CRYSTALLINE TRYPSIN : II. GENERAL PROPERTIES

A method is described for isolating a crystalline protein of high tryptic activity from beef pancreas. The protein has constant proteolytic activity and optical activity under various conditions and no indication of further fractionation could be obtained. The loss in activity corresponds to the decrease in native protein when the protein is denatured by heat, digested by pepsin, or hydrolyzed in dilute alkali. The enzyme digests casein, gelatin, edestin, and denatured hemoglobin, but not native hemoglobin. It accelerates the coagulation of blood but has little effect on the clotting of milk. It digests peptone prepared by the action of pepsin on casein, edestin or gelatin. The extent of the digestion of gelatin caused by this enzyme is the same as that caused by crystalline pepsin and is approximately equivalent to tripling the number of carboxyl groups present in the solution. The activity of the preparation is not increased by enterokinase. The molecular weight by osmotic pressure measure is about 34,000. The diffusion coefficient in ½ saturated magnesium sulfate at 6°C. is 0.020 ±0.001 cm.² per day, corresponding to a molecular radius of 2.6 x 10^–7 cm. The isoelectric point is probably between pH 7.0 and pH 8.0. The optimum pH for the digestion of casein is from 8.0–9.0. The optimum stability is at pH 1.8.     

CRYSTALLINE TRYPSIN : V. KINETICS OF THE DIGESTION OF PROTEINS WITH CRUDE AND CRYSTALLINE TRYPSIN

The rate of digestion, as determined by the increase in non-protein nitrogen or formol titration, of casein, gelatin, and hemoglobin with crystalline trypsin preparations increases nearly in proportion to the concentration of protein, but with crude pancreatic extract the rate of digestion becomes independent of the protein concentration in concentrations of more than 2.5 per cent. With both enzymes the rate of digestion of mixtures of 5 per cent casein and gelatin is greater than would be expected from the point of view of a compound between enzyme and substrate. The rate of digestion of 5 per cent casein in the presence of 5 per cent gelatin is exactly the same as that of 5 per cent casein alone. This result is obtained with both enzymes. The digestion of casein with crude trypsin follows the course of a monomolecular reaction quite closely while with purified trypsin the velocity constant decreases as the reaction proceeds. In the case of hemoglobin the monomolecular velocity constant decreases with both purified and crude enzyme. When the reaction is followed by changes in the viscosity of the solution the abnormal effect of changing substrate concentration disappears and the reaction is in fair agreement with the monomolecular equation. The results as a whole indicate that the abnormalities of the reaction are due to the occurrence of several consecutive reactions rather than to the formation of a substrate enzyme compound.     

CONCENTRATION AND PURIFICATION OF BACTERIOPHAGE

1. A method for isolating a nucleoprotein from lysed staphylococci culture is described. 2. It is homogeneous in the ultracentrifuge and has a sedimentation constant of 650 x 10^–13 cm. dyne^–1 sec.^–1, corresponding to a molecular weight of about 300,000,000. 3. The diffusion coefficient varies from about 0.001 cm.²/day in solutions containing more than 0.1 mg. protein/ml. to 0.02 in solutions containing less than 0.001 mg. protein/ml. The rate of sedimentation also decreases as the concentration decreases. It is suggested, therefore, that this protein exists in various sized molecules of from 500,000–300,000,000 molecular weight, the proportion of small molecules increasing as the concentration decreases. 4. This protein is very unstable and is denatured by acidity greater than pH 5.0, by temperature over 50°C. for 5 minutes. It is digested by chymo-trypsin but not by trypsin. 5. The loss in activity by heat, acid, and chymo-trypsin digestion is roughly proportional to the amount of denatured protein formed under these conditions. 6. The rate of diffusion of the protein is the same as that of the active agent. 7. The rate of sedimentation of the protein is the same as that of the active agent. 8. The loss in activity when susceptible living or dead bacteria are added to a solution of the protein is proportional to the loss in protein from the solution. Non-susceptible bacteria remove neither protein nor activity. 9. The relative ultraviolet light absorption, as determined directly, agrees with that calculated from Gates'' inactivation experiments in the range of 2500–3000 Å. u. but is somewhat greater in the range of 2000–2500 Å. u. 10. Solubility determinations showed that most of the preparations contained at least two proteins, one being probably the denatured form of the other. Two preparations were obtained, however, which had about twice the specific activity of the earlier ones and which gave a solubility curve approximating that of a pure substance. 11. It is suggested that the formation of phage may be more simply explained by analogy with the autocatalytic formation of pepsin and trypsin than by analogy with the far more complicated system of living organisms.     

THE MECHANISM OF THE INFLUENCE OF ACIDS AND ALKALIES ON THE DIGESTION OF PROTEINS BY PEPSIN OR TRYPSIN

1. The effect of the addition of acid on the amount of ionized protein has been compared with the effect on the rate of digestion of gelatin, casein, and hemoglobin by pepsin. 2. A similar comparison has been made of the addition of alkali in the case of trypsin with gelatin, casein, hemoglobin, globin, and edestin. 3. In general, the rate of digestion may be predicted from the amount of ionized protein as determined by the titration curve or conductivity. The rate of digestion is a minimum at the isoelectric point of the protein and a maximum at that pH at which the protein is completely combined with acid or alkali to form a salt. 4. The physical properties of the protein solution have little or no effect on the rate of digestion.     

Northrop''s phenomenon; the digestion of proteins by enzymes in the presence of gelatin

1. Evidence has been found that Northrop's phenomenon (so called by us) is produced in the digestion of casein or hemoglobin brought about by trypsin, papain, and pepsin either crude or crystalline in the presence of gelatin. 2. Anson's and Kunitz' methods permit the measure of proteolytic activity of any protease on casein or hemoglobin substrate in the presence of gelatin, even in very small quantities and with prolonged digestion time.     

THE PRESENCE OF A GELATIN-LIQUEFYING ENZYME IN CRUDE PEPSIN PREPARATIONS

A protein fraction has been isolated from crude pepsin preparations which is about 400 times as active as crystalline pepsin in the lique-faction of gelatin. The activity as measured by the digestion of casein, edestin or egg albumin is less than that of crystalline pepsin. It is more resistant to alkali than the crystalline pepsin.     

The action of enzymes on rhodopsin

The effects have been examined of chymotrypsin, pepsin, trypsin, and pancreatic lipase on cattle rhodopsin in digitonin solution. The digestion of rhodopsin by chymotrypsin was measured by the hydrolysis of peptide bonds (formol titration), changes in pH, and bleaching. The digestion proceeds in two stages: an initial rapid hydrolysis which exposes about 30 amino groups per molecule, without bleaching; superimposed on a slower hydrolysis which exposes about 50 additional amino groups, with proportionate bleaching. The chymotryptic action begins at pH about 6.0 and increases logarithmically in rate to pH 9.2. Trypsin and pepsin also bleach rhodopsin in solution. A preparation of pancreatic lipase bleached it slightly, but no more than could be explained by contamination with proteases. In digitonin solution each rhodopsin molecule is associated in a micelle with about 200 molecules of digitonin; yet the latter do not appear to hinder enzyme action. It is suggested that the digitonin sheath is sufficiently fluid to be penetrated on collision with an enzyme molecule; and that once together the enzyme and substrate are held together by intermolecular attractive forces, and by the "cage effect" of bombardment by surrounding solvent molecules. The two stages of chymotryptic digestion of rhodopsin may correspond to an initial rapid fragmentation, such as has been observed with many proteinases and substrates; superimposed upon a slower digestion of the fragments. Since the first phase involves no bleaching, this may mean that rhodopsin can be broken into considerably smaller fragments without loss of optical properties.     

Digestive proteinases of Brycon orbignyanus (Characidae,Teleostei): characteristics and effects of protein quality

Juvenile piracanjuba, Brycon orbignyanus, in the wild consume protein from both plant and animal sources. Digestion of protein in piracanjuba begins in the stomach with pepsin, at low pH, and is followed by hydrolysis at alkaline pH in the lumen of the intestine. The digestive system in piracanjuba was evaluated to characterize the enzymes responsible for the digestion of feed protein and their composition. The gastric tissue synthesizes pepsin and the intestine tissues trypsin and chymotrypsin. Operational variables were evaluated and defined for future studies of the digestive system physiology. The enzymatic activity in the intestine and the relative concentration of enzymes were heavily influenced by the composition of the feed and the feeding regime, as detected by substrate-SDS-PAGE. Piracanjuba possess a mechanism of enzyme adaptation responding to food quality and regime, by varying the amount and composition of digestive proteases. This is a requisite study to determine the enzymes digesting protein in food and their characteristics and to gain some clues about the possible regulation mechanisms of enzyme synthesis in piracanjuba.     

FORMATION OF TRYPSIN FROM TRYPSINOGEN BY AN ENZYME PRODUCED BY A MOLD OF THE GENUS PENICILLIUM

1. A powerful kinase which changes trypsinogen to trypsin was found to be present in the synthetic liquid culture medium of a mold of the genus Penicillium. 2. The concentration of kinase in the medium is increased gradually during the growth of the mold organism and continues to increase for some time even after the mold has ceased growing. 3. Mold kinase transforms trypsinogen to trypsin only in an acid medium. It differs thus from enterokinase and trypsin which activate trypsinogen best in a slightly alkaline medium. 4. The action of the mold kinase in the process of transformation of trypsinogen is that of a typical enzyme. The process follows the course of a catalytic unimolecular reaction, the rate of formation of a definite amount of trypsin being proportional to the concentration of kinase added. The ultimate amount of trypsin formed, however, is independent of the concentration of kinase used. 5. The formation of trypsin from trypsinogen by mold kinase is not accompanied by any measurable loss of protein. 6. The temperature coefficient of formation of trypsin from trypsinogen by mold kinase varies from Q _5–15 = 1.70 to Q _25–30 = 1.25 with a corresponding variation in the value of µ from 8100 to 4250. 7. Trypsin formed from trypsinogen by means of mold kinase is identical in crystalline form with the crystalline trypsin obtained by spontaneous autocatalytic activation of trypsinogen at pH 8.0. The two products have within the experimental error the same solubility and specific activity. A solution saturated with the crystals of either one of the trypsin preparations does not show any increase in protein concentration or activity when crystals of the other trypsin preparation are added. 8. The Penicillium mold kinase has a slight activating effect on chymo-trypsinogen the rate being only 1–2 per cent of that of trypsinogen. The activation, as in the case of trypsinogen, takes place only in an acid medium. 9. Mold kinase is rapidly destroyed when brought to pH 6.5 or higher, and also when heated to 70°C. In the temperature range of 50–60°C. the inactivation of kinase follows a unimolecular course with a temperature coefficient of Q ₁₀ = 12.1 and µ = 53,500. The molecular weight of mold kinase, as determined by diffusion, is 40,000.     

ISOLATION,CRYSTALLIZATION, AND PROPERTIES OF SWINE PEPSINOGEN

1. A method is described for the preparation of pepsinogen from swine gastric mucosae which consists of extraction and fractional precipitation with ammonium sulfate solutions followed by two precipitations with a copper hydroxide reagent under particular conditions. Crystallization as very thin needles takes place at 10°C., pH 5.0 and from 0.4 saturated ammonium sulfate solution containing 3–5 mg. protein nitrogen per milliliter. 2. Solubility measurements, fractional recrystallization, and fractionation experiments based on separation after partial heat or alkali denaturation and after partial reversal of heat or alkali denaturation failed to reveal the presence of any protein impurity. 3. The properties of the enzymatically inactive pepsinogen were studied and compared with the properties of crystalline pepsin. The properties of pepsinogen which are similar to those of pepsin are: molecular weight, absorption spectrum, tyrosine-tryptophane content, and elementary analysis. The properties in which they differ are: enzymatic activity, crystalline form, amino nitrogen, titration curve, pH stability range, specific optical rotation, isoelectric point, and the reversibility of heat or alkali denaturation. 4. Conversion of pepsinogen into pepsin at pH 4.6 was found to be autocatalytic; i.e., the pepsin formed catalyzes the reaction. Conversion of pepsinogen into pepsin is accompanied by the splitting off of a portion of the molecule containing 15–20 per cent of the pepsinogen nitrogen.     

PURIFICATION AND CRYSTALLIZATION OF DIPHTHERIA ANTITOXIN

Purified preparations of diphtheria antitoxin have been obtained by digestion of the toxin-antitoxin complex with trypsin, followed by fractional precipitation with ammonium sulfate. The various fractions obtained in this way are all 90 per cent or more precipitated by diphtheria toxin but combine with different quantities of the toxin. The fraction precipitated between 0.33 and 0.5 saturated ammonium sulfate is homogeneous by electrophoresis and ultracentrifuge but does not have constant solubility. A small amount of a more soluble fraction has been obtained which does have constant solubility and satisfies the criteria of a pure protein. This protein crystallizes readily in poorly formed thin plates. It is very unstable and reverts to a less soluble non-crystallizable form. It has a sedimentation constant of 5.7 x 10^–13 and a molecular weight of 90,500. It has an antitoxic value of 700–900 flocculation units per mg. protein nitrogen and has an antitoxic value by the protection test of about 700 units per mg. protein nitrogen. The precipitation range of the purified antitoxin with purified toxin is much wider than that with crude preparations.     

Studies on the Acid-protease of Paecilomyces varioti Bainier TPR-220

The crystalline acid-protease of Paecilomyces varioti Bainier TPR-220 is most active toward casein as substrate, at pH 3.0 and 60°C, and stable at pH 3.0 to 6.0 below 40°C. The enzyme decomposes protein molecules into smaller fragments than pepsin does and is inhibited by p-chloromercuri-benzoate, monoiodoacetate, sodium lauryl sulfate, iodine, potassium permanganate, N-bromosuccinimide, bacitracin, nitrofurylacrylamide, and Hg⁺ ion, but affected neither by metal ion except Hg⁺ ion, nor metal chelating agent, soy bean trypsin inhibitor, potato-protease inhibitior, cysteine, diiso-propylfluorophosphate, cyanogen bromide, and heparin. The presence of Ca⁺⁺, Co⁺⁺, Cu⁺⁺, Mg⁺⁺, Sr⁺⁺, and Zn⁺⁺ ions prevents heat inactivation of the enzyme.     

COMPARATIVE HYDROLYSIS OF GELATIN BY PEPSIN,TRYPSIN, ACID,AND ALKALI

A comparison has been made of the relative velocity of hydrolysis of the various peptid linkings of the gelatin molecule when hydrolyzed by acid, alkali, pepsin or trypsin. It has been found that: 1. Those linkages which are most rapidly split by pepsin or trypsin are among the more resistant to acid hydrolysis. 2. Those linkages which are hydrolyzed by pepsin are also hydrolyzed by trypsin. 3. Trypsin hydrolyzes linkages which are not attacked by pepsin. 4. Of the linkages which are hydrolyzed by both enzymes, those which are most rapidly hydrolyzed by pepsin are only slowly attacked by trypsin. 5. Those linkages which are attacked by trypsin or pepsin are among the ones first (most rapidly) hydrolyzed by alkali. In general it may be said that the course of the early stages of hydrolysis of gelatin is similar with alkali, trypsin, or pepsin and quite different with acid.     

THE KINETICS OF TRYPSIN DIGESTION : I. EXPERIMENTAL EVIDENCE CONCERNING THE EXISTENCE OF AN INTERMEDIATE COMPOUND.

1. The rate of hydrolysis of a casein solution by trypsin is not affected by the addition of gelatin. The trypsin, therefore, is not combined with the gelatin unless there is a separate enzyme for casein and for gelatin. 2. The presence of casein protects the gelatin-splitting power of trypsin from heat inactivation, and the presence of gelatin protects the casein-splitting power from heat inactivation. 3. It does not seem possible to account for both the above results by the assumption of an intermediate compound between enzyme and substrate, since, in order to account for the first result, a different enzyme must be assumed for each protein, while, to account for the second result, it must be assumed that the same enzyme attacks both.     

AN ATTEMPT AT PEPTIC SYNTHESIS OF INSULIN

1. Synthesis of plastein from the products of peptic hydrolysis of small quantities of egg albumin can be demonstrated with amorphous or crystalline pepsin. 2. Synthesis of plastein from the products of peptic hydrolysis of amorphous or crystalline insulin can be demonstrated with amorphous or crystalline pepsin. 3. The plastein synthesised by pepsin from the products of peptic hydrolysis of insulin is physiologically inactive. 4. The plastein formed in the insulin experiments could not be crystallised by the methods used for the crystallisation of insulin. 5. The physiological activity of insulin is not destroyed by repeated freezing (at about –50°C.) and melting of an aqueous or an alcoholic solution of this hormone. 6. No marked decrease in the physiological activity of insulin after incubation at 37°C. with pepsin at pH 4.0, in dilute or concentrated solutions, was detected.     

INACTIVATION OF PEPSIN BY IODINE : II. ISOLATION OF CRYSTALLINEl-MONO-IODOTYROSINE FROM PARTIALLY IODINATED PEPSIN

1. Pepsin solutions were iodinated at pH 5.0–6.0 until 10–20 per cent of the activity was lost and 1/20 (0.7 per cent) of the saturating amount of iodine had been introduced into the protein molecule. After alkaline hydrolysis 65 per cent of the original iodine was accounted for as mono-iodotyrosine although only 42 per cent was isolated as a crystalline product. No evidence was obtained to support the possibility that any group other than tyrosine in pepsin was iodinated. 2. Some of the properties of the crystalline l-mono-iodotyrosine were determined and compared to those of di-iodotyrosine. 3. One iodinated pepsin preparation was crystallized. The crystal form was the same as that of the original pepsin. A solubility curve of the crystals demonstrated that it was very different from pepsin and had nearly constant solubility.     

Staining Glycol Methacrylate Embedded Cartilage with Triethyl-Carbocyanin Dbtc (“Ethyl-Stains All”) With Special Reference to the Interlacunar Network

The dye, triethyl-carbocyanin DBTC, was tested for differential staining of cartilage structures. Femoral head articular cartilage from neonatal rats was processed for histology to demonstrate the interlacunar network. Sections of glycol methacrylate (GMA) embedded cartilage were stained at pH 2.8, 5.4, 6.1 and 8.0 to determine the optimal staining conditions. Only at pH 6.1 were all cartilage structures stained and the best contrast achieved. Streptomyces hyaluronidase, chondroitinase ABC, pepsin, trypsin, and pronase digestions were carried out prior to staining at pH 6.1 to evaluate the selectivity of the stain. Undigested chondrocyte nuclear chromatin stained dark purple; staining intensity was reduced slightly by pepsin or trypsin digestion. Undigested chondrocyte cytoplasm stained light blue but stained purple after hyaluronidase digestion. Undigested extracellular matrix stained light violet; staining was almost entirely eliminated by chondroitinase ABC digestion, was unaffected by hyaluronidase, and was either unaffected or increased after proteinase digestion. Staining of a narrow zone of matrix adjacent to the network was prevented by proteinase digestion while the network element appeared as a thin dark line. The network appears to be a trilaminar structure; a core element of hyaluronic acid and protein surrounded by a protein sheath. Triethyl-carbocyanin DBTC staining of cartilage offers slightly more selectivity and contrast than methylene blue, toluidine blue or safranin O. At pH 6.1, DNA, perhaps RNA, and hyaluronic acid stained deep purple; chondroitin sulfate, light violet; protein (collagen), stained very light violet if at all.     

The effect of pancreatic procolipase and colipase on pancreatic lipase activation

Intestinal fat digestion is carried out by the concerted action of pancreatic lipase and its protein cofactor colipase. Colipase is secreted from pancreas as a procolipase and is transformed into colipase by the trypsin cleavage of the Arg5-Gly6 bond during liberation of an N-terminal pentapeptide. The kinetic parameters for the lipase-colipase system compared to the lipase-procolipase system has been compared using trioctanoin and Intralipid as substrates. It was found that at pH 7.0 the Kmapp using Intralipid as substrate was the same for procolipase and colipase, 0.06 mM and 0.05 mM, respectively. At pH 8.0, however, the Kmapp were different-0.23 mM for procolipase and 0.08 mM for colipase. In a similar way the binding between colipase and lipase had a dissociation constant of 2.4 x 10(-6) M at pH 7.0, while for procolipase--lipase binding the dissociation constant was 4.1 x 10(-6) M with no significant difference. At pH 8.0 the binding between colipase and lipase was stronger, Kd being 2.0 x 10(-7) M, while weaker for procolipase and lipase, Kd being 1.0 x 10(-5) M. It is concluded that at the physiological pH value as is found in the intestine, the activation of procolipase to colipase has no influence on the hydrolysis of trioctanoin or Intralipid in the presence of bile salt.     

Hydrolysis of proteins by immobilized-stabilized alcalase-glyoxyl agarose

This paper presents stable Alcalase-glyoxyl derivatives, to be used in the controlled hydrolysis of proteins. They were produced by immobilizing-stabilizing Alcalase on cross-linked 10% agarose beads, using low and high activation grades of the support and different immobilization times. The Alcalase glyoxyl derivatives were compared to other agarose derivatives, prepared using glutaraldehyde and CNBr as activation reactants. The performance of derivatives in the hydrolysis of casein was also tested. At pH 8.0 and 50 degrees C, Alcalase derivatives produced with 1 h of immobilization time on agarose activated with glutaraldehyde, CNBr, and low and high glyoxyl groups concentration presented half-lives of ca. 10, 29, 60, and 164 h, respectively. More extensive immobilization monotonically led to higher stabilization. The most stabilized Alcalase-glyoxyl derivative was produced using 96 h of immobilization time and high activation grade of the support. It presented half-life of ca. 23 h, at pH 8.0 and 63 degrees C and was ca. 500-fold more stable than the soluble enzyme. Thermal inactivation of all derivatives followed a single-step non-first-order kinetics. The most stable derivative presented ca. 54% of the activity of the soluble enzyme for the hydrolysis of casein and of the small substrate Boc-Ala-ONp. This behavior suggests that the decrease in activity was due to enzyme distortion but not to wrong orientation. The hydrolysis degree of casein at 80 degrees C with the most stabilized enzyme was 2-fold higher than that achieved using soluble enzyme, as a result of the thermal inactivation of the latter. Therefore, the high stability of the new Alcalase-glyoxyl derivative allows the design of continuous processes to hydrolyze proteins at temperatures that avoid microbial growth.     

A PROTEOLYTIC ENZYME OF SERUM: CHARACTERIZATION,ACTIVATION, AND REACTION WITH INHIBITORS

1. Fibrinolysin-activated lysin factor and chloroform-activated serum protease of serum and plasma are one and the same enzyme, differing only in their mode of activation. 2. The enzyme as it normally occurs in serum or plasma is not inactive because of combination with serum inhibitor. It is present as an inactive precursor or zymogen and may be activated from this state by streptococcal fibrinolysin. 3. The activation of serum protease by streptococcal fibrinolysin is a catalytic reaction, analogous to the kinase activation of trypsinogen by enterokinase. Treatment of serum or plasma with chloroform apparently results in removal of serum inhibitor which may allow autocatalytic activation of the serum protease. 4. The serum enzyme differs from trypsin in its pH of optimum activity, in its reactions with specific protease inhibitors, and in its action on casein. 5. A revised nomenclature for the serum enzyme system is suggested which more accurately describes its properties than the terms in current use.