. 137 186 THS A STUDY OF SOME OF THE PROPERTIES OF SUGARS AND PROTEINS AT THE DROPPING MERCURY ELECTRODE Thesis for the Degree of M. S. MlCHiCAN STATE COLLEGE Amos Clark Griffin 1941 AA...—b -- “A __..—... ,"__._...‘-_.__+47 > L , , Mfich‘ga“ State Univer‘sny - ----»-._—'. ”u -4, -,’(wv . c . a o - - . .r—, Irv-4' “an In I‘hfgwM'CB’f-V“ ' 1 xi...” \ ‘. ‘ a u: “11-"! :mW 1‘ 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 6/01 cJClRC/DataDuepss-sz "'0 VMOWWWW'VW‘ V” V" vm * «rm-Ir 'rmmmmmtmnv- ‘9- ‘!- ‘7‘" m' * A STUDY OF SOME OF THE PROPERTIES OF SUGARS AND PROTEINS AT THE DROPPING MERCURY ELECTRODE by Amos Clark Griffin A THESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of MASTER OF SCIENCE MICHIGAN STATE COLLEGE Department of Chemistry 19h1 ACKNOWLEDGMENT I wish to express my sincere appreciation to Professor C. D. Ball for the encouragement and assistance he has given me during the course of this work and in the preparation of this thesis. II. III. IV. VI. T B “ O CONTEITS INTRODUCTION HISTORICAL A. The Determination of Sugars at the DrOpping Mercury Electrode. B. The Determination of Proteins and Amino Acids at the DrOpping Mercury Electrode. C. Evidence Pointing to Protein- Carbohydrate Combinations. EXPERIMENTAL A. Apparatus and Explanation of Method. B. The Determination of Sugars. C. Proteins and Related Compounds. D. Combinations of Sugars and Proteins. DISCUSSION CONCLUSIONS BIBLIOGRAPHY Page No. 15 18 22 26 INTRODUCTION The dropping mercury electrode method of analysis has proved to be of value in the study of sugars and pro~ teins. Considerable work has been recently reported in the literature, and from these investigations we have been presented with a better knowledge of these very important compounds. The sugars have been studied only to a limited extent by this method, and further work is necessary in this field. The proteins have been subject to more exten- sive investigation. The protein investigations have indi. cated that some very pronounced changes occur in the tissue proteins during the course of cancer and other diseases. Other important preperties of proteins are also being investigated. Since many proteins and several of the common sugars have been characterized at the dropping mercury electrode, a further study of the properties of sugars and proteins by this method should prove to be of value. An eXplan- ation of the behavior of proteins when in the presence of sugars might be derived from studies of this type. The protection that sugars give proteins against heat coagul- ation, and the hardening or resistance that plants may acquire against heat and cold has been suggested by many investigators to be due to a sugar-protein combination. In addition to combinations that apparently exist when proteins and sugars are in solution together, evidence has been presented to show that carbohydrates are an integral part of many, and possibly all, isolated proteins. Rimington (1) has compiled a table of the carbohydrate content of proteins, and many of the proteins are shown to contain large amounts of sugars. The type of combination, however, that exists between carbohydrates and proteins in many cases still remains to be solved. Since the drOpp- ing mercury electrode method of analysis is well adapted for sugar and protein investigations, the application of this method to a study of the properties of these biolog- ically important compounds should prove to be a source of valuable information. HISTORICAL A. The Determination of Sugars at the Dropping Mercury Electrode. The successful polarographic investigations of Shikata and Smoler (2, 3, h) on the e1ectro~reduction of aldehydes at the dropping mercury electrode influenced Heyrovsky and Smoler to try a similar experiment with glucose and other aldoses. Heyrovsky and Smoler (5) reported the results of their investigations in 1932. They reached the conclusions, however, that none of the following aldoses showed any re- duction: glucose, galactose, mannose, rhamnose, 1-arabinose, lyxose; and none of the disaccharides: sucrose, maltose, lactose. Heyrovsky and Smoler did report an electro- reduction of fructose and sorbose. Their investigations showed that fructose could be determined to a high degree of accuracy and that it was possible to determine invert sugar in quantities of about 0.000002 gram or 2 ,‘by the polarographic method. Polarographic research has found application in the sugar industry. Sanders and Zimmerman published two papers- in 1929 (6, 7), and Zimmerman two more in 1930 (8, 9). Their researches dealt mostly on the decomposition of sugars. They found sucrose to undergo a decomposition at 70°C. in the dry state. Sanders (10) reported that heating solutions containing sucrose three hours at lOO‘C., or eight hours at 70°C. caused a permanent depression of the oxygen maximum when the solutions were determined polarographically, the suppression of the oxygen maximum being due to organic substances produced by the sugar decomposition. A very complete review of the application of the polarograph to research in the sugar industry was made by Semerano (11) in 1956. From the results of the extensive research of Heyrovsky and workers on the application of polarographic methods to the reduction of aldehydes and ketones, Cantor and Peniston (12) extended the procedure to the study of the aldehyde form in sugar solution systems. These investigators showed that, contrary to the results of Heyrovsky and Smoler, aldoses are reducible at the dropping mercury cathode. They attributed the reduction of the aldoses to the presence of an aldehyde form in the equilibrated solutions. The amount of this form was estimated under various conditions of pH and concentration for the pentoses: d-xylose, learabinose, d-lyxose, and d-ribose; and the hexoses: d-glucose, d-mannose, degalactose, and l-allose. B. The Determination of Proteins and Amino Acids at the Dropping Mercury Electrode. As in the case of the sugars, Heyrovsky was respon- sible for the initial polarographic research on proteins. Heyrovsky and Babicka (13, lh) in 1930 presented evidence showing that certain proteins produce a characteristic uprotein wave". Human serum was analyzed in the presence of aqueous solutions containing ammonium chloride and lithium chloride. The height of the "albumin wave" in- creased proportionally to the amount of serum added, pro- vided ammonium ions were present along with the protein. The protein wave began at, ~1.2 to -l.6 volts, or at a potential about 0.2 volts more positive than that of the free ammonium ions. They explained the "wave" as being due to a deposition of a hydrogen ion from the ammonium ion. The albumin was assumed to associate with the ammonium ions, thus loosening the bond between the hydrogen ion and ammonia. The deposition of this loosely bound hydrOgen ion then takes place more readily or at a more positive poten- tial than that due to ammonium ions. In 1933, Babicka (15) applied this method to the determination of crystalline flour proteins. Brdicka in 1933 published the procedure for a new test for proteins (16). He showed that proteins in the presence of cobalt salts in ammoniacal solutions of ammonium salts, produced a'mofgsmr in? characteristic protein effect, or a double wave, at the dropping mercury electrode. This work of Brdicka's was partially the basis for the subsequent polarographic investigations on carcinomatic sera and proteins (19). The effect of buffer solutions on the reaction of proteins was reported by Brdicka in 1936 (17). He inves- tigated the polarographic wave due to proteins in buffer solutions of pH h to 11. The substances examined were: egg white, Witte's peptone and the amino acid, cysteine. The Fwave" was found to be higher in solutions of lower pH values. Proteins not containing cystine gave no wave. Brdicka claimed at this time that the sulfhydryl groups of the cysteine nucleus of the protein reduces the potential at which hydrogen deposits, thereby producing the wave. In an article published in 1938, Brdicka (18) again reported that only the sulfur-containing proteins are polarOgraphi- cally active. In 1937, Brdicka (19) published the first of his polar- ographic investigations on serological cancer diagnosis. The serum of cancer patients and of normal individuals was compared. Both sera were treated with alkaline iodo- acetate for seventy minutes and determined polarOgraphically in the presence of ammonium ions and cobalt chloride. Serum of cancer patients gave an abnormally low protein wave. The activity of the serum was ascribed to the sulfhydryl groups of the proteins present, or the sulfhydryl groups catalyzed the reduction of hydrogen, which was responsible for the "wave". They assumed there were fewer available sulfur groups in the pathological serum, thus a lower protein wave resulted. Brdicka (20) in continuation of this work showed the hydrolysate of carcinomatic serum also produced a very low "protein wave" in comparison with the hydrolysate of normal serum. Brdicka reported the polarographic test was of value in cancer diagnosis, although some acute cases of inflamation and fever would give a similar serum reaction. At this time Brdicka reported the groups mainly responsible for the polarographic reaction were the disulfide groups, which was somewhat in contrast to his earlier statement (17). One of the outstanding aspects of Brdicka's inves- tigations was the very convincing "protein waves", or the current-voltage curves he obtained. Brdicka (21) published an account of his polarographic studies on serum proteins and their significance in the diagnosis of cancer in 1938. This report was very similar to that which has already been discussed. The fact that certain inflammatory conditions would produce the protein wave was of interest to Crossley, (22), who extended the method to the study of lobar pneumonia in dogs. Using the polarograph and the Brdicka technique, he found there was a definite and continuous change in the serum protein until a maximum.was reached at the height of the disease. The protein wave due to the pneumonia serum was smaller than the corresponding wave of normal serum. The wave from the protein degradation products, or peptone fraction, was higher than the normal. The cystine content of the whole serum was found to reach a minimum when the pneumonia had reached its intensity. 0n the basis of Brdicka's protein test which indicates the presence of disulfide or sulfhydryl groups, KotlJar and Podrouzek (23) developed a polarographic method to study the proteolysis during serological enzymatic reactions. Wenig and Jirovec (2A) in a study of the polarographic reaction of proteins irradiated with ultraeviolet light, showed the proteins became irreversibly denatured. They stated there was an increase in the number of sulfhydryl and disulfide groups which were apparently masked in the undenatured protein solution. Schmidt (25) made studies on the ultra-violet action upon varying concentrations of albumin solutions. The ultra-violet radiation increased the number of sulfhydryl groups set free from the protein molecule. Globulins did not show this effect to as great an extent as the albumins. Several investigations have been made on the hormones of protein structure by this method. Thomassen (26), in a study of insulin and other hormones, attempted to corre- late biological activity with the polarographic activity effect of these compounds. No correlation for insulin could be determined. However, the polarographic effect produced by the pituitary growth hormone appeared to be related to its biological activity. Handovsky and Hauss (27) made a study of the extracts of endocrine glands con- taining hormones of a protein nature. From the anterior pituitary lobe of cattle they obtained an extract and measured polarographically the reducible substances present. The reduction potentials of these compounds were deter- mined and a comparison between these reducible groups and physiological activity was made. Tropp and co-workers (28, 29, 30) made a series of investigations on various proteins. Their studies included the polarographic effect of fibrinogen, albumins, globulins, plasma and serum. The polarographic effect of proteins has been demon- strated as being undoubtedly due to the active sulfur groups present. This protein effect was discussed by Jurka (3h). His studies were carried out on protein from fresh human serum. When the protein was determined polarogra- phically in buffered solutions of cobalt chloride and ammonium salts, two waves were produced, one being a double wave. The heights of these double waves were sufficiently separated so that the height of each could be determined. The protein double wave, according to Jurka, is caused by adsorbable sulfhydryl groups activated by the cobalt ions, the single wave representing a catalytic effect of adsorbed, non-activated sulfhydryl groups. Because of the direct association of cystine and the protein effect, a part of the work reported on the amino acids cystine and cysteine should be included in the review. Brdicka (31) in 1933, reported the micro-determination of cystine and cysteine. He studied these compounds in protein hydrolysatesand showed cystine and cysteine produce distinct -10- and reproducible polarographic curves. More recent inves- tigations have been reported by Stern, Beach, and Macy (32) in 19h0, and by Kolthoff and Barnum (33), 19h1. C. Evidence Pointing to Protein-Carbohydrate Combinations. Many of the preperties exhibited by proteins have been shown to be due to the presence of sugars or carbohydrates. A most important illustration of this prOperty is that concerning the immunological role of the plasma proteins and carbohydrates. Heidelberger, Avery and co-workers (35) isolated polysaccharides present in bacterial cultures and showed them to have the general prOperties of haptens. This distinctive group of carbohydrates were capable not only of combining with antibodies but also of precipitating them. Since the discovery of these immunologically spec— ific polysaccharides, the field has been subject to exten- sive investigation (36). Why these carbohydrates act as haptens, or what groups are responsible for their serolo- gical activity is still a point of consideration. It has been shown that proteins and carbohydrates do combine in these immunological reactions, a significant feature in this discussion. Varied evidence has been presented showing that certain sugars protect protein solutions against denaturation and coagulation. Newton and Brown (37) reported that the presence of sucrose or dextrose would protect the proteins of presSoJuice from leaves of winter wheat against frost precipitation. A complete explanation of the sugar pro- tection was not given, but the possibility of a sugar- protein combination was regarded as probable. Beilinnson (38) demonstrated that proteins were stabilized against heat by the presence of sucrose. Egg albumin was reported by Duddles (39) to be completely stable against heat coag- ulation to a temperature of 70°C. when in a saturated solution of glucose or fructose. The coagulation was also inhibited by addition of sucrose, mannose or mannitol against the action of heat or ultra-violet light upon the protein. In a continuation of this work, Hardt (DO) extended the«investigation to most of the common hexoses and pentoses. The course of denaturation was followed by estimation of sulfhydryl groups. All these sugars decreased the amount of sulfhydryl groups liberated during denatur- ation and protected egg albumin against heat coagulation. ThermOphilic organisms in the sugar industry and cer~ tain of the canned food industries have been observed to withstand extremely high temperatures (hl). The spores of certain obligate thermOpiles have been found to withstand. continuous boiling at 100°C. in corn Juice of pH 6.1 for twenty one hours. The cell proteins of most organisms would have been coagulated at a much lower temperature. Since many investigators have reported protoplasmic coag- ulation as the actual cause of bacterial death, it is not -12- unlikely that these organisms in the sugar and canning industries owed their survival to the presence of the sugar, or perhaps to a sugar-protein combination. In reviewing the subject of carbohydrates and proteins, Rimington (1) made the statement: "It is becoming increas- ingly evident that carbohydrate groups enter into the struc- ture of a wide variety of proteins, not only the so-called glycoproteins (a term of doubtful value) but also into such materials as ovalbumin, casein, the serum of proteins, etc.". Rimington prepared a table of the carbohydrate content of a wide variety of proteins. From this com- pilation, he concluded that carbohydrate structures may have to be recognized as an integral part of the majority, if not all, proteins. Many of the proteins were shown to contain rather large amounts of sugars, especially mannose. Karl Meyer (A2) divided protein and carbohydrate combinations into two classes: 1. MucOpolysaccharides- occuring in nature as poly- saccharides or as protein salts. 2. Glchproteins- proteins or polypeptides containing hexose amines and other sugars in an unknown combination. Numerous investigations have been made to determine the exact mode of sugar-protein combinations. Very adequate reviews of the prOgress that has been made in this work are given by Rimington (1) and Meyers (hé). No work in this respect has been reported from the polarographic method. -15- EXPERIHENTAL A. Apparatus and Explanation of Method. The drOpping mercury electrode arrangement used in this investigation was a modification of the arrangement as described by Kolthoff and Lingane (AS) in a very com- plete review of the method and field. The essential circuit necessary for measurements by this method includes a source of variable E.M.F. to an electrolysis cell and a means of measuring the current across the cell. With such an arrangement it is then possible to control the E.M.F. across the cell. If the total voltage applied across the cell is determined at increments over the desired voltage range and corresponding current readings observed for each voltage setting, the necessary data for a current-voltage curve of the substance in the cell is obtained. Current-voltage curves have the general shape as shown in Figure 1. This illustration represents the cathode reduction of a solution containing only one cation in the presence of an excess of a second cation which is reduced at a higher potential. The lower horizontal por- tion of the curve shows only a residual current flowing across the cell. With increasing applied voltage, the de- composition potential (E) of the reducible substance is reached. it this point the reduction of this substance causes a rapid increase in the current which gradually ”HAN/Ian 6f leltflj (“7"”f J¢€Oflok ,\ ----- A 4 r 3‘; \ ‘3 § " : 3 I3: \5‘ c f u "H‘If‘H/avc Mat-Z ([15) ‘3‘}. R. 3 $ s ‘K Q ~‘- : g E f (Inf of K) ”(tiling Cuvrcnf ._ erflflpfi‘fang,‘ ("n.l‘ ._ I/a/f: VO/faje ——: _ __ Irv/lure“! ._ A (hr—ruf- Vo/fyc; (“pvt -1“- approaches a limiting value on further increase of applied E.M.F.. The second vertical portion of the curve is due to the reduction of the second cation. The vertical portion of the curve, representing a rapid increase in current, is caused by a diffusion of the reducible substance into the dropping mercury cathode. The limiting current is reached because the diffusion rate approaches a near constant value. The diffusion current is roughly proportional to concentration of reducible material, provided a relatively high concentration of an indifferent ion is present to carry the migration current. If certain factors, which will be considered later, are kept constant the height of the current step is a direct measure of con— centration. The position of the current-voltage curve on the voltage axis is a characteristic constant for each ion and thus may serve as a means of identifying the substance. This voltage is commonly called the "halfewave potential", and is designated "3%". The half-wave potential is usually determined by a point on the voltage axis which corres- ponds to one half of the current step produced by the re- duction of the substance being analyzed. The position of the half-wave potential is shown in Figure l. The detailed arrangement of the apparatus used in this work is shown in Figures 2 and 3. The galvanometer was a product of the Rubicon Company, CatalOg No. 3hlu, having the following constants: Sensitivity: 0.001365%%; R: h50 ohms Period- h.6 seconds CODOROXO: 10,1400 Ohms figure 2 onff", ”trcew/ Fla/70A 4’7“" 01" c"'¢l0~¢r¢-f 3/»...7‘ flit-floater; 7' (M A {.73 a, flr.f/°;1] ”(may [/ecfroo/a. 4ff0f‘nf -15- The shunt used in connection with the galvanometer was a Leeds and Northrop, No. 32793h, with an internal resistance of 10,000 ohms. This shunt made it possible to make current measurements over a wide range by this very sen- sitive galvanometer arrangement. The potentiometer was a Student type, Leeds and NorthrOp, No 7651. The usual range of this instrument is 0 to 1.6 volts. In this work it was desired to reach voltages above -2.0 volts. The range of the potentio- meter was extended by standardizing the instrument to one-half of the standard cell voltage. All voltage read- ings obtained were then multiplied by the factor 2. The E.M.F. was maintained so as to retain at least 100 ohms in the resistance box. With this potentiometer and galvan- ometer it was possible to determine current-voltage curves over a wide range of reducible substances and also over a wide concentration range. I _The electrolysis cell and the electrodes are of great importance in this method of analysis. For this reason, they were constructed and maintained so as to keep all variable factors due to the instrument as near constant as possible throughout the investigation. Capacity of the electrolysis vessel as shown was 15 ml.. The anode con- sisted of a large pool of mercury at the bottom of the cell. From Figures 2 and 3, a relative idea of the dropping mer- cury cathode arrangement can be obtained. This electrode was made of a Special high vacuum tubing and was connected -16- to the mercury reservoir by neoprene tubing, so as to prevent contamination of the mercury by sulfur which is present in ordinary tubing. All contacts with the mercury were made with platinum wire. By varying the height of the mercury in the reservoir, the dr0p rate of the mercury was controlled. Since the size of the mercury drop and the drop rate are a function of the current-voltage curves, these values were kept constant during the entire inves- tigation. The mercury drOp rate was maintained at 10 drOps per thirty seconds. The position of the dropping electrode in the solution in the electrolysis cell was kept as near constant as possible. Mercury used was C.P., redistilled in vacuum. There were many other factors to be considered in this work. Temperature must be considered; therefore, all determinations were made between the temperature range Zl‘to 23.0.. The presence of dissolved oxygen in the sol- utions being tested must in some cases be removed. In.any such cases, dissolved oxygen was removed by bubbling puri- fied nitrogen through the solution in the electrolysis cell Just before starting the determination. Other factors such as age of the solution, pH, buffer capacity of sol- utions, source of indifferent ion, will be considered as the results of this investigation are described. All potentials will be referred to the saturated cal- omel electrode. The iR drop across the cell was kept low by maintaining a high concentration of an indifferent ;17- ion in the solutions being analyzed. All pH determinations were made with the Cameron glass electrode apparatus. The procedure followed in making a determination was roughly as follows: The solution was placed in the cell and the dropping mercury electrode adjusted as to position and preper dr0pping rate. The potential arrangement was checked against the standard cell. The E.M.F. supplied to the cell was regulated by the position of the slide wire resistances. Voltage and corresponding current readings were taken over the desired voltage range. It was necessary to obtain sufficient readings so that the entire curve could be accurately determined. Readings were taken as rapidly as possible since equilibrium at the dropping mercury electrode is established very quickly. The current readings were plotted against the corresponding voltage readings and the current-voltage curve of the substance in the cell was obtained. B. The Determination of Sugars. In working with sugars at the drOpping mercury elec- trode the following factors were considered: 1. Buffer capacity of the solution, 2. Effect of dissolved oxygen, 3. Effect of toluene, h. pH, 5. Age of solution. Sugars investigated included the hexoses: fructose, Pfanstiehl C.P., Sp. Rot. ~91; glucose, Pfanstiehl, C.P. anhydrous, Sp. Rot.+ 52.53 and the disaccharides: maltose, Pfanstiehl, C.P. Sp. Rot.1~13l‘and also Eastman No. 15h, lactose, Baker and Adams reagent quality, and also Eastman quality. Since glucose was to be used during later investigations, the pr0perties of the Pfanstiehl source of this sugar at the dropping mercury electrode were checked against U. S. Bureau of Standards glucose, Sample No. hi. The two sugars proved to be almost identical in every respect as far as this investigation was concerned. Unbuffered solutions of glucose or fructose showed a distinct decrease in pH during the course of a determin- ation. Using a volume of 15 m1. of sugar solution, the pH showed a decrease of approximately 0.5 units when the determinations were made near the neutral pH range. The current-voltage curves obtained from the unbuffered sol- utions were irregular, and consistent values could not be obtained. Phosphate buffers were used in the greater part of the investigation.~ Lithium hydroxide and phosphoric acid of C.P. quality were used in the preparation of these buffers. Lithium hydroxide was selected because of the -19- high reduction potential of the lithium ions. Figure A represents the pH of solutions of varying composition. To provide for constancy, the concentration of the buffer was kept the same in all solutions investigated in which buffer capacity was desired. Concentration of lithium in the buffer was maintained at 0.1 M.. This high concen- tration of lithium also served as a source of "indifferent electrolyte". Typical curves as obtained from varying concentrations of fructose in buffer solution are shown in Figure 5. These determinations were made within four hours after the fructose solutions were prepared. Glucose curves obtained under similar conditions to those of the fructose are illustrated in Figure 6. Because of the greater activity of fructose at this electrode system, it was necessary to make determinations on glucose using more concentrated solutions in order to obtain significant current-voltage curves. One undesirable feature of the phOSphate buffers was observed throughout the entire investigation. The addition of glucose to this buffer system always lowered the eff- ective pH of the buffer. For example, a potassium hydroxide- phOSphoric acid or a lithium hydroxide-phosphoric acid buffer solution of pH 7.0, when made 0.5 M. with respect to glucose, had a pH of 6.9. If the buffer solution was made 1.0 M. with respect to glucose, the pH dropped from 7.0 to 6.8. For this reason the buffer solutions were made m/ of o-IM 00/! «(Jul A 20 a.) Hang—14° ‘ ._ g .a 4/3» a ' fi 7‘ f_‘b/9:r (In rycgut‘ffl‘wfi- "‘°“ (bf/(u 7"“ ”((70 tppjrfrr /.= a-os’M Fr-J'o'n "" 04M 1., o//- ”3’04 buli’er- I": 7-0 My: ”t'}1/‘ /J" Mitfo- lmfficrcr (In-g.) ?- 0-02M {ruins In. [fimva Pl 3 7-0 14/”: 425’ m-l- 3- 0w M Fm 2:". In Jar/3’" fluid W” : 23 M4 . 4390/1- IJ/Iy lain oar/)1. [0,9, 70 " VO/fiJQ. ,0)qu 5‘ [ntfit‘ (“""'7l'%//1)¢. (an/cs" (’(arffn ’- "_’ 6/acose oil/03.»;- In} Zldfl—flgflo Agfir ffl «77-0 1‘ [0’1 Jo/fifiom “(#3244 Murray/1'"! 2-/""1 fOIs In». W”' 1'0 M4 3' 4-5/7 J. ,- fies w.” ’.f,,..4. 4- 0.5244 3./.1$.... V”: ‘.II mu. 5- 0-25MJo/w75’... Mll- 'd-Ilh-c- ffx—lld l/ Qv-I-ar Effigy [fa/{4V 14/51.- -———.—-.-.i ,r/Jan 5'. 67....“ [arren/-%//*j< (“7’35 __-_ _ #4...-——~ -...__.. _ _ _W -20- at a higher pH than was desired so that after the addition of the glucose the resulting solution was of the desired pH. The glucose, when added to water solution, pH 7.0, did not lower the pH. Since the hexose sugars are known to form hexose phosphates, glucose was analyzed in the presence of other buffer systems. The glucose curves obtained in a lithium hydroxide-lithium citrate buffer solution were almost identical with those obtained in the phosphate buffers. This proved that the presence of the phosphates was in no way reaponsible for the sugar curves obtained. Glucose in buffer solutions and also buffer solutions alone were analyzed both in the presence and absence of dissolved oxygen. The effect of dissolved oxygen is shown in Figure 7. The oxygen curve does not interfere with the glucose curve, as is illustrated. As it was necessary to keep the protein solutions used in later investigations saturated with toluene, the effect of toluene on the glucose curves was determined. Identical glucose solutions were determined at the dropping mercury electrode both in the presence and absence of toluene. The presence of the toluene exerted no noticeable effect on the current-voltage curves obtained. The amount of the reducible form present in glucose solutions was shown by Cantor and Peniston (12) to be a function of the pH. Glucose solutions varying in pH from 6.5 to 7.5 were determined in this investigation and the ‘ Wh' ’F' ‘V‘T-u- 01],". (one )6”: “L y'fl')‘ .II: Af/Ulo“ 0‘53.” ffcfcnf VI//'J‘ 70y! l'0- /»f ”/7: ’5 upper :o/aflonf 9/Dhlt. (Inc. 5:: - I'd n/f. [3 - 2’ “If: our, 5!“ 01,7“! (am (out. (aflfd/ if: —/0 V. ”I A, w. yyaa, my" ”.4, LI: (Ll/v] p”: 70 asWy/um. II‘I Lily-”3P0, “fly" ”I: In {Out 50/1760“ 0" 5+ =-/( “/4 04”“ - 02 Trun/tJ Sun. So/O/“K t: (:‘2'014/[1 (Ion - 0! fra- at" my. .__ .-_ ,_ _ ”jar: 7. 6700:: ("7“, Ff"), ‘f Oijcq ww-__ 6% use. . 4.5” [A 4””- l6”? [It/yer: oft/Hy”? f” /- ,0//- 74 My. ”'71/' /‘2 0m". 'h’f T E-M- 7.0 \: W'VC //¢Ilz/-’ 0.3 47.1. E 37. fill: 64 6 Mre lfg‘/—‘ 0'6‘5’ fl... ’- "/53 Va”: { £{:-/‘1 16/5 (3) K/fgc Aj‘lrc 8. [ff—eff of,” an {/uc'ofc (til/(J. -21- current-voltage curves are illustrated in Figure 8. It is clearly evident that the height of the current-voltage curves increase with increasing pH. The aging effect of glucose upon the height of the current-voltage curves was thoroughly investigated. Deter- minations were made on the solutions immediately after the glucose was dissolved and also at varying time intervals. Most of the determinations were made on more concentrated glucose solutions at pH 7.0 in the presence of lithium hydroxide-phOSphate buffers. Some investigations were also made at a higher pH. Figures 9 and 10 represent the aging effect of glucose on the current-voltage curves. There was a rapid decrease in the wave height in the first few minutes and then the wave height approached a constant value. An attempt was made to correlate the change of the wave height with age of solution against mutarotation velocity. The results are shown in Figure 11. The disaccharides, sucrose, lactose and maltose, were determined at this electrode system. These sugars were determined in the presence of lithium phosphate buffers and the investigations were carried on similar to those reported for the hexoses. Figure 12 is a graphic illustra- tion of the behavior of some of these sugars. Lactose at pH 7.0 shows no distinct wave, but the voltage position of the rapid change in current is different from that of the buffer solution alone. At higher pH (8.0), the lactose gave some indication of a distinct current-voltage curve Corn-7" (l;f-fla/- /-o MJ/uolv. ml [Mm/6,0,, ‘uflr‘r, fly-'10 l‘fOlr 56mm," 0” VIII-'2'? "I... 2" 34 luv-0:70! 0” W”‘ 2/ non- 3‘ ,5"?! by W-fl t /-9 M4- 45” ha". "In [IIH'FJNII Ahfl’cr. P”: 7-0 #- Sol-ton . 2- 8 hc-qf'.‘ o/J ,‘rl ”cal/3 [f ””1" cuflonf .5 ' 50/11. 30 pap-7:! a H W”‘ = Nita. 4.- 65m“. 24am: ./J W”: [I ".0. . 77F. fgu'r‘ug. (“of of J , AJ¢ 0!! 670010. t'arn- 'Mflyc rower ' w- A5M /an:c It.» ' («flu/3M, Mr”, p/No - /-.r./;/,.. J-MM-Z, .A/ 14/3" l/uj'l/ : /26' mtr. 65-,"- 2- Jul" 30 h’lQ/ll' a/c/ ”I”: /02./fl( . 3- So/q. 20 A." 01/ W H? die? m «- '0.5H)/“(01¢ [K ZIJH'MPIY “$7. (pl/:7" f' Jolt/Ion 1" hut-Z! O// Wort. ”(717,: /'6‘6’ ”Mirna-v.1. \5'. 50/“. {Alufl O/J WM = [3607.4- 6'50“! . June I! 445’- fit/Uni] Mill] 70/V‘QG “VII/'3 [3/ mv (. Var, .. _._..- _ —..__. , ”Jun /0, [yet]. affljc an 6’7”.“ (urn-f'M/z/t [:7fo _- _._ _ w__ _ _ 62".? Ah... Me"? i y ”'9‘” ”(7%:— - 2:..1 , ':‘__ a — // . ”‘15 353W sci-"g" -22- on about the same position on the voltage axis as glucose. This was checked several times using lactose of different qualities. Sucrose was almost non-reducible at this elec- trode system (Figure 12). Maltese solutions show a rapid rise in the current at a more positive value than the buffer solution alone. The position of this rapid change (Figure 12) was somewhat dependent upon the maltose concentration, and different qualities of this sugar also showed a somewhat different effect. C. Proteins and Related Compounds. The experimental procedure as followed in this inves- tigation was carried out in three parts. The first part consisted of the characterization of proteins in buffer solutions only. The second part was a study of certain of the amino acids and ribonucleic acid. The final work on proteins constituted a study of the Brdicka "protein wave" or the current-voltage curve as obtained from solutions of protein in the presence of cobalt salts. Proteins used were egg albumin and casein. The casein was of Merck quality (according to Hammarsten). The egg albumin was prepared according to the method of Keckwick and Cannan (uh) and the modification of Westfall (AS). Dialysis of the preparation was carried out under reduced 09- - Yo”: m___ Apgrg13_ QItudu-olcr -I-C 4.7 I - O — - I V0”! I H -).. -2 '6 4-1 +0 4-: -24) -24 I: )erc {3. F” Alla-i1 Jo/ofioqf OI¢O ~01 -l-3 .14 . Volt! pressure until the diffusible ions, etc. were removed to the extent that the solution gave a negative test for sulfates upon the addition of barium chloride. The stock solution of albumin was kept saturated with toluene and in the refrigerator throughout the investigation. Protein concentration was determined by the micro Kieldahl pro- cedure according to the method of Niederl and Niederl (ho). At regular intervals the stock solution was filtered and the protein concentration redetermined. 388 albumin solutions were analyzed in the presence of the usual lithium phosphate buffer. The dissolved oxygen was not removed from any of the protein solutions examined. Age of the solution could not be considered in this phase of the work. The effect of protein concentration was studied and the behavior of egg albumin solutions at pH 7.0 is shown in Figure 13. The egg albumin did not show any indication of producing distinct current voltage curves. This protein did, however, result in a shifting of the rapid rise in current to a different position on the voltage axis. The more concentrated protein solutions showed this effect at more positive values than the more dilute solutions. Figure 13 illustrates this protein effect. The effect of pH on the protein current change Just described was investigated. Egg albumin solutions of vary- ing pH were analyzed by this method. With concentration the same, solutions were determined over a wide pH range. The results of some of these investigations of effect of “Eh- pH are illustrated in Figure 13. The position of the protein reduction effect was a function of pH as well as concentration, being at a more positive value at lower pH values. Similar investigations as those carried out on egg albumin were extended to casein. The results obtained were very much like those already reported for egg albumin. Investigations were carried out on several amino acids. Determinations were made for each amino acid at several pH values. All solutions of amino acids were unbuffered and were maintained at 0.1 M with respect to potassium chloride. Quality of all amino acids used was Pfanstiehl C.P.. Concentration of alanine and leucine solutions studied was 0.1 M and tryptophane solutions were 0.05 M. The results of the amino acid determination are shown in Figure 1h. TryptOphane was found to produce a distinct current voltage curve. Leucine and alanine showed a re- duction effect, the position of the effect depending upon the pH. A one per cent solution of ribonucleic acid in lithium phosphate buffer was determined at pH 3.5 and 7.2. The current-voltage effect of this substance is shown in Figure 1h. The ribonucleic acid was prepared according to the method of Johnson and Harkins (h7). This part concerned a study of the Brdicka protein waves. This wave is produced by proteins in the presence of cobalt ions. Concentration of cobalt chloride (C.P.) in all solutions analyzed was 0.005 M. The solutions were 41 -l-e roll; "‘7 . - -l'l - ---M-H— Flier; H A~:oo 40/: ca-J plélnou/Lu 0:11 0 -lO on | __..‘__,_. (urn. f 5:41!" V fo/a. an I/ 2- /-o 71;, , o/(..,.-. I ll! "' 'IIOI-[ar /— Mot/fie (I; - omit/11,4 ”:14 fi“"’ J’h-a. 3’ 0‘5,£}) 4/56."; 5! a II. a, 4/1.- . 510' ”75),, Al‘nbcn (OI/’rJna Io/efuqr) 0'4“” ('0 dz 0',” lief) (0H €{:-/'2'V "I'V‘ I‘bbrc Ar. 6; A/‘bnu. “/0": IR 610/, ("/n/c 50A” to!!! —‘ "‘_—"r . w—v f“ _ ——' +— -25- also maintained 0.1 M with reapect to ammonium chloride so as to provide a source of indifferent ion and for buffer capacity. The pH of the solutions was controlled by the addition of varying amounts of ammonium hydroxide. . Preliminary determinations were made on the supporting solution containing the cobalt chloride and the ammonium chloride only. This was done so that the individual effect of these constituents could be established. Cobalt exhibited a very marked maximum (Figure 15). The ammonium ions showed reduction at about ~2.0 volts. When egg albumin was added to this solution, this maximum was suppressed and a normal current-voltage curve for cobalt was obtained. The egg albumin in the presence of the cobalt ion produced' the characteristic protein wave as is shown in Figure 15, the height of this wave being pr0portional to concentration of protein added. The protein waves were determined over a wide range of concentration using egg albumin solutions. All curves obtained were in close conformity with those as shown in Figure 15. Determinations were made over the pH range 6.0 to 8.0. The protein wave obtained from solutions of higher pH appeared to show a slight decrease in height, the decrease being preportional to pH increase. This effect was so slight over the pH range investigated that it is not poss~ ible to show by illustrations. A solution 0.5 per cent with respect to egg albumin at pH 6.0 would show a curve very nearly like a similar solution at pH 8.0. -26- D. Combinations of Sugars and Proteins. This study was divided into two parts. The initial work was a determination of the effect of proteins upon the current~voltage curves of sugars. The second part was devoted to a study of the influence of sugars upon the protein wave. Glucose was used in the major portion of the study of possible sugar-protein combinations. Fructose could not be used because the current-voltage curve from this sugar was found to be in nearly the same position on the voltage axis as the reduction current resulting from protein sol— utions. This can be verified by examining the curves in Figures 5 and 15. Glucose has a lower reduction potential than fructose and was used as the sugar source. In studying the protein effect upon glucose, first the current voltage curve of a glucose solution was estab* lished; second, the reduction current of a protein solution was established; and third, the sugar and protein were mixed and the combination effect determined. Figure 16 shows a glucose curve, the protein solution effect, and combination curves. The protein reduction current obtained from solutions of higher concentration interfered with the normal glucose curves. In such cases, the effect of the protein upon the sugar curve could not be determined. When protein solutions of lower concentrations were added to the glucose, distinct sugar curves were obtained as shown in Figure 16. From these curves it was pOSsible to determine (‘ffoaf' )- 5 . ,1 JIM 6- '1' «MW l-rm- 7. 4-5» l“... {/Ma“ 2.1:?- 2” .4 j [7 f “”511 4"” 041:7! "hm-4 I} . . ‘mu. .’ . 5’”: 0-79 And. 2' 0/1 e l/‘bI-M; an d-{Mj/0nft 8- [lfijrfilolg 3" od/Z I}, 4/‘60-4 ""fl(’, Alva-av [-5, f}, l/[bo-aq W.” . _. 0", pl. f/‘Colc (61" Cu”, 6'. 0.5” /.,',f¢, ‘1 .7th 4‘73 )1 "“"4 (‘ ’/ Io/ot." “L (2) 0) (1) W‘fls 0") MI. Ila/L ”’fl’y I‘m" ) ' (4) M: 7.4 {67 (c) (7) (g) ((r-mu fiz-MN’ qfl-ljy I/o/fijc 5);" ’6‘. [m‘/ #6, A/Jg~,‘ on j/“COI‘ (’firftaf— “/foj‘ (lift/Cr /' 0-57 ()7 lab-'4 3— 0.57!” 4/6.“; 5- 4-57“); 4/04"}; 2-0-5H,‘nl¢ [A d‘/M}/br9:c /-0M ,/‘(01& ‘Ufltf fi‘fl.‘ a", 4' 05,67 l/l‘hua' {din/a”... 3 l-J'MJ an“. 0-005/‘1 C. (/._ /A’: {-I-r 4-”! Mi. cl ) ' if. 4.2{1’ Vol/.1. fl).r¢ /7. chcf of g/acora ”I £1; 4/590"; W“ l, - , , _.i .. -27- the effect of the protein on the sugar curve and also to determine if evidence was conclusive enough to indicate a possible protein-sugar combination. Presence of the egg albumin did indicate a lowering of the sugar curve. This same effect of proteins upon sugar curves was studied at pH 7.5. The results obtained were very much like those shown in Figure 16. Solutions of higher protein concentration completely covered up the glucose current- voltage curves, while solutions of lower protein concen- tration gave some evidence of decreasing the height of the glucose wave. When casein was used as a source of protein, no indi- cation as to whether or not the casein had any effect on the glucose curve could be observed. In the investigations on the influence of sugars on the Brdicka "protein wave", egg albumin was the only source of protein used. The cobalt chloride and the ammonium chloride were maintained in all solutions at the same concentration as that already described. In determining the protein wave at this electrode system it was possible to make the current readings at a lower galvanometer sensitivity than that required for glucose solutions. When this lower sensitivity was employed, it was noted that the curve of glucose was almost completely eliminated. At this same galvanometer sensitivity, the protein wave was very pronounced. The glucose and protein curves shown in Figure 17 illustrate that the glucose curve -28- in no way interferes with the protein wave. Glucose was added directly to egg albumin solutions, and the current-voltage curve of the sugar—protein solution was determined. The effect of the glucose on the egg albumin was studied using: (1) various combinations of sugar, (2) various protein concentrations, (3) influence of the age of the sugar-protein solution, and (h) pH. The presence of glucose was found to reduce the height of the egg albumin waves. By increasing the concentration of glucose added, the height of the protein wave was acc- ordingly decreased. Figure 18 clearly illustrates the decrease in the egg albumin wave by the presence of glucose. The effect of glucose on the protein wave was investigated, using a wide variety of both sugar and protein concen- trations. This was done to determine if there was a point at which increased sugar concentration would produce no further decrease in the protein wave, and also to determine the minimum amount of glucose that would cause a change in this wave. The results are shown in Figure 18. The more concentrated solutions of glucose almost completely supp- ressed the egg albumin wave. Glucose solutions below 0.1 M showed little effect on this wave. The solutions of egg albumin and glucose were examined immediately after mixing and also at varying time intervals thereafter. The age of the solution seemed to have little effect upon the height of the protein wave. The solutions of twenty four hours standing did show a slightly lower Carreaf' F" AIIOQM' Mic. /- d'/ 7 (J7 A/IQOOOO' 2° l-Iflj/burc IA log, J‘(“/I.I\ 2V 5:; 4/1..“ MO" 3- "Wt... O-IM j/u (01¢, ' J'/7’ 6) A/‘DAIK f offlj/ «are. (4) (AI/ :o/b/hnf aw 3'” C. d; o-I/v mg (I Pl; : {-0- 7-5' \ p)". I8. emif .f 7%...“ 0h m ,5, 4/5.»; K/orw. I- /- 4-515” Allen/it 2° 057537 Allen’s J'IMj/auro. mm, . 3. 0.5x [3, l/Ibhlh. 0'9!” truer-n, ¢. 0575’), A/‘baua 0'5” X’v/olc. :F' 0!, I}, 4/10.“: MM m'. . .‘n. (C// fi/htouf ’00!” (‘0 (It ‘O'IMWIvC’ flirt-7) [98" /’. Inf/Hun: «3/52;er on #9. £7] 4/561-13‘ MVL -29- protein wave when compared with solutions of only a few minutes age. 'This change was not enough to show by 111- ustration. The influence of glucose on egg albumin was investi- gated over the pH range 6.0 to 8.0.. The protein waves obtained were almost independent of the pH of the solution. Sucrose, Pfanstiehl C.P., xylose, Pfanstiehl C.P., and mannose, Eastman quality, were used in place of glucose, giving resluts as illustrated in Figure 19. -30- DISCUSSION The current-voltage curves obtained from the sugar solutions are of unusual interest. These curves indicate that sugars exist in a reducible form,this form being responsible for the curve. The height of the curves is a function of sugar concentration. Cantor and Peniston (12) in a study of the aldose sugars by this method, re- ported that glucose exists in a reducible form to the extent of about 0.02u per cent of the total sugar in sol- ution. They attributed this as being due to an aldehydo form. From the curves obtained from glucose and fructose solutions of varying concentrations, it can be observed that fructose is a more active sugar at this electrode system. The height of the current step for a 0.5 H glu- cose solution is about 1.0 micro-ampheres, while that of a corresponding fructose solution is over 100 micro- ampheres. The height of this current step is a direct measure of the concentration, and indicates that fructose exists in a reducible form or forms in a concentration about 100 times greater than a corresponding glucose solution. If 0.02h per cent of the glucose in solution is in a reducible form, then fructose would exist in a reducible form to the extent of 100 times this value, or approximately 2.h per cent. This is based on the assump- tion that the value for glucose reported by Cantor and Peniston is correct. The half-wave potential of fructose is shown to be -l.8 volts, while that of glucose is more positive, being at approximately -l.60 volts depending upon concentration and pH. These values are in close agreement with those reported in the literature._ The amount of the reducible form of glucose is a function of pH. This was shown by Cantor and Peniston (12), and their results are confirmed in this work. The height of the.current-voltage curves from glucose solutions shows a rapid increase at pH values above 7.0, indicating that the amount of the reducible form is greater. Cantor and Peniston stated that the aldehydo form was a possible intermediate in mutarotation. From the reaction Q3153? it appears that the amount of this inter- mediate form should be some function of mutarotation velocity. When the glucose used in this investigation is placed in solution, the specific rotation shows a rapid decrease until the constant rotation off 52.5'is reached. This decrease in positive rotation would indicate that the sugar exists in the alpha form when placed in the sol- ution, the equilibrium between the alpha and beta forms being gradually reached. The intermediate form or forms should then be at a relatively high concentration immed- iately after the alpha form is dissolved, because the alpha form is rapidly changing to this intermediate in the establishment of the equilibrated solution. As the inter- mediate changes to the beta form and the equilibrium is -52- established, the concentration of the intermediate should gradually decrease until a constant value is reached. In other words, the amount of the intermediate or reducible form is roughly a function of mutarotation velocity. This was actually observed in a large number of cases. The height of the current-voltage curves, which is a function of the amount of reducible form or forms present, is greatest immediately after the sugar is dissolved. Further, the height of the curve shows a gradual decrease until a constant value is reached. This effect is com- pared with mutarotation velocity in Figure 11. Mutaro- tation at PH 7.0 almost reaches a constant value after one hour. The wave height also approaches a constant after fifty to sixty minutes. Due to the difficulty in getting accurate readings by the dropping mercury electrode method, small changes that occured after thirty to forty minutes could not be detected. The aging effect of glucose upon the current-voltage curve does indicate that the intermed- late or reducible form is a function of mutarotation vel- ocity. Cantor and Peniston show the relation between muta- rotation velocity and the reducible form. They assume that the aldehydo form is an intermediate in mutarotation, and show that sugars having a more rapid mutarotation velocity exist to a greater extent in the reducible form. They did not show, however, that the amount of the intermediate form in any one sugar solution decreased with mutarotation. -33- The extension of this same investigation to other sugars should prove to be of interest, although some obstacles may be encountered. Almost all of the common sugars have a more rapid mutarotation velocity than glucose. This rapid change may be difficult to follow by the drOpping mercury electrode method. The investigation of the disaccharides indicates that these sugars possess some activity at this electrode system. Lactose appears to form a distinct curve in about the same position on the voltage axis as the glucose current-voltage curves. The purity of this sugar used was not very high, and this effect may be due to an impurity. Maltose shows a rapid current change at a position on the voltage axis at a more positive value than the buffer solution alone. Different qualities of maltose show this reduction at different points. The best maltose used, Pfanstiehl C.P., has a specific rotation of-fljl: or S'below the value recognized for this sugar, indicating the possibility of the presence of impurities. Sucrose did show some activity at this electrode system, but not enough to be considered of any significance., If the reducible form of a sugar is the intermediate between the alpha and beta forms, it seems 10gical to assume that any sugar showing mutarotation should exist in the intermediate form. If this be true, then lactose and maltose should both be capable of showing current-voltage curves to about the same degree that glucose does, while sucrose should show no curve. -314- Proteins in the presence of the phosphate buffers did not show a distinct current-voltage curve although they did produce a very interesting effect. The presence of egg albumin in the buffer solution causes a rapid rise in the current at a position on the voltage axis more positive than the buffer alone, the value being more posi- tive in more concentrated solutions. The position of this current change is almost an exact function of protein con- centration, and could possibly be used in analytical det- erminations. The position on the voltage axis of this protein effect is also shown to be a function of pH, being at a more positive point at lower pH values. It is of interest to note that the amino acids, leucine and alanine, and also ribonucleic acid show a similar effect. Ribonucleic acid shows this reduction at even more positive values. The pronounced effect at lower pH values as exhibited by these compounds may be due in part to a hydrogen discharge or reduction. TryptOphane, when determined by this method, gives a distinct current voltage curve. A procedure for the quantitative estimation of this amino acid could poss- ibly be deveIOped. More work of this type from the stand- point of the proteins and amino acids is necessary. The Brdicka protein wave need only be mentioned. This is a well known effect, and many proteins have been investigated using the Brdicka technique. The effect is undoubtedly due to a catalyzed reduction of hydrogen by the protein, the cobalt, or possibly a protein-metallic complex. The protein wave is shown to be a function of the concentration. However, the wave height does not increase prOportionally to concentration. The study of sugar-protein combinations produced some results of unusual interest. The presence of egg albumin is shown to decrease the height of the sugar current-voltage curves. This is very difficult to observe in more concentrated protein solutions, as the protein reduction current interferes with the normal sugar curves. When lower protein concentrations were used, their influence on the sugar wave could be studied. The presence of 0.01 per cent egg albumin in a 0.5 M glucose solution reduces the height of the sugar curve by twenty five per cent. A 0.05 per cent concentration of the protein reduces the glucose curve by approximately fifty per cent. The fact that the presence of the protein reduces the height of the glucose wave can possibly be accounted for in several ways: (1) That the presence of the protein interferes with the ordinary diffusion of the glucose into the drOpping mercury cathode; thus less of the sugar is reduced; (2) That the aldehydo form of the sugar does exist and this reducible form combines with some of the groups present in the protein, such as aldehydes are known to do in the case of the proteins and amino acids. This would remove part of the reducible form from the solution, thus account- ing for the decrease in the curve; (3) That the protein -55- and the glucose undergo some "surface combination", or perhaps the glucose is adsorbed by the protein molecule. Thus the sugar is not as free to be reduced at the drOpping mercury electrode. In the investigations on the study of the influence of sugars on the protein wave, some very convincing curves were obtained. The presence of the sugar is shown to reduce the protein wave in prOportion to the amount of sugar added. This protein wave appears to be a catalytic effect, or possibly a "maximum". Certain of the sugars are known to possess the ability to suppress maxima and it may be that a similar effect occurred in this investigation. What is responsible for this sugar reaction on the protein wave cannot be explained. It is not unlikely that the sugar and protein undergo some combination, thus changing the protein so that the protein wave is reduced. It has been reported that the active sulfur groups present in proteins are responsible for this wave. Perhaps the sugars combine with, or in some way change these sulfur groups. 0f the sugars used, mannose shows the greatest influence on the egg albumin wave, xylose next, and sucrose and glucose least. The Brdicka protein wave and technique has been exten- sively studied from the standpoint of cancer diagnosis. This disease apparently causes some change in the proteins. This change reduces the height of the protein wave. If this test is to be used for this type of work, the possible -37- influence of sugars and other interfering material should always be considered. It is even possible that during the course of the cancer growth substances are formed which affect the protein wave as the sugars do. -33- CONCLUSIONS 1. The current-voltage curves for several of the common sugars have been studied under various conditions at the dropping mercury electrode. 2. The relative amounts of the reducible forms of glucose and fructose solutions have been shown. 3. The current-voltage curves of glucose have been shown to decrease with age of solution and the relation of this change to mutarotation has been indicated. h. Proteins and related compounds have been inves- tigated under various conditions and their effect at this electrode determined. 5. Egg albumin in the presence of cobalt chloride has been demonstrated to produce the characteristic "protein wave" as reported by Brdicka. 6. Egg albumin has been shown to reduce the height of glucose current—voltage curves, and the possibility of a sugar-protein combination indicated. 7. The presence of several of the common sugars has been shown to cause a change in the egg albumin wave. -39- BIBLIOGRAPHY l. Rimington, C. Proteins and amino acids. Annual Rev. Biochem. V., 137, (1936). Stanford U. Press, California. 2. Smoler, I. Electroreduction of acetaldehyde at the drOpping mercury cathode. 'Chem. News, lh2, 97. (1931). 3. Shikata, M. and ShoJi, K. Microanalysis of reducible substances in fermentation products. Mem. Col. Agr. Kyoto Imp. Univ. No. h, Art. III, 75, (1927). C.A. 22, 2895, (1928). h. Smoler, I. Polarographic studies with the drOpping mercury cathode. Part XVI. The electro- reduction of acetaldehyde. Collection Czech. Chem. Commun. 2, 699, (1930). 5. Heyrovsky, J. and Smoler, I. PolarOgraphic studies with the dropping mercury cathode. Part XXX. The electroreduction and estimation of fructose and sorbose. Collection Czech. Chem. Commun. h. 521. (1932)- o. Sanders, K. and Zimmerman, B. Polarographic measure- ments in sugar research. Listy Cukrovar, A7, 377, (1929). C-A- 23. h09h. (1929). 7. Sandera, K. and Zimmerman, B. Polarographic measure- ments in sugar research. Zukerind, 2., Cech- oslovak. Rep.. 53. 375, (1929). 8. Zimmerman, B. Methodical remarks on the application of polarography in the sugar industry. Listy Cukrovar, ha, 277, (1930). C.A. 2h, 2628, (1930). 9. Zimmerman, B. The influence of the kind of sugar and non-sugar on the change of the oxygen maxima in polarOgraphic waves. Listy Cukrovar, h8, 367, (1930)- CoA. 2h, 3917, (1950). 10. Sanders, K. Polarographic studies with the drOpping mercury cathode. Part XII. Investigations on the beginning of the decomposition of sacc- harose. Collection Czech. Chem. Commun. 2,363, (1930). C.A. 2h, h206, (1930). ‘MO- 11. Semerano, G. Polarographic research in the sugar in- dustry. Ind. saccar. ital. 29, 190, (1936). c.A- 50. 6971. (1936). 12. Cantor, Sidney M. and Peniston, Quintin P. The reduc- tion of aldoses at the drOpping mercury cathode: Estimation of the aldehydo structure in aqueous solutions. J. Am. Chem. Soc. 62, 2113, (19h0). 13. Heyrovsky, J., and Babicka, J. Polarographic studies with the dropping mercury cathode. Part XIII. 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