 1.95 750 2.10 0.310 62 Table XXI Changes of Turbidity and Viscosity During Rennin Digestion of a Solution of Citrate Buffered Sodium Caseinate at 30° Centigrade Digestion time Absorbancy Flow time minutes 360 mg seconds 1 0 2 0 122.1 8 ° 5 0.00 5 0.008 121.6 8 0.013 120.8 9 0.015 10 0.022 11 120.3 120. i% 0.052 17 0.051 120.0 20 0.080 120.0 25 . 0.130 120.0 30 0.203 120.1 35 0.317 121.0 10 0. 5 15 0.5 o 1 .o 50 0.712 12 .0 55 0.902 128. 60 .1.05 131. '35 1'233 115 6 1. . 80 1.3% 9g 1.39 165.2 9 1. 108 191.1 120 215.0 130 258.5 1 0 297.2 5° 5% 0 .2 150 192.0 63 Table XXII Change of Viscosity During Rennin Digestion of a Solution of Citrate Buffered Sodium Caseinate at 00 C J-l-fi Digestion time Flow time Digestion time Flow time minutes seconds minutes seconds 3 251.7 1615 306.9 8 252.5 1650 307. 21 25 .0 1655 307. 26 2 .9 1815 317.0 105 8.3 2007 326 125 gil.8 2730 368 130 9.7 2930 373 18 252.1 3000 376 19; 250.5 3230 388 20 2 0.9 3510 110 330 7.5 1320 70 335 5.8 1575 190 616 2 7.7 18 0 520 631 268.2 57 0 636 1395 296.5 6120 716 00 297.9 6120 763 1110 299.2 7080 clot Table XXIII Change of Viscosity During Rennin Digestion of a Solution of Citrate Buffered Sodium Caseinate in 1.6 M Urea and pH 5.20. 30° Centigrade Digestion time Flow time Digestion time Flow time minutes seconds minutes seconds With 0.0003 % Rennin With 0.0063 5 Rennin 3 210.3 185.7 8 210.8 . 8 186.1 12 209.5 12 180.E 16 209.0 16 166. 20 208.8 20 161.7 30 210.1 25 151.8 35 208.2 30 151.6 60 207.2 10 1 0.1 90 207.0 50 7,0 110 197.0 61 Table XXIV Changes of Turbidity, Viscosity and N.P.N. in a Citrate Buffered Solution of Enriched a-Casein During Rennin Digestion _ h Digestion time .Absorbancy Flow time N.P.N. minutes 360 mg seconds milligrams 0 0.270 2 0.060 120.3 3 0.063 1 0.063 119.9 5 0.280 6 0.065 7 0.072 119.8 9 0.085 10 120.1 0.308 11 0.093 12 0.101 13 0.130 122.0 11 0.155 15 0. 326 16 0.171 123.7 17 0.195 . 18 0.220 . 19 0.255 128.8 20 0.338 21 0. 223 33g}; 2 0. 3% O°h25 152 9 0 351 26 0.188 27 0.505 28 0.535 29 0.512 30 219.8 o. 353 31 0.562 32 0.568 33 0.57 clot 35 0.588 12 0.2965 0. 9 0 0.608 65 IV, RESULTS AND CONCLUSIONS The Effects g£.Ageing EBQ.9£ heating. It is well known that as milk, or a casein solution, is aged it becomes more susceptible to rennin clotting. It has also been shown that the nitrogen soluble in 12 % trichloroacetic acid (N.P.N.) increases upon storage, even at zero degrees Centigrade. Since N.P.N. is liberated in the primary reaction of rennin clotting it became necessary to know whether the increased sensitivity to rennin was a result of the uncatalyzed cleavage of the bond(s) whose hydrolysis is specifically catalyzed by rennin. If this were the case, the difference between the initial N.P.N. concentration of the solution and that at the clotting point should be decreased upon storage of the solution. A cursory examination of a plot of initial and final N.P.N. concentration as a function of storage time (see Fig. III and IV) might lead one to believe that this is not indeed the case, because these lines are nearly parallel in all cases. But upon careful examination one can notice that the rate of N,P,N. liberation in the original solution (zero digestion time) is more rapid for the first few days, whereas the line representing the "final" N.P.N. concentra- tions (clotting point) is essentially straight. This means that the difference between these values actually decreases 66 nouaaegaoo you eohoaso need» msapaoao cannon Mo defined on» Aemasumsv genus one Asauaoacagv 656666 .o oo on oopoon case coon can nouns soap: on opcsaooeo nouuaeafioo no“ vehoaao need» mcappoao .adnaoa no coupes one Azfiacahev henna one Agaaapaea=e opaque . .o 666 6» cocoonpnu noon pea we: deem: soaasaou opusuomeo _as«uou a Mo accuses az.m.z .>H ohsmum. asauou a Mo penance .z.m.z .HHH oazmam whee .owd as ma oa m1 m w m ghee .om< ma on m, m a m ‘ ‘ I q d J :Hdupacas .ONN . aw 0.". odN . 1 com 1 . omN .eoo .sfi meas»OHe N e N a: e- so 1n 2* O O O O O BWUJQIIIFU "H 'g ° 0‘ e 67 est first. It should also be noted that the decrease of clotting time is much more rapid in the first few days, after which it levels off to a more or less constant value, and finally eventually increases again. This behavior may be interpreted as showing not only that this increased rennin sensitivity is due to uncatalyzed hydrolysis of the renninrspecific bonds, but also that at least some of these bonds are "weaker", or more susceptible to proteo- lysis. Since it was recommended by Nitschmann and Varin (10) that casein solutions be heated to 90° C. for ten minutes in order to destroy the proteolytic enzyme normally iso- lated with the casein, this precaution was carried out in early work. However, it became convenient to work at a temperature of 20° C. instead of 30° C. for certain experi- ments, and then it was noticed that the heating had adver‘ sely effected the clotting properties of the solution, i.e. the clotting time was greatly increased. For example, in one case two casein solutions were prepared in exactly the same manner, except that one was heated in boiling water for ten minutes, When calcium caseinate substrates were prepared from these solutions, and the same enzyme added to each, the one which had been heated required 81 minutes to clot, whereas the unheated sample required only eight minutes and 10 seconds. This amounts to a rate difference of nearly a factor of ten: Later it was demonstrated that 68 this difference is almost nil at 30° C., and therefore it was attributed to a difference in the shape of the rate- temperature curve. This has not yet been rigorously proven, but probably such a difference would be found, if experi° ments were made to relate effect of heating to effect of temperature. The difference in clotting time is not a result of a change in the primary reaction, as is shown in figure I. The slopes of these lines are almost exactly identical. These data were taken on solutions of phosphate-buffered sodium caseinate, about pH 6.5, which had been prepared from heated and unheated stock solutions. To make sure that the heating was the only difference, a casein solu- tion was divided into two parts, one being placed in boiling water for ten minutes and the other refrigerated immediately. In view of the presence of a proteolytic (caseinolytic) ol'lzy'me in casein preparations, it is interesting to note .. that there is very little difference in clotting behavior between the heated and unheated casein solutions upon stor-age. Although the N.P.N. increased more rapidly in the unheated sample, and the heated sample produced a 1itiale more consistent results, there was very little °rfect either on the rate of decrease of clotting time upon storage, or on the convergence of the ”initial" and "final” N.P.N. line. This would indicate that this caseino- lytic enzyme has no function in the clotting reaction. 150 x 1 e N.P.N., 10° 0. o ' r ‘ N.P.N.. 30° C. 1:: 17.8 30 _ 26 . 1o - ‘ S3 . A8 . g7 . g A \\'6 ' 5 h. ‘ k a: 2.9 G 3 C) o O 2 . ° 0 ‘2‘0 1‘0 69 I 35 15° Digestion time, minutes Figure V. Plot of a/(a-x) for N.P.N. liberation from casein solution which had not been previously heated to 90° C., as a function of digestion time. 7O 50 - o N.P.N., 10° 0. 110 I. I N.P.N.. 30° C. k a 18.0‘ 30 1 A 20 - A 10 - 9 , 8 . r 7 . 3 6 \ F ‘ e5 _ 1. * I 2.30 3 Q 0 o 2 o 0 ° . 0 Figure v1, Plot of a/(a-x) for N.P.N. liberation from casein solution which had been previously heated to 90° C., as a function of digestion time. 1 L . 20 10 6b ‘ 8‘0""'—IO‘F Digestion time, minutes 71 Nitschmann and Bohren (11) have reported that there is no change in the casein solutions upon heating except for a slight decrease in viscosity, which is slowly reversed upon standing. This has been attributed by these authors to a reversible dissociation of the protein complexes. Such an effect would also be observed if the extent of hydration were decreased by the high temperature. If it could be established which of these possibilities is lctually responsible for the observed heating effect, perhaps by studies of the viscosity changes during rennin digestion of heated and unheated substrates, then it might be possible to obtain valuable information about the postulated intermediate destabilization step. It might be particularly valuable to correlate such infor‘ Ination with the temperature dependence curves of heated and unheated solutions, since a significant effect of heating was found upon the clotting rate at lower tempera- tures . 72 Conmarison 9_f_ Electrophoretic and Sedimentation gghalior. Nitschmann and Lehmann, (12) in 1911? showed that the electrophoretic pattern of paracasein displays a split cr- peak, and were able also to demonstrate that this split would occur either in the absence or in the presence of calcium ion. However, no concrete evidence has been pre- sented to indicate that the change in pattern was a result of the primary reaction alone, and was not dependent upon a temperature above 15° C. In the present investigation, three separate substrates were prepared from the same stock solution, and treated simultaneously with the same enzyme solution at two diff- erent temperatures, zero and thirty degrees Centigrade. O Examination of the resulting patterns (Figs. VIII, XII I ) clearly shows that this change in the electrophoretic pattern is a result of the primary reaction. This is particularly apparent in the patterns obtained on solutions digested at zero degrees, in which clotting does not occur within the time periods or the experiment, but the primary reaction proceeds to completion. It will be observed that for any single substrate the electrophoretic pattern is almost the same at the clotting point for solutions at 30° C. as it is for the calculated "and of the primary reaction", based on the temperature-coefficient observed for the N.P.N.-liberation reaction. Actual N.P.N. analysis 73 shows that at the clotting time the primary reaction is not yet quite finished (Figs. VII, IX, XI ). Accordingly, there are slight differences between patterns of samples taken at this time, and samples taken several hours later. However, an additional one hundred hours digestion makes no detectable difference in the appearance of the pattern oven though N.P.N. continues to be released at a slow rate. This would seem to indicate that the electrophoretic change is related only to. the primary reaction. When the calcium- rree, phosphate-buffered solution is allowed to digest for several hours beyond the end of the primary reaction, a turbidity develops, and figure XV displays the pattern obtained~ after sixty hours digestion, men general proteo- lysis has proceeded to a large extent. If the mobilities of the original as- peaks are compared with the mobilities of the new 0.1 and (:2 components, it may be seen that the cause for the resolution of these compon- ents is a decreased mobility of the (:2 fraction, while the 0.1 component remains essentially the same as the original 0. It may also be noted that, while'the proportion of 0.1 to (:2 seems to differ somewhat from one substrate to an- other, the proportions do not seem to change during the digestion in any one given substrate. This would indicate that the progressive change during the digestion does not represent a conversion of one of the components to the other, but rather indicates that a component originally 7% having a mobility identical to that of a1 is being changed so as to decrease its charge, causing it to appear as (12, This tends to substantiate the findings of Cherbuliez and Baudet (114), who maintained that a- casein is in reality two separate components, (1.1 and (re, which are converted by rennin to para0 (1.1 and para- (1.2, respectively. The description by laugh and Gillespie (#3) is not so readily supported by these data, however. According to the most recent report by the latter workers, casein consists of a complex which is 55 fl "(1,“, 25 % B‘, 15 % k, and 5 % ”m“ oaseins, and depends upon the 1: fraction for its stability. In the clotting reaction, k is supposed to be the substrate for the primary reaction, and thus the complex is destabilr 1zed, causing precipitation. Presumably, therefore, the new component, the az- peak, is the residual Piece of the 1:- molecule after the "glycomacropeptide" has been removed. 'There is also the possibility that the high mobility or the a, *- 1: complex (the original 0:- component) was due to the 1:. portion, and when this is broken up, the residual a,- component migrates at a lower, innate, rate. A clear picture of the mechanics of the reaction is not possible from this electrophoresis data because there seems to be no consistant stoichiometry between the al- and’the (:2 components from one substrate to another. In fact; the proportions seem to be almost reversed in figure X and in figure XXXV. 7S coma courses .eaap soaaueman 83 oofi coma 83 com .nsoaae hp oeaadwuueo upsaon on» newness» nauhasaa eupeaonaoauoeao you neagaem ocean scannemuo no doaaoasu a as om.m ma .aoapsaoe eaaaaeueo.asuoom oeaeuuanueaeauao a sea 33.933 .z.m.z .Hg 983m :fioaasm coo 8: com cl. 0. o. n o ..u . J m a m 0. u n T. n m. . m G d a. w I T o. . m 0.0 _.m shepuam m 76 Pattern 602. Citrate-buffered Pat‘tern 608. Citrate-buf- casein at the clotting point, fered casein at time ° C., in Veronal, pH = 8.h calculated to be equivalent 9 - 0.05, protein 2 1.5 , to the clotting point 0° current = 9.3 m.a., time = C., in Veronal, pH = 5. 2700 seconds. a = 0.05, protein a 1.5 % current = 9.0 m.a. time = 2700 seconds. Ascending ‘ Ascending Pattern 610. 'Digestion time Pattern 616.’ Digestion time == .5 hours, 0 C. 1.5 % a 125 hours, 0° C.' 1.5 Iarotein in Veronal, pH = 8.h protein in Veronal, pH = 8.h is = 0.05, current a 9.2 m.a. u a 0.05, current 2 9.0 m.a. time = 2700 see. time a 2900 sec. I’lgure VIII. Electrophoretic patterns of citrate-buffered sodium caseinate solution, after various periods of digestion with crystalline rennin. 77 saunas .08.: so.“ paeman oops col: 83 8.3 8.0 So 83 cam poo- .I 9 . m u _ . h. .8393 hp monogamoo nonwon on» Java on some» husbands oupoaosmoaaoeae Ms no.“ means—am .25“. doauuemao W. No nouuoasu a on .omoo mm. #3333 . opoduouao gfionso ooaeuudnlopmnfionn m a spa censuses: .z.m.z .fi 283a . 4 km. a on m . u .Wt. w v s. cm .36 shopaem . a {0| A: 78 JV. N- l V47 Ascending ‘ Ascending Pattern 620. Rennin-free Pattern 60h. Clotted para- in Veronal, pH 8.h, p = caseinate, 30° C. . 0.05, time = 2700 see. protein in Veronal, pH = 8.h, u = 0.05, current = I 8.5 m.a., time = 2700 sec. T_4 Ascending ‘ Ascending Pattern 607. Digestion time Pattern 606. Digestion time s 10 minutes, 0° c. 1.5 % = Lin minutes, calculated protein in Veronal, pH = to be equivalent to clot. 8.h, p a 0.05, current = 1.5 % protein in Veronal, 8.5 m.a., time = 2700 sec. pH = 8. , u = 0.05, cur- rent = .9 m.a., time = 2700 seconds M Ascending <7 Ascending M I ‘— Pattern 611. Digestion time Pattern 617. Digestion time = . hours. Electrophor‘ = 125 hours. Electrophor- esis conditions identical esis conditions identical to those above. to those above. Figure X. Electrophoresis patterns of phosphate-buffered calcium caseinate solution, after various periods of digestion with crystalline rennin. 79 oceans .oeae coapeemeo BB] BB oofi coca coo coo ensoaao hp oepmnwficoo condom on» as none» machine capoaosaoavoofle so.“ commando 38.3 aoauoewao no coupons.“ a no .035 max. escapades ease—«once Eamon oeaehusnieposno can s acne 83233 i.e.z .2 93m; co: com 03' N moans-non "'3' if smasfiunm ‘ueSoaa; if? 80 t... A». ‘ Ascending ‘ Ascending Pattern 603. Digestion time Pattern 605. Digestion time a 25 minutes, 30° C. 1. 5% s hll minutes, 0° C. 1. 5 grotein in Veronal, pH = protein in Veronal, pH = .h, u a 0 .05, current : 8.h, u = 0. 05, current = 8.9 m .a., time = 2700 see. 8.9 m .a., time = 2700 see. Ascending V Ascending Pattern 609. Digestion time Pattern 618. Digestion time .5 hours, 0° C. 1.5 % = 125 hours 0. 1.5 rotein in Veronal, pH: rotein in Peronal, pH = .h ., u = 0 .05, current c .h, p = 0. 05, current a 9 m.a., time s 2700 see. 8.7 m.a., time a 2700 sec. A; ' Ascending Pattern 612. Digestion time = 60 hours, 30° C. Electrophoresis conditions identical to those above. Figure XII. Electrophoretic patterns of phosphate-buffered sodium caseinate solution, after various periods of digestion with crystalline rennin. 81 30° c. k a 27.3 O C. k = 1.76 Figure XIII. Plot of a/(a-x) as a function of digestion.time for N.P.N. liberation from a citraterbuffered solution of sodium caseinate, pH 5.20. 20 [Co 60 ‘ 1‘0 160 150 Digestion time, minutes 82 9W 8 . 7. 6’ k 8 21:14. 51’. ,ehT 7 V3 / : 1:221:36 Figure XIV. Plot of a/(a-x) as a function of digestion time for N.P.H. liberation from a'phosphate buffered solution of calcium caseinate, pH 6,20. ' 72% no 60 ' 80 100 120 Digestion time, minutes 9. 30°C 8' k=6.88 7. 6. S. h. F 33. \ 0°C. a k81.10 2 /I/ Figure XV. Plot of a/(a-x) as a function of digestion time. 20 no 60 ‘ 80 100 120 Digestion time, minutes. NW on Rate Constant F libornti N.P.N. a_4n____4hL—4EZ—¥1—SL:L£E¥!: e42____ia_2 83 hosphate-caseinate m = 2.22 calcium.caseinate 1:182. \ citrate-caseinate m.= 3 30 IFigure XVI. Arrhenius plots of rate constants for N.P.N. liberation from three different substrates under identical conditions. 3"; l " . 2‘6 2 3‘3 1 / sail? ’1; J n “to tsmnsraturs- 8h The sedimentation data were found to be somewhat less revealing than the electrophoretic analysis. The only consistant change seemed to be a marked decrease in the fastmmoving component of the original solution, presumably the a- fraction, with a concomitant appearance of a larger slow component having a skewed side. This skewed appear- anee was probably attributable to the formation of a whole series of new components of varying molecular size. This is consistant with the findings of Alais, g; g_1_. (us), who reported that the amount of N.P.N. found in the tri- chloroacetic acid filtrate at any given time increased as the concentration of the trichloroacetic acid was decreased. This would indicate that a series of different peptides was being liberated by rennin, and would presumably result in a series of residual protein fragments of varying size. These patterns were made on the entire digestion mixture after rennin action, and perhaps more unambiguous results would be obtained if the paracaseinate clot were isolated, redissolved, and its pattern compared to that of the original casein solution. Preliminary studies were made to determine the com- position of the N.P.N. being liberated by rennin. One- dimensional paper chromatography, using butanol-acetic acid-water solvent, indicated that there was one major ninhydrinrsensitive component and two minor, but never. theless significant, components. Twordimensional 85 chromatography of a hydrolysate of the T.C.A. filtrate showed about the same amino acid content as was found by Nitschmann, gt 5;. (7') with the exception that proline seemed to be absent in this hydrolysate, whereas the pub- lished analysis included quite a large quantity of proline. Since there was so much similarity, and it appeared that the N.P.N. had been fairly well characterized, no further tbme was spent on this project. 86 N,P,N, ggberation and Clotting Rate If it is assumed that in any given solution a certain quantity of reaction must occur in order to achieve a clot, and that this endrpoint is the same, irrespective of other conditions, then it is possible to define Rate of Clotting as being equal to the inverse of the clotting time, and then this clotting rate may be studied as a function of temperature by relating these quantities according to the Arrhenius equation: rA/RT K 8 Z e . where K equals the rate constant, A is the activation energy per mole, R is the gas constant in calories per degree per mole, T is the absolute temperature, and Z is a steric factor which for bimolecular gas reactions is about equal to the number of molecules colliding, but has not yet been completely worked out for reactions in solution. If this equation is conwerted to its logarithmic form, one obtains: inxsinz-A/R-l/i' which is the slope-intercept equation for a straight line if 1n K is plotted against l/T, the slope being equal to A/R. When the clotting rates at different temperatures of normally clotting solutions, (e.g. casein in calcium phosphate at about pH 6.2 or citrate buffered casein solu- tions at about pH 5.2) are treated in the manner described . eurons omens 0p Hoofipdooa ecofiuaoaoo .mao>aoacfl opsaae 0H pd coma neasnoaxm .:.m mm .Hanoao> CH R m.H edoausHom Gaomeo mo mahopuan soauauaoafioom exHx oaswum 87 .0 0mm kaopqaaxoamnc enaou .eS.n.a ooo.om u oeomm .nHe>AoasH opsqaa ed as coca sesamomxm edem mm .aa:oae> CH R oem ecoapsHom cfioneo Ho meaoupam GoapmpCoanom eHH>x oaswwm 88 once» on Heoneoeoa nnoaesonoc .:.w ma .Heaoae> as R m.H .Qeop s.c no . .e>ona — ‘— .0 0mm haouosfixoaoma ..S.Q.a ooo.om u oooam .mHa>aouaw oascafi pH no code monomomxm Hadoao> ca R oem .GOHnsaon cfiomaoeaam no anaconda coaucpnoaaoem .nHe>aopc« commas pH on some uoasnooxm ecowpzfiou naomaocaam we announce dofiuapcesaoom .Nx oaswam .HHH>x enemas 89 90 r 80 70' 60’ 50 ' MO' 20 ' A. 16.5° c. ’ k I: 13.50 uA l v " 0 0° c. o O k = «Bel-l5 o o O 0 Figure XXI. Semi-log plot of a/(a-x) for N.P.N. Liberation from Citrate Buffered Casein Solution for Rate Constant Determination 00 to 16.50 C. 1.0 so 39 ‘ to 50 (20 Digestion time, minutes 90 a / (a-X) 90 - 80 ' 70‘ 60 > So - ho * (3 37.2986. 300 k.= 10 0. Bar In: .9 22.5° C. X Ira-20.5 20 F A A. 10' 9. 8; 7? C) 6* (3 X o 0 5. h C) o 3' /° ,0 2' Figure XXII. Semi-log plot of a/(a-x) for N.P.N. Liberation from Citrate Buffered Casein Solution for Rate Constant Determination 22.5° to 37.2° C. __10 20 30 ‘yo 59 69 ‘Digestion time, minutes 5000' 1000 500 H O O U'l 0 Reaction Rate Constants 91 I 90 | o o Eslope = 37 0 ‘ I Q10 3 1.6 \\‘ | 0 ‘~\ ‘.‘ \\‘\ I ‘\ O | ‘\‘ ‘ ‘~\slope = 2.05 O z ‘ Q10 z 1.06 \ | \ | O N.P.N. Rates slope = 2.h1 o = 1.07 I 0 Q1 ’ Figure XXIII Clotting Arrhenius Plots of Clotting Rate Rate and N.P.N. Liberation from Calcium Caseinate in Phosphate Buffer at pH 6.20 o 3. 1 3,2 3.3 3.1; - 3.15 3.6 l / Absolute Temperature, deg. K 5000 x I | , \\ | "\ ‘slope = 3h.8 O O O ‘ Q10 = 1e 6 C O | o o x o | o l I 1000' 0 . | . \ , a O \\ ‘ r ‘\ | t \I 500 ‘\\ a r ‘. \slope = 2.16 43 b ‘ o | \\Qlo = 1eo7 3 .P.N. Rates . *3 ‘ ‘ a i o I 0 r 0 4.) i m 5100. g : slope = 2.52 2 ' Q10 = 1.07 ,2 r 50* Figure XXIV A ’ Arrhenius Plots of Clotting Rate and N.P.N. Liberation from Sodium Caseinate in Citrate Buffer , at pH 5.20 A Clotting Rate 10M 0 L F s. o F 3,1 3. g 3. 3 3,1; 3,5 3,6 1 /.Absolute Temperature. deg. K 92 ‘ ‘ ' ' 93 V V V t w 5000 iooq ‘ t 500- p 0 Reaction Rate Constant U'l O 10 t ‘V w f—fl— Arrhenius Plot of Clotting Rate of Sodium Casein Figure XXV Clotting Rate in Citrate Buffer at pH S.h5 SO 3 3 u c 3 empera ure, BIS degrees RT""2‘§ 9h .asoauaocoo uaeaeuuaa eeama neon: wafi»»0Ho sou ne>a50 asuaenaac Ho doefiaegaoo .HBQ enemas o. a (sh 3.34.. < n _ 0.. hi v.0 ad a *6 ad «.0 3 vi a n «.0 Ion i,.mn eeaso c0333: i.e.. no one: «:35 s N a x eeeoeee .. .v. on «o useeeeacxa ue.m so o~.m no snatch meezeso 2H mkdzummco lbmnom xmhham uH13 1: [>L . :Eéti . rEain. and it was found that the diffusion rate at 30° C. is about 2.30 times the diffusion rate at zero degrees. This is too small a difference to explain the difference in reaction rate. Such factors as density difference, kinetic effects, and other similar minor corrections have been neglected in this calculation and the viscosity has been assumed to be proportional to flow time in the 0stwald See Appendix 98' viscosimeter. In addition, if this effect were important it would be expected that agitation would increase the clotting rate, whereas actually the clotting point is de- layed by agitation. The possibility of a microscopic viscosity change, i.e. an intramicellular increase, is not obviated by results discussed here because such an effect would not necessarily be apparent in macroscopic measure- ments. Indeed, they would be very difficult to measure at all. If the high temperature coefficient of the clotting reaction is to be attributed to a non-enzymatic secondary reaction, it would be expected that the clotting rate at low temperatures would be independent of enzyme concentra- tion, particularly at 1ow concentrations of rennin. That this is not indeed the case is shown in Figures XXX, XXXII and XXXI, which show clotting rates as a function of rennin concentration at 30°. 12.50 and zero degrees Centigrade, respectively. The shape of the curve on rectilinear graph paper was roughly the same in each case, although the absolute rates differed by one or more orders of magnitude. These data may be more profitably treated by plotting the clotting rate (inverse of clotting time) as a function of enzyme concentration on logrlog graph paper (i.e. log of clotting rate versus log of enzyme concentration. See Figs. XXXXI, XXXIII, and XXXIV). When this is done, three different results are obtained, depending upon the tempera- 99 mm 4m Tm ~.m Hem .o com Godomaom cassaoneo assoon oeeeoesm eooeeao no teem moaeeoac no me no access HHH>xx enemas 0 J A I} o 0 \D O O . N H H squeqsuog eqsg uopqoaeg N e N 100 Figure XXIX NH HA Ascending Descending Pattern 593. Clotted Citrate Buffered Sodium Caseinate pat =pH WE.25. 1. S % protein in Veronal. = 0. 05, current: 9 m.a., time = 700 Sec. N. M. Ascending Descending Pattern 592. Citrate Buffered Sodium Caseinate after 12. 75 minutes digestion, equivalent to the clotting time in the above solution. 1. 5 % protein in Verenal, pH: 8.h, p = 0.05 current = 9 m.a., time = 2700 Sec. M! M. Ascending Descending Pattern 59h. Citrate Buffered Sodium Caseinate, pH 5.h5 after 8 hours digestion without clotting. 1.5 m protein in Veronal, pH: 8 .h, u = 0. 05 current: 9 m. a., time = 2750 Sec. 101 .HE \ needs :asnom .noapeApnoccoo cabana N o .c on on noapsawm daemon oeeooucm econeec co seem moaoeoeo so cospcapdeocoo cahunm no oceans .xxx enemas m new w 02 0‘11 0‘9 09 001 SUI: 4°10 eqau 102 .HE \ moan: saccem enoausapcoocoo oshncm on ea ma ca . c o is .o om.me no noeocaom epscuonmo azaoom oeaehusm eootoso oo teem mnaoeoao no . o soapaApnoosoo ashesm uo poohum ,Hxxx enemas oat 09 on 02 outs 3UI440I0 09 021 103 eHS \ mafia: :«cnom .coauwApCoosoo oshncm ca :4 we om . m o c m 2 i0 0 W .o co co noaecaom oponfienco.as«oom oeaemmsm o eeeeeeo no open moaeooao no soapsapaeonoo Schema no vacuum 9 Au HHNXK oaswam @ 9 .0 T. no no I 7. no Crew-n n n-rn AA us! -flm'ww. -A- Clot ting, Ra to er \n (D'fl ahpfj _ “4' 9C) on» TC) 60 So he. 20 w 10h \ Figure XXXIII Effect of Enzyme Concentration on Clotting Rate of Citrate Buffered Sodium Caseinate Solution at 30° 0. log - log plot ..!+.é.-§.1. - .2 . 4 .§; Enzyme Concentration, R.u. / ml. .U .)- I.) 11’ I!" . I... t In Ill-s. Ill. uol:.l fists Clotting Q34 I __ _ ---——--—-v 'r ._... g- we“ , ‘. we‘re _‘*n g _ 7 6 S h 105 N Y O f w 0 0 4r v:a~ Figure XXXIV Effect of Enzyme Concentration e on Clotting Rate of Citrate Buffered Sodium Caseinate Solution at 12.5 log r log plot A l A L .2 .h .6 .81 2 ti 6‘ 8 10 20 to 60 Enzyme Concentration, R.u. / m1. 106 801 60- M’ 20* pr 0“_ Clotting Rfit° N Figure XXXV Effect of Enzyme Concentration on Clotting Rate of Citrate Buffered Sodium Caseinate Solution at 0° 6. log - log plot I I j .2 .h .6 .8 1 2 h 6' 8 lo 20 Enzyme Concentration, R.u. / ml. . ho . 60‘. . 107 ture at which the experiment was carried out. At zero degrees, a straight line is obtained (Fig XXXV'), the slope of which was found to be 0.72. This line represents an equation of the form: a logysnlogx+ logB Where y equals the clotting rate, x equals the enzyme cons centration and the slope is equal to n/a. Since the above equation is merely the logarithmic form.of an equation: ya 8 B In then the slope of 0.72 found at zero degrees indicates , that at this temperature the clotting rate increases as the 1.39 power of the enzyme concentration. At 12.5° C. (Fig. XXXIV') a similar plot produces a set of points which apparently cannot be Joined by a single straight line, but rather at higher enzyme concentrations a line of slope 0.68 is obtained, whereas at the lower concentrations a line of slope 0.98 is obtained. ‘When the same variables are plotted on logrlog paper for the reaction at 30° 0., again a fairly good straight line is obtained, this one haveing a slope of about 1.0 (See Fig. XXXIIIL Of course, at 30° C. an enzyme concen. tration greater than four Rennin units per ml. could not be used because the clotting rates are too high to measure accurately. These results seem to substantiate the contention by Berridge, and the modification by Heinicke that there is 108 a destabilization step involved which may be brought about either thermally or enzymatically, or by a combination of these two effects. At higher temperatures, above 15° C. the thermal mechanism should be completely operative and the primary reaction, which is enzymatic, would be rate controlling so that an essentially linear enzyme-concentra- tion dependence would be expected. At lower temperatures, on the other hand, the secondary reaction should be rate limiting and the proportion of enzymatic to thermal effect in accomplishing this reaction should depend largely upon the enzyme concentratiOn. At higher enzyme concentrations the secondary reaction would be more inclined to be enzym- atic; at lower concentrations of enzyme the thermal destabilr ization would be most effective. If this explanation is applied to the results obtained in the logrlog plots described, it becomes possible to rationalize them. At high temperatures the clotting rate is limited by the primary reaction and consequently is proportional to enzyme concentration. At zero degrees, the thermal destabilization rate is too low to make much of a contribution so that enzymatic destabilization is the factor which controls the rate; and at 12.5° C., the thermal destabilization is more important at low enzyme concentra- tion while at higher enzyme concentration the enzymatic effect is more prominent. 109 This does not serve to explain the clotting behavior of a citrate buffered solution at pH S.h5, however. If the hydrogen ion concentration of the citrate buffered system is decreased by one-half, to give a solution of pH S.h5 the behavior with rennin is much different. In such a system, no clot is formed unless much higher enzyme concentrations are used; of the order of 30 units per ml. instead of the usual concentration of about one unit per ml. used with the normally clotting systems. The high temperature coefficient which was so striking in the case of other substrates is no longer noticed; the Arrhenius plot (see Fig. XXV) exhibits a straight line down to zero degrees, with a slope of 3.6. This is fairly close to the slope of the N.P.N. liberation rate line and one is tempted to attribute this effect to an enzymatic secondary change in the protein in lieu of a thermal change, which allows it to coagulate. There are two facts which render this con- clusion untenable, however. In the first place, the tem- perature coefficient for the temperature hypersensitive step seems to be the same whether it is being affected by the msyme or thermally. Thus, even if the high enzyme concentration is capable of bringing about the postulated destabilization step, the same temperature effect should be observed. Secondly, at temperatures between 15° and 35° C., the clotting rates are markedly slower than would be exhibited by one of the "hermal" substrates at this enzyme 110 concentration. If the clotting rate were being limited by the primary reaction, then this would not be observed, be- cause the optimum.pH for the N.P.N. liberation (determined at lower enzyme concentrations) is near S.h (see Fig. XXVIII), so that if anything the rate of the primary re- action should be higher in this substrate. In fact, at low temperatures there is an indication that perhaps the clot- ting rate is really faster in the substrate of higher pH. Although it is difficult to compare absolute clotting rates between two different substrates, and the relationship be- tween enzyme concentration and rate is not linear, it might nevertheless be interesting to draw a rough comparison of this sort. In a substrate of pH 5.25 with an enzyme con* centration of 0.1 5, a digestion period of 72.000 seconds was required for clot formation. In the substrate at pH S.h5, however, with an enzyme concentration of 0.2 %, only 16,800 seconds were required. This is a rate difference of about a factor of four, though the enzyme is only twice as concentrated. That the primary reaction was proceeding normally is further indicated by comparison of the electro* phoretic patterns of identically treated substrates of pH 5.20 and at S.h5 (see Fig. XXIX). The pattern of the unclotted sample is very similar to that of the clotted one, even though the former remains fluid even eight hours later. 111 These facts lead to the conclusion that in the less acidic substrate an entirely different mechanism is operative and that one cannot explain the "normal" mechanism on the same basis as the "abnormal" one. If it were known how the rate of N.P.N. liberation is effected by enzyme concentration, and if the temperature dependence of the primary reaction could be determined at different pH values, then it is possible that the differences between the reactions in these two substrates could be explained. 112 Changes 2; Turbidity and Viscosity. Turbidity (ho) and viscosity (#7, RB) changes due to rennin action have been previously measured on milk and on calcium caseinate solutions, but there have been no such measurements on citraterbuffered sodium caseinate substrates, nor have these measurements been correlated on the same solutions. Therefore it was considered advisable to carry out, on the same solutions and at the same temperature and enzyme concentration some measurements of viscosity and turbidity changes during the clotting reaction. These were correlated with increase of N.P.N. in an aliquot portion digested under identical conditions. As can be seen from the figures (XXXVI, KXXII, XXXIXL), and as would be ex- pected, the turbidity reaches a maximum.at about the same time as does the N.P.N." The solution also undergoes a sharp increase in viscosity as the N.P.N. approaches a maximum, which would also be expected in the light of the fact that clotting has been shown to commence at this point. The shapes found for the two curves were quite interesting, however. It will be noticed that there is in both cases an initial period during which practically no changes can be detected, either in the turbidity or in the viscosity. In fact, the viscosity was seen to decrease slightly before it rose sharply to infinity as a clot formed. This is the type of behavior one would expect if one were dealing with con- secutive first order reactions, and if turbidity were a true 113 measure of the concentration of the final product. Although turbidity is often proportional to concentration if the particle size is uniform, there is little liklihood that these particles are of uniform size. In fact it has been reported that the number of particles actually decreases, with the small particles disappearing in favor of larger ones (kg). The quantity actually being measured here, then, is the increase in particle size, and this is presumed to be a function of the extent to which the reaction has proceeded. Claesson and Nitschmann have obtained similarly-shaped curves (kg), but with much less symmetry, in experiments in which they measured the development of turbidity in milk by the action of rennin, and also in light-scattering measurements on the same substrates. These authors have attributed this shape to an autocatalytic behavior of the coagulation step in the reaction due to the initial poly- dispersity of the casein, though this explanation accounts for neither the leveling off of the curve, nor for the failure to obtain the sane results at low temperature. It is interesting to note that the steeply-rising portion of the turbidity curve coincides with the beginning of the increase in viscosity, and the leveling off coincides with the clotting of the solution. This is at variance with the results of Claesson and Nitschmann, who report that flocculation occurred near the beginning of the sharply 114+ mouscfis .oEup coapmewan ow ms 3 mm 3 am am me .I . Do a .2. O .0 com soapmomam dascom wcaaso . soapsaom :«oneo monomhfim ch' opsauao a CH hquHnasa use .aa .z.m.z no omcsso .95? 83mg m .L.e um, T... on J H r m a. .7; Oz 7 O .2. 11 333.89 c 9 OZOmOz 9' ‘ ilff I . Alx K..I\I.!fi .! .vtnlll‘fl' 9t, ZIOI 095 ousqaosqv flm Z O 8 9 I 115 spuooes ‘emtm notg 06I OLI CI? of? ogt OSI mopscafi .oSau Coapmouwa pmH mfia one pea pm inc H.o N.o .0 COM .0 Goapmemam magnum madman haacanasa one hpunoouw>, .:.o no omssnu .HH>xax enemas m.o . r o om. o J . m a on O ”A 0.0 m" A 0. c’: H 4&0 H e H Nod m.H hufinoomw> mpficanase o 0 LL ‘v ‘2!!!"8 116 mouscae .oaau coaumowfia omme osem cam: 83 8mm omem can: magnum a mooo.o eosoeesm oneness .o 00 pa cede no soauossm s as hpauooua> we ommsno HHH>NNN ohfimam till. 1’“. .Ill ,ll'fialul llIIIIIIIIII 1 com 005 oofl’ 0' 4 meta 009 spuooes 00L 009 ‘am; 117 mopscfis areas GOfipmomHQ a: mm mm mm mm . .m as poao . . a N as Goapspmosanom m N H as mamoaonmoapooam .nouusm obscene an naomsole venowacm no coupsflom a ma newcsmo mmauooufl> was assessess z.m.z .NHxxx shaman O wacanhfis WV haunooua> 118 omN hHopsafixoaaas .Qfiop .. 5.9. a coo omu uoeam .aHs>aopmH ousnfia 0H us soxapo o>onu Amy as case coaumowfiu masses moussaa humor» nevus :Honsold venouamo he ncaoupaa coapauneefinow j e T T .nouscfis mm. M oSHB .d d.w ma .nohhfin Hacoao> .2 mo.o CH R m.H ma moapahpCoocoo naepoam .oEHu ho encased sneaks» hog Coaunowfiu Gammon monks cfionsolu monoaaso no unpopusa empeaonnoapoofim 2.: a 333.8 .m mcfivcoou< newscas humoke .N msfivmoen< opsmas oco .H mcHomoonm J I'll ax oaswam _ a . Ill‘lllllll? I. 2‘ 119 rising portion of the curve, rather than.at the end. This Ininor discrepancy may possibly be due to the fact that the light used was of a different wavelength, and the floccu- lation point makes little difference to the discussion. The cause of the slight viscosity decrease at the beginning of the digestion is not clear. The decrease in the viscosity of’sodium.caseinate solutions has been attri- buted by Hankinson.and Palmer (33) to a decrease in the ex. tent of hydration. However, calcium.caseinate solutions, which these investigators found to be initially hydrated to a lesser extent, exhibit this decrease to about the same extent as do the citrate-buffered sodium caseinate solu. tions. The relatively minor decrease in average molecular weight, as estimated from.the N.P.N. determination, seems inadequate to explain this viscosity decrease. It is possible that a combination of these two effects are op- erative in causing the observed results. What ever the precise cause, however, it is probably that this decrease is related to the primary reaction, whereas the subsequent increase is probably controlled by the secondary reaction. These conclusions are based upon a comparison of the rela- time rates of the viscosity decrease and rise in solutions digested at 30° C. and at zero degrees (Fig. XXXVII and XXXVIII). The absolute rates may not be compared because the enzyme concentration is much higher in the low-tempera- ture sample, in order to bring about a measurable rate of 120 change. However, the shapes of the curves are significant The clotting time for the same enzyme - substrate combinar tion at 300 as was used for the zero temperature measure- ments, was fbund to be about nine minutes. By reference to other measurements under similar conditions (see Fig. VII) it may be estimated that the primary reaction at zero de- grees was essentially complete after about 100 minutes digestion time. Although the viscosity data are slightly _erratic at this point, it would appear that a minimum is not reached until after about 300 minutes; at least the curve does not begin to rise until well after the end of the primary reaction. At 30° C., however, clotting has occurred before the primary reaction (as measured by N.P.N. libera‘ tion) has been altogether completed. In the absence of . calcium.ions at pH values about b.6, the viscosity continues to decrease with time (hB) and in the presence of h.6 M urea at about pH 5.2 in citrate buffer, the same results are obtained, though the data were slightly erratic. This would indicate that the reaction responsible for the via. cosity increase (i.e. the aggregation) will not take place at higher pH in the absence of calcium ions (as is obvious by its failure to clotl-and is also inhibited by urea, though a fine granular precipitate eventually was seen to settle in the bottom of the viscosimeter reservoir. These observations might be interpreted as being indicative of a polymerization reaction, such as has been found in the 121 clotting of blood, occuring subsequent to the N.P.N. lib- eration and dependent upon either calcium phosphate or else free hydrogen’bonding sites for crossrlinking. That hydro- gen bond formation is involved in this polymerization is indicated by the fact that there is no viscosity increase in the h.6 M aqueous urea solvent, though some reaction is apparently taking place to produce the granular precipitate. It will be noted that with the exception of one point, the viscosity curve at zero degrees C. between 800 minutes and the clotting time is a smooth function. At 2,730 min- utes a significantly high value was measured which was considered not to be simply anomolous because the flow time was reproduced twice to within a few tenths of a second. Just prior to this measurement, the temperature of the bath had risen briefly to four degrees C., but had been returned to ice—water temperature for 15 minutes before actual flow time determinations were made. If an irreversible change had been produced due to this temperature increase, it would be expected that subsequent points on the curve would be displaced to higher values. This was not found to be the case. All other points are found to conform to the values which would have been expected if the warming had not occurred. This would tend to indicate that a reversible change had occurred upon heating slightly, and that the gradual viscosity increase being measured under these con- <31tions was not the result of the same reaction which 122 produced the clot, but rather was due to an unfolding of the protons molecules. If this were true it would be exe pected that under conditions where clotting can occur, an irreversible clot should be formed, but if conditions are chosen so that clot formation is not possible, then it should be possible to reverse the secondary reaction, even if the primary reaction is carried out under conditions which would theoretically allow the secondary reaction to proceed, 0.8. at 30° C. in phosphate buffer near neutral pH. This experiment was attempted and, although the con- ditions were inadequately controlled, there was an indies- tion that reversion did take place. A solution of phos- phate-buffered sodium caseinate was prepared by adding twenty ml. of 0.1 M phosphate buffer, pH 6.38 to 70 ml. of six per cent sodium.caseinate stock solution. ‘This was then divided into four portions of 20 ml. each, two were cooled to zero degrees and two were equilibrated at 30° C. Two ml. of 0.0003 % crystalline rennin solution were then added to each portion and those at zero degrees were allowed to digest overnight. The samples at 30° were allowed to digest for 15 minutes, at the end of which time one was placed in the refrigerator to stand overnight, while the other was cooled to about ice-water temperature and two ml. of cold 0.21 l calcium.chloride solution was added (making a total calcium ion concentration equal to that used for the usual calcium.caseinate substrate). A coagulum was 123 immediately formed. After the remaining sampleshad been held.overnight at temperatures below ten degrees, one of them was placed in the bath at 30° for fifteenminutes, then.cooled in an ice - water bath for an hour, along with the other two samples. Then two ml. of cold 0.21 M calcium chloride solution were pipetted into each, in the same manner as with the first sample. In no case was a precipir tate immediately formed, although after standing for a short while, about fifteen or twenty minutes, a clot appeared in each of these, also. The fact that clotting did not occur right away seems to indicate that the secondary reaction had been reversed in those samples which had been brought to 30° C., though it may never have occurred in the one kept in the cold. The clotting time for the calcium caseinate substrate at this enzyme concentration is usually in the vicinity of ten minutes at 30° 0,; about 100,000 minutes at zero degrees. The primary reaction should have been com- pleted at zero degrees after about 200 minutes digestion time. Therefore, if an irreversible change had occurred, the three samples which had experienced a temperature of 30° for fifteen minutes or more should have clotted upon the addition of calcium ion, while the fourth should not have clotted for several days unless it was warmed. If the secondary reaction were completely reversible, then none of the three tubes held below ten degrees overnight would be expected to clot right away, except perhaps the one 121+ warmed just previously to the calcium chloride addition. .Although the clot formation in the unwarmed tube and the failure of the recently-warmed tube to clot immediately tend.to render these results slightly suspect, and prevents drawing positive conclusions without several repetitions of experiments similar to this, at least the indication of reversibility from the viscosity data is supported by these results. There was also a third incidental observation which inclines one to believe that the thermal change which allows clotting is reversible. This too was the result of a fail’ ure of the ice . water bath to last overnight, this time during the study of clotting rates as a function of enzyme concentration. It will be noted (see Fig. XXVIII) that the sample containing 0.5 Rennin units of enzyme exhibits a high clotting rate, as compared to the previous and subsequent samples. This relatively fast clotting time has been exr plained to be the result of the bath warming to 9.50 c, overnight. However, the samples containing 0.25 units and 0.125 units of enzyme also had warmed to this temperature, and would therefore be expected to clot earlier than initially anticipated if an irreversible secondary reaction had been speeded up for a period of time. This was not found to be the case. The sample containing 0.25 Rennin units clotted at the initially expected time, based onpa smooth clotting-rate versus enzyme-concentration curve, 125 and the last sample, expected to clot at the end of 800 hours, was discarded after hZO hours, unclotted, because of lack of sufficient time to complete the experiment. These results suggest that there are two separate types of der stabilization, both of which result in clot formation, but which are basically different reactions. The thermal de- stabilization might be a reversible process, while the enzymatic counterpart is perhaps a more drastic, irrever. sible change. If the enzymatic conversion has proceeded to a sufficient extent than a relatively small increase of thermal energy will bring about the necessary change to form a clot, but if the enzymatic reaction has not yet proceeded far enough, then the change wrought by heating is reversed upon cooling, making it necessary for the relatively temperature independent enzymatic reaction to affect the entire destabilization step. It would probably be rewarding to test this hypothesis by carrying out several series of controlled experiments similar to the one described. 126 Effect 9; Different Buffers 9g Clotting. Nest explanations of the clotting phenomenon have implicitly involved the participation of calcium phosphate in the clot formation, and Pyne (214.) has stated that ”Calcium . . .(is) indispensible for the second stage (of the clotting reaction).” However, the citrate-buffered systems used in these present investigations were free of calcium ions, and in fact experiments were carried out which showed that calcium ions up to 0.00h M in these solutions had practi- cally no effect on the clotting time. Therefore it became important to determine whether the observed_clotting was a result of a peculiarity of the citrate ions, or if actually clotting would occur in other buffers at a similar pH. Calcium free casein solutions were prepared in malate, acetate, and phthalate buffers, as well as in citrate, at different values of pH and ionic strength, in order to compare clotting rates under these various conditions. As can be seen in Table XXV, clotting occurs in all of these solutions if the pH is in a favorable range, i.e. below 5.h. The ionic strength also seems to have a marked in fluence, which might possibly explain the differences be- tween solutions at the same pH. It was noted that in every case where a clot occurred (within reasonable clotting times) it had the normal appearance of a soft rennin curd. There was an initial, sudden heterogeneity which was taken as the 127 clotting point, followed by a fairly firm gel formation. If this gel was allowed to stand undisturbed, retraction of the clot proceeded in the typical fashion. These observa- tions would seem to contradict previous assertions that calcium phosphate is essential to clotting, but an alter- native explanation is not obvious. The clot might be simply a precipitation of isoelectric paracasein, as has been suggested by Lundsteen (h), but there are a number of facts which tend to refute this possibility. The principle one is the nature of the clot formed. An isoelectric pre- cipitate would not be expected to form a gel, and if a gel did form, there would be no explanation for the clot re- traction observed. A less arbitrary criterion for disr counting the isoelectric precipitation theory is the original solubility data of Pertzoff (50) on which Lundsteen based his explanation. Pertzoff shows that at any given temperature paracasein is less soluble than casein, as judged by the base-binding capacities of these proteins. But in the same paper it is noted that the solubility in distilled water is the same for paracasein and casein at low temperatures, and that the isoelectric point of para- casein is lower than that of casein._ This means that in the change from casein to paracasein, a product of lower isoelectric point is formed, so that in a solution of con- stant pH there should be a slightly decreased tendency for precipitation to occur, if solubilities of the proteins are Table XXV 128 Effects of pH, Ionic Strength, and Specific Ion Buffer Malate Malate Malate Malate Acetate Acetate Acetate Acetate Acetate Acetate Acetate Phthalate Phthalate Phthalate Phthalate Phthalate Phthalate Phthalate Citrate Citrate Citrate Citrate Citrate Citrate Citrate 'U m myunm ¥runon> 000 O tfinnnvroxnvx U'IUIUly‘lU'LU‘l O\ on Clotting Ionic OOOO . O O . 0000 0.0 ooooooo . O O O . . ooooooo wwww w Emwmwfim 0 go 0 w \nmmmmgo eeee aqrfifig 0000000 00 Strength (apprOX.) Initial Turbidity (Klett) 582 212 (std.) 21 o 2h9 90 333 8- 1+2 17 216 the 20%.8 .9 55.1 155 no clot no clot gfi'glot 00 66.3 no clot 80.9 no clot no clot no clot 6k 800 93.3 103.6 26.9 Clotting time seconds (10 enz.) h08 (lo enz.) 38 3 no clot no clot no clot 76.8 27.1 129 similar. Furthermore, although casein is about 1.5 times as soluble as paracasein in a given amount of base, and at any given temperature, these data show that paracasein is more soluble at 25° C. than casein is at five degrees. Thus if the protein solution is near enough to saturation to permit isoelectric precipitation at 25° upon conversion to paracasein, then a supersaturated solution should be obtained upon reducing the temperature to five degrees. The citrate buffered casein solutions at about pH 5.2 were found to be quite stable for periods up to two months at five degrees Centigrade, but at the end of this time clot- ting occurred after digestion for a few days with rennin. Since the critical pH above which no clotting occurred was in the neighborhood of the ionization constants of the free carboxyl residues of the protein, i.e. 5.h5, it was considered a possibility that the clotting might involve hydrogen-bonded polymer formation between unionized carboxyl groups at low pH. To test the importance of free carboxyl groups, an attempt was made to esterify them with methanol. The product obtained was practically insoluble in 0.2 N sodium.hydroxide, and the comparatively dilute solution finally obtained was unstable to 0.0h M calcium chloride. This question remains unresolved, therefore. 130 Optical Rotatoq Studies. The high temperature coefficient of the secondary reaction in the rennin clotting scheme led Berridge to lassert that this step must be a thermal denaturation of the rennin-altered casein, since denaturations character- istically possess temperature coefficients of the order lof 1.3 to 2.0, about the same as for the clotting reaction. Such a hypothesis is somewhat supported by the reversible viscosity increase noted at low temperature. Since de- 2naturations have been accompanied by changes in optical rotation in all cases studied so far, this measurement was deemed essential. After obtaining several measurements on a casein solution diluted to h.2 % with phosphate buffer, pH 6.38, one ml. of casein solution was removed from the tube, replaced by one ml. of rennin solution, and mixed by inversion of the tube several times. Readings were taken as rapidly as possible over a period of ninety minutes. Not only was there no significant change of rotation, but the readings were extremely erratic (Table XXVI), so that it could not be positively stated whether or not there might have been some small increase of levorotation. The turbidity of the solution dictated the use of a ten cm. tube with as large an aperture as possible, and even then the measurements were most difficult. That the reaction had gone to completion was demonstrated by adding calcium 131 Table XXVI Measurement of Optical Rotation of a Phosphate Buffered Sodium Caseinate Solution During Rennin Digestion Without Enzyme With Enayme Readings Digestion time Reading 12' 653 0 time 3.5180 2 O .7 0 h.39 5 min. 3.8ho %.1%6 3.712 .9 6 10 min. 3.805 h.06o 3.710 h.050 60 min. 3.815 h.02u 3.798 %.080 90 min. 3,730 ..965 3.816 Ave. h.05h Table XXVII Measurement of Optical Rotation of an Unbuffered Sodium Caseinate Solution During Rennin Digestion Without Enzyme With EnZyme Readings Digestion time, 22:2- Readings 5.728 h. n.786 s. 00 5 5.150 S. 66 5.2 5.250 5.777 6 S. 52 5.773 7 S. 72 8 5.278 Ave. 5,729 t.o,ozu 8.5 S.h86 9 5.575 - 10 5.265 (G)D = 100° 10.5 5.378 11 5.286 12 5.302 132 Table XXVIII Optical Rotation Measurements of Casein and Paracasein in the Presence and Absence of Urea Native Casein 5.61 % BNO 00 0‘0 H nrd\90\ O O O raviunncon) toknunacnu HxOI-‘O‘ON \flAnURRUfiflAnUUflNfl Average 5.391), t 0.27 Probable error = 5.00 % (G)D = 96°is° Denatured Casein 5.61 % in 6.6 M Urea Average Probable error = 3.85 % O O O O O O O 0 3002322153.: \» {TUtEknfincouHU\en3 O‘O‘O‘N O‘ O‘O‘O‘O‘O‘ 6.778 (ME = t 0.26 121°‘1 5° Native Paracasein 2.81 % Average 2.221 1 0.111 Probable error'= 6.35 % (“ID = 790 t 5° Denatured Paracasein 2.81 % 1 n6.6 M Urea Average Probable (MD x» 0‘ l 80 wNwwwNNNNN O O O O O O O O O C HOOOOSOCDCDCD H 2.971 OI‘I‘OI‘ ‘1 0.091 = 3.06 % a 106° 1 3° 133 chloride, forming a coagulum. It has been shown that at least in some instances phosphate is capable of reversing the increased optical rotation upon denaturation (by urea, guanidine hydrochloride) so the experiment was repeated in the absence of phosphate (Table XXVII). Again a great deal of variation was found between successive readings, but there was no distinct change in the rotation during the reaction. Although this evidence tends to refute the denaturation hypothesis, there is still a possibility that the lack of a change is merely the result of two opposing effects which compensate one another. To test this possi- bility, casein and paracasein solutions of known concentra- tion were prepared, and their specific rotations were com- pared with the specific rotations of comparable solutions prepared in 6.6 M aqueous urea. These results are shown in Table XXVIII. It will be noted that both casein and paracasein are "denatured" by urea, and that the specific rotation of "native" casein is quite close to that of "denatured" paracasein, within the rather large experimen° tal error. The values for "native" and "denatured" casein are calculated to be 96° and 121° respectively, while equivalent values for paracasein were found to be 79° and 106°. These findings conflict with those of Wright (51) who has reported that the specific rotation of paracasein is initially the same as that of casein, and that'racemiza‘ tion" rate is also identical. The reason might possibly 13h ‘be due to the fact that his solutions were of rather high ijQ Ulmquist and Greenburg have found a specific rotation <3f 97.5 for native casein at pH 7.3, which compares favor- ably with the values reported here. Assuming the differences described are not a result of changes during isolation, it may be suggested that 'perhaps upon the action with rennin at 30°, "native" casein is converted to "denatured" paracasein, thus account- ing for the failure to observe a rotation change during the reaction at this higher temperature. Such an explana- tion would implicitly require the postulate of a "re- naturation" of the paracasein during the isolation pro- cedure, which is quite unlikely. An alternative explana- tion could be that the N.P.N. products, probably having a higher specific rotation than the protein, obscure the decrease due to the formation of paracasein. If the first postulate were true, the formation of native paracasein could be observed as a decrease in levorotation, if the rotation were measured during the enzymatic‘reaction at temperatures below 15° C. If the second explanation were actually the correct one, and it undoubtedly makes some contribution, it will be most difficult to resolve the question of whether or not denaturation occures in the rennin clotting reaction. 135 V. Summary 1. It was shown that the increased sensitivity of. aged solutions to rennin clotting was a result of un- catalysed hydrolysis of rennin specific bonds, and that probably some of these bonds are more susceptible to proteolysis than the average for casein. 2. Heating the sodium caseinate solution to 90° has little effect on the results of ageing, except for a diminished rate of spontaneous N.P.N. liberation. Apr parently the temperature dependence of the clotting re- action is changed, while the primary reaction remains essentially uneffected. 3. Arrhenius plots of the clotting rates and N.P.N. liberation rates of solutions of calcium caseinate and citrate buffered sodium caseinate showed that in each case the primary reaction was probably rate‘limiting at higher temperatures while a different, more temperature sensitive reaction controlled the clotting rate at lower temperatures. The low-temperature limiting reaction was found to have a temperature coefficient about equal to that reported by Berridge for the clotting step, i.e. about 1.3 - 1.6 per degree. ' h. Solutions in citrate buffer S.h5 failed to exhibit the extremely high temperature dependence found with 136 similar solutions at 5.20, but required very high enzyme concentrations to clot. 5. There is probably an intermediate destabilization step between the primary reaction and the clot formation whiéh.may be affected by thermal energy or by enzyme action, but the mechanisms of these two processes are jprobably basically different. The thermal destabilization Inight be reversible, while the enzymatic destabilization probably is not. 6. Comparison of the electrophoretic patterns ob- tained at various points during the rennin reaction leads to the conclusion that the splitting of the a-peak is related directly, and only, to the primary reaction. 7. There are indications that the a-peak split is due to the decreased mobility of a component originally present, rather than the partial conversion of a-casein to al- and a2- caseins. 8. The pH optimum of 5.h for the clotting reaction, as found by Lundsteen, has been supported by determining primary reaction rate constants at different ph values. 9. Turbidity changes in citrate-buffered, calciwm free substrates were found to be similar to those reported by Claesson and Nitschmann from their studies on similar changes in milk. 10. Viscosity changes in citrateebuffered, calcium free substrates were found to be similar to those in milk 137 CH1 calcium-caseinate solutions, as reported by Sfihngen, .g§_§;4_ The viscosity changes seem to reflect the progress <1f the secondary reaction to a greater extent than they do 'the primary step. 11. Casein solutions at low pH, prepared in Malate, Acetate, and Phthalate, as well as in citrate buffers, were found to clot normally, indicating that the citrate clot was not the result of a specific ion effect. 12.. A decrease of ionic strength seems to facilitate clotting, even at slightly higher pH values (up to S.h0). 13. Optical Rotation studies, while generally un- definitive, gave indications that changes in rotation might be occurring, but are being compensated by concomi- tant changes in the reaction mixture. 1h. Suggestions are made to pursue further studies in (a) effect of heating on the Arrhenius curve, (b) determination of whether or not thermal destabilization might be reversible, (0) Optical Rotation and Rotatory dispersion. 9. 10. 11. 138 BIBLIOGRAPHY Hammersten, 0., Nova Acts Regiae Soc. Sci. Upsaliensis in Memorium Quattuor Saec. ab Univ. Upsaliensi Peracr torum; "Casein and its Industrial Applications", E. Sutermeister and F. L. Browne. Reinhold Publishing Co., New York, 1939. Berridge,’N. J., The PurificatIOn and Crystallization of Rennin, Biochem. J.,.29, 179, (19h5). Heinicke, R. M.,'Comp1ementary Enzyme Actions in the Clotting of Milk, Science, 118, 753, (1953). Lundsteen, E., fiber das pHeOptimum der Chymosinaktiven Enzyme. (Chymotry sin, Pepsin, Chymosin). Biochem. z., 235, 191, (19 ). weugh, D. F., A Process for'Producing a Water-Soluble Casein. .U. 5. Patent No. 2,7th,891, May 9, 1956. Berridge, N. J. The Second Phase of Rennet Coagula- tion., Nature, :43, 19h, (19h2). fl Nitschmann, Hs., H. Wissmann and R. Henzi, Uber ein Glyko-Makropeptid, ein Spaltprodukt des Caseins erhalten durch Einwirkung von Lab, Chimia 11, 76, (1957), Mulder, G. J. Ann. der Pharm., 28 73, (1938); McMeekin, T. ., Milk Proteins. n "The Proteins", H. Neurath and K. Bailey, editors Academic Press, Inc., New York, (195A) vol.,§, pp. 3892611. Nielsen, H. C. Molecular Weight Studies on Acid‘ Precipitated, Calcium Precipitated, Alpha, and Beta Caseins by Osmotic Pressure Measurements in 6.66 M Urea, WPh. D. thesis, Michigan State University, 1957, 113 numb. leaves. Linderstrdm-Leng, K. and S. Kbdama, Is Casein a HomO‘ geneous Substance? Compt. Rend. Trev. Lab. Carlsberg, 1'10 NO. 1' ’48, (1925)e Mellander, 0., Electrophoretische Untersuchung von Casein, Biochem. Z. 300, 2h0 (1939). 12. 13. 15. 16. 17. 18. 19. 20. 21. 22. 23. 25. 13? warner, R.'C ‘Separation of a- and 9* Casein, J. Am. Chem, Soc., 66, 1725 (l9hh). Hipp, N. J., M. L. Groves, J. H. Custer, and T. L. McMeekin, Separation of Gamma Casein, J. Am. Chem. SOC., 1g. 4928 (1950). Cherbuliez, E. and P. Baudet, Recherches sur la caseine V. Sur les constituents de la caseine, Helv, Chim. Acta,‘22, 398 (1950). Long, J., Q. van Winkle, and I. A. Gould, Isolation and Identification of l-Casein, J. Dairy Sci., XLI, 317, (1958). McMeekin, T. L., M. L. Groves and N. J. Hipp, The Separation of a New Component of Casein, Abstracts of Papers, 132nd. National Meeting, American Chemical Society, Miami, Florida, April 7-12, 65c (1957). Osborn T. B. and A. J. Wakeman, Some New Constituents of Milk; A New Protein Soluble in Alcohol, J. Biol. Chem.,'22, 2MB (1918). Linderstran-Lang, K., On the Fractionation of Casein, Compt. rend. lab. Carlsberg, ll» 1M9 (1929). Hankinson, C. L., The Preparation of Crystalline Rennin, J. Dairy Sci., gé, 53 (19MB). Berridge, N. J., Pure Crystalline Rennin, Nature, .151. N73 (1911.3). Schwander, H., P. Zahler and He. Nitschmann, Das Lab und seine Wirkung auf das Casein der Milch, V. Ana- 1ytische Untersuchungen an Kristallisiertem Lab. Helv. Chim. Acta, 22, 553 (1952). Berridge, N. J., Milk Coagulation. In "The Enzymes", J. B. Sumner and K. Myrbach, editors, vol 1, part 2. Academic Press, New Ybrk (1951) pp. 1079‘1105. Berridge, N. J., Rennin and the Clotting of Milk, Advances in Enzymology,‘§!, h23 (19Sh). Pyne, G. T., The Chemistry of Casein, A Review of the Literature, Dairy Science Abstracts, 11, S32 (1955). n Holter H., Uber die Labwirkung. Biochemische Z. 2gg, 160 (1932). 26. 27. 28. 29. 30. 31. 37. 38. Inc Cherbuliez, E. and J. Jeannerat, Recherches sur la Caseine. Sur le fractionnement de la Caseine et de la Paracasein an Chlorure d'ammonium. Helv. Chim. Acta, :23, 952 (1939). Nitschmann, H3. and P. Zahler, Das Lab und seine Wirkung auf das Casein der Milch, III. Entstehen bei der Labgerinnung der Milchgerinnungsaktive Stoffe? Helv.,tnin. Acta, _3__3, 851; (1950). Alais, C., Etude des Substances azotees non-proteiques (N.P.N.). Separees de la Casein du Lait de Vache sous l'action de la Presure. lhth. International Congress of Milk Industries, Rome, g, 823 (1956). Lundsteen, E., The Influence of the Hydrogen-ion Concentration on the Transformation of Casein to Paracasein by Chymotrypsin. Carlsber Laboratoire Comptesrrendus, Serie Chemique, 22, 3 8 (1938). McMeekin, T. L. Milk Preteins. In "The Proteins", H. Neurath and k. Bailey editors, Academic Press, Inc., New York (19511) voi. _2_, pp. 389 - £111. Pyne, G. T., Casein Heterogeneity and the Rennet Coagulation, Chemistry and Industry, March 3, 1951, p. 171. Nitschmann, Hs., and W. Lehmann, Zum Problem der Labwirkung auf Casein, Helv. Chim. Actalgg, 80h (19h?). Hankinson, C. L. and L. 3. Palmer, Rennin Action in Re- lation to the Water Bindin Properties of Caseinate 8015 J. Ddiry'Sci. _2__(_:_, 10MB (19.3). ' Beau, M., La Caseine et la Presure, Le Lait 21, 113, (lth). Cherbuliez, E., and Meyer, F., Recherches sur la Caseine II. Helv. Chim. Acta, _1_{>_, 600 (1933). Dunn, M. 8., Preparation of Casein. In "Biochemical Preparations", H. E. Carter, editor. John Wiley and Sons, Inc., New York, vol. 1, p. 22 (1919). Fox, K. K., Separation of a Calcium-Soluble Fraction of Casein from Isoelectric Casein. J. Dairy Sci., 41, 715 (1958). Hawk, P. E., B. Deer and W. H. Summerson, "Practical Physiological Chemistry", 12th ed. The Blakiston Co., Philidelphia, Pa. (19u7). ' -a—h Wk... ‘<.<‘.-..‘___ _- 4_ “'"‘“‘~'fi—._ _____ 1M1 39. waugh D. F., and P. H. von Hippel, k-Casein and the Stabilization of Casein Micelles, J. Am. Chem. Soc., IQ. 11576 (1956). No. Nitschmann, Hs., and R. Varin, Das Lab und seine Wirkung auf das Casein der Milch IV. Die Proteolyse des Caseins durch kristallisiertes Lab. Helv. Chim. Acts, 24, lh21 (1951). N1. Nitschmann, Hs., and H. v. Bohren, Das Lab und seine Wirkung auf das Casein der Milch, X. Eine Methode zur girekten Bestimmung der Geschwindigkeit der Primarreaktion der Labgerinnung der Milch. Helv. Chim. Acta, 2Q, 1951 (1955). AZ. Nitschmann, Hs. and W. Lehmann, Electrophoretische Differenzierung von Saure- und Labcasein. Experientia,‘2, 153 (19h7). h3. waugh, D. a., and J. M. Gillespie, Structure of the Stoichiometric Complex of 03‘ and k- Caseine, Abstracts of the l3hth National Meeting of the American Chemical Societ§, Chicago, Illinois, September, 1958. Paper N0. .12 . hh. von Hippel, P. H., and D. F. Waugh Casein: Monomers and Polymers, J. Am. Chem. Soc., Ii, M311 (1955). ks. Alais, C., G. Mocquot, Hs. Nitschmann, and P. Zahler, Uber die Abspaltung von Nicht-Protein-Stickstoff (N.P.N.) aus Casein durch Lab und ihre Beziehung zur Primarreaktion der Labgerinnung der Milch. Helv. Chim. Acta 29, 1955 (1953). M6. Claesson, O. and He. Nitschmann, Optical Investigation of the Renne Clotting of Milk, Acta Agriculturae Scandinavica, VII, h (1957). A7. Hankinson C. L, and D. R. Bri gs. The Electroviscous Effect, I . J. Phys. Chem., 9L3 A8. Sghngen, N. L., K. T. Wieringa and A. Pasveer, The Curdling of Milk. A Study of the Curdling Enzyme. Recueil des travaux Chimiques de Pays‘bas 56, 280 (1937). M9.. Christiensen, L. E., The Action of Proteolytic Enzymes on Casein Proteins, Arch. Biochem. and Biophys.,‘52, 128 (1954). 50. Pertzoff, V., The Effect of Rennin upon Casein, J. Gen. Physiol., 1Q, 987 (1927). ._ w— mes—##- 51. 52. 1&2 Wright N. C., The Action of Rennet and of Heat on Milk, Biochem. J., _1_§_, 2115 (192b,). Djang, S. S. T. "The Isolation, Fractionation, and Electrophoretic Characterization of the Globulins of Mung Bean". W Ph. D. Thesis, Michigan State College, pp. 75-80 (1951). Almquist, H. J. and D. M. Greenberg, The Influence of pH on the Optical Rotation of Proteins. J. Biol. Chem., 105, 519 (193u). APPENDIX I., The symbols used in the equation on page 97 represent the following quantities: DT = Diffusion rate at the given temperature. (13 = Viscosity of the solution at that temp. T a The absolute temperature at which the measurements were made. II. A sample calculation of activation energy from the slope of an Arrhenius curve: E 1081‘ = 1°82 ‘m’% E rm == slope of curve :2 m E = 2.303 R (- m ) if the slope, m , = -35 (x 103): E a 2.303 x 1.987 x -35,000 E = 160.125 Calories T‘FRHIS'I’RY DEERE? Date Due Demco-293 "77177771777MM)M“