Course Of Proteolysis Research Articles

Development of technology for the production of all-purpose buckwheat malt using plasmochemically activated aqueous solutions

The result of the conducted research is the development of buckwheat malt production technology using plasma-chemically activated aqueous solutions. Buckwheat grain with high starch content became the object of research. The main technological problem is obtaining high-quality brewing malt suitable for the production of gluten-free beer. The expediency of using plasma-chemically activated aqueous solutions as an intensifier of the process of germination of buckwheat grains and an effective disinfectant of buckwheat malt has been experimentally proven. It is shown that the use of plasma chemical activation of technological solutions allows to speed up the process of moistening of buckwheat grain by more than 2 times. The energy and germination capacity of buckwheat grains, when using plasma-chemically activated aqueous solutions, underwent positive changes. The effect of increasing the energy of germination was from 8 to 14 %, and the ability to germinate was 2–9 %. The amylolytic activity of buckwheat malt was analyzed, an increase in amylolytic activity in the range of 30–77 units/g was noted. The extractability of buckwheat malt was investigated separately. This indicator increased by 2–9 % depending on the concentration of peroxides in the solution. The Kolbach index also increased by 2–10 %, which indicates an intensive course of proteolysis. The total amount of amino acids in the experimental samples increased by 619 mg/100 g. There was a significant decrease in the viscosity of the wort, which indicates the high solubility of the obtained buckwheat malt. The technology can be applied in the industrial production of brewing malt and sprouted buckwheat grain of functional purpose. The developed buckwheat malting technology will receive priority in the production of environmentally friendly buckwheat malts of universal purpose that are not contaminated with pathogenic microflora

Precision Interferometry as a New Method for Studying the Conformational State of Protein and Its Interaction with a Solvent

A new method for studying the conformational state of a protein and its interaction with a solvent has been developed. The method is based on measuring the refractive index of a protein solution using a precision laser interferometer with an error of 10–7. The interferometer was calibrated on the basis of the known dependences of the refractive index change with a change in water temperature, and the refractive index change of a NaCl solution with a change in concentration. Using the interferometer, the refractive index changes were first recorded in the course of proteolysis (during pepsin-catalyzed hydrolysis, an increase in the refractive index of solutions of bovine serum albumin and egg lysozyme by 9 × 10–6 and 2.4 × 10–6, respectively) and denaturation (an increase in the refractive index by 4.5 × 10–5 during the reaction of egg lysozyme with guanidine hydrochloride and dithiothreitol). Based on direct interferometric measurements, we have disproved the widespread assumption that the refractive index of protein solutions is determined only by concentration of the protein and its amino acid composition and does not depend on the state of protein fragmentation. The increased accuracy of the interferometer makes it possible to study the refractive index increment (dn/dc) at low concentrations of dissolved compounds ( $$ \ll $$ 1%), as well as to examine the processes leading to changes in the conformation of macromolecules in solution.

Simultaneous quantification of the degree of hydrolysis, protein conversion rate and mean molar weight of peptides released in the course of enzymatic proteolysis

This paper describes an original analytical methodology for a simultaneous measurement of the protein conversion rate, the mean molar weight of peptide and the degree of hydrolysis in the course of proteolysis by Size-Exclusion High-Performance Liquid Chromatography. Peak area of dead volume eluents reflects the non-converted protein. The protein conversion rate is thus determined by comparing the area at a given time to the initial area. The peptide signal allows determining the peptide molar weight distribution and degree of hydrolysis of hydrolysates. As a first step, the approach was tested on the hydrolysis of bovine serum albumin, lysozyme and rapeseed albumin by Alcalase 2.4L. Values of degree of hydrolysis were also determined by TNBS and pH-stat methods. Most of the hydrolysate obtained showed relative differences < 20% with the reference methods. The method was also adapted to fit the TNBS assay. 39 experimental validation tests were analyzed by size-exclusion chromatography, TNBS and pH stat methods. 90% of the validation data show non-significant differences between the degree of hydrolysis predicted and the degree of hydrolysis measured by TNBS method. Hence, the proposed methodology can be efficient to study the process of enzymatic proteolysis while minimizing time and quantity of sample assay required.

Proteolysis and lipolysis of white-brined (Beyaz) cheese during storage: Effect of milk pasteurization temperature

Beyaz cheeses were produced from milk pasteurized at 65 °C for 20 min, 75 °C for 5 min, or 85 °C for 5 min, and investigated at 7, 30, 60, and 90 days of storage. Pasteurization temperature affected significantly the parameters of total solids, fat, titratable acidity, pH, salt, and total free fatty acid of Beyaz cheese. Water-soluble nitrogen fraction (% WSN/TN), caseins (αs1- and β-casein), and individual free amino acids were analyzed to evaluate the proteolysis of Beyaz cheese during 90-day storage period. Pasteurization norm did not affect % WSN/TN levels in direct proportion to pasteurization temperature. αs1-Casein hydrolysis of Beyaz cheeses increased with elevated pasteurization temperature. As the pasteurization temperature increased, β-casein hydrolysis grade showed a slight decrease. Leucine, lysine, phenylalanine, and valine were found to be dominant in Beyaz cheeses. The total free fatty acid content in cheeses decreased as the pasteurization temperature increased. Practical applications High pasteurization temperature could be used to incorporate more whey proteins into white-brined cheeses. This process directly affects the course of proteolysis and lipolysis in white-brined cheese. Thus, innovative cheeses which have desired proteolytic and lipolytic characters may be obtained by this process.

Building phenomenological models that relate proteolysis in pork muscles to temperature, water and salt content

Throughout dry-cured ham production, salt and water content, pH and temperature are key factors affecting proteolysis, one of the main biochemical processes influencing sensory properties and final quality of the product. The aim of this study was to quantify the effect of these variables (except pH) on the time course of proteolysis in laboratory-prepared pork meat samples. Based on a Doehlert design, samples of five different types of pork muscle were prepared, salted, dried and placed at different temperatures, and sampled at different times for quantification of proteolysis. Statistical analysis of the experimental results showed that the proteolysis index (PI) was correlated positively with temperature and water content, but negatively with salt content. Applying response surface methodology and multiple linear regressions enabled us to build phenomenological models relating PI to water and salt content, and to temperature. These models could then be integrated into a 3D numerical ham model, coupling salt and water transfers to proteolysis.

Growth and Proteolytic Activity of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus in Reduced Sodium Kashkaval Cheese

Reduced sodium Kashkaval cheese was produced from cow’s milk. Mixtures of NaCl, NaCl:KCl (1:1, 2:1) and NaCl:KH2PO4 (1:1, 2:1) were used for hot brining and salting of the cheddarized cheese curd. There were no significant differences (P < 0.05) in the count of Lb. delbrueckii ssp. bulgaricus after aging of Kashkaval samples. At the end of the ripening process the counts of Lb. delbrueckii ssp. bulgaricus reached 106 c.f.u./g and the counts of Streptococcus thermophilus varied from 104 to 105 c.f.u./g. Proteolysis during ripening of reduced sodium Kashkaval cheese, initiated by the starter microorganisms Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, was studied through the changes in the levels of non-casein and non-protein nitrogen. It was observed that non-casein and non-protein nitrogen increased significantly (P < 0.05) during ripening. The amounts of non-casein and non-protein nitrogen accumulated in the studied Kashkaval samples were similar. That indicates that the partial replacement of NaCl with KCl or KH2PO4 does not cause significant changes in the course of proteolysis of Kashkaval cheese by Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus.

Identification of the Geometric Requirements for Allosteric Communication between the α- and β-Subunits of Tryptophan Synthase

The pyridoxal 5'-phosphate-dependent tryptophan synthase alpha2beta2 complex is a paradigmatic protein for substrate channeling and allosteric regulation. The enzymatic activity is modulated by a ligand-mediated equilibrium between open (inactive) and closed (active) conformations of the alpha- and beta-subunit, predominantly involving the mobile alpha loop 6 and the beta-COMM domain that contains beta helix 6. The alpha ligand-triggered intersubunit communication seems to rely on a single hydrogen bond formed between the carbonyl oxygen of betaSer-178 of beta helix 6 and the NH group of alphaGly-181 of alpha loop 6. We investigated whether and to what extent mutations of alphaGly-181 and betaSer-178 affect allosteric regulation by the replacement of betaSer-178 with Pro or Ala and of alphaGly-181 with either Pro to remove the amidic proton that forms the hydrogen bond or Ala, Val, and Phe to analyze the dependence on steric hindrance of the open-closed conformational transition. The alpha and beta activity assays and the equilibrium distribution of beta-subunit catalytic intermediates indicate that mutations do not significantly influence the intersubunit catalytic activation but completely abolish ligand-induced alpha-to beta-subunit signaling, demonstrating distinct pathways for alpha-beta-site communication. Limited proteolysis experiments indicate that the removal of the interaction between betaSer-178 and alphaGly-181 strongly favors the more trypsin-accessible open conformation of the alpha-active site. When the hydrogen bond cannot be formed, the alpha-subunit is unable to attain the closed conformation, and consequently, the allosteric signal is aborted at the subunit interface.

Proteolysis of Non-phosphorylated and Phosphorylated Tau by Thrombin

The microtubule-associated protein tau aggregates intracellularly by unknown mechanisms in Alzheimer's disease and other tauopathies. A contributing factor may be a failure to break down free cytosolic tau, thus creating a surplus for aggregation, although the proteases that degrade tau in brain remain unknown. To address this issue, we prepared cytosolic fractions from five normal human brains and from perfused rat brains and incubated them with or without protease inhibitors. D-Phenylalanyl-L-prolylarginyl chloromethyl ketone, a thrombin-specific inhibitor, prevented tau breakdown in these fractions, suggesting that thrombin is a brain protease that processes tau. We next exposed human recombinant tau to purified human thrombin and analyzed the fragments by N-terminal sequencing. We found that thrombin proteolyzed tau at multiple arginine and lysine sites. These include Arg(155)-Gly(156), Arg(209)-Ser(210), Arg(230)-Thr(231), Lys(257)-Ser(258), and Lys(340)-Ser(341) (numbering according to the longest human tau isoform). Temporally, the initial cleavage occurred at the Arg(155)-Gly(156) bond. Proteolysis of the resultant C-terminal tau fragment then proceeded bidirectionally. When tau was phosphorylated by glycogen synthase kinase-3beta, most of these proteolytic processes were inhibited, except for the first cleavage at the Arg(155)-Gly(156) bond. Furthermore, paired helical filament tau prepared from Alzheimer's disease brain was more resistant to thrombin proteolysis than following dephosphorylation by alkaline phosphatase. The results suggest a possible role for thrombin in proteolysis of tau under physiological and/or pathological conditions in human brains. They are consistent with the hypothesis that phosphorylation of tau inhibits proteolysis by thrombin or other endogenous proteases, leading to aggregation of tau into insoluble fibrils.

質量分析によるタンパク質線維状凝集体のキャラクタリゼーション

Fibrillar aggregates observed in vivo and in vitro are part of “abnormal” structures of proteins. Since many proteins are capable of forming such fibrillar aggregates that share characteristics of the cross-β structure at an atomic level, we consider the fibril structure as one of general states of proteins. It is thus profoundly important to reveal the fibril structure and mechanism of its formation. To this end, mass spectrometry is used as an essential tool by making full use of the inherent sensitivity and high information content. Especially, combination of proteolysis and mass spectrometry is a powerful technique to characterize structural features of the aggregated state of proteins. By identifying fragments produced in the course of proteolysis, it is possible to discern differences in local stability within the analyzed protein and to reconstruct the structure of the mature fibril. In this review, we explain several useful methods to characterize fibrillar aggregates by the combined use of proteolysis and mass spectrometry.

Vitellogenin-derived yolk proteins in a hybrid sturgeon, bester ( Huso huso x Acipencer ruthenus): Identification, characterization and course of proteolysis during embryogenesis

Vitellogenin (Vg) and its corresponding yolk protein (YP) products, YP1, YP2 and YP3, were isolated from serum of estrogen-treated hybrid sturgeon (bester; Huso huso X Acipencer ruthenus) and eggs from untreated fish, respectively. Vitellogenin had an apparent molecular mass of 580 kDa and appeared as two major bands corresponding to 180 kDa and 120 kDa after SDS-PAGE. Apparent molecular weights of YP1, YP2 and YP3 were 370 kDa, 88 kDa and 19 kDa, respectively. After SDS-PAGE, YP1 appeared as a main band of 110 kDa, while YP2 was resolved as a single band of 94 kDa and 29 kDa band under non-reducing and reducing conditions, respectively. Yolk protein 3 appeared as a diffuse band corresponding to 16 kDa and two faint bands below 14.4 kDa after SDS-PAGE. However, the 16 kDa band alone was observed after dephosphorylation with alkaline phosphatase. The course of cleavage of yolk proteins in bester embryos and alevins was observed by SDS-PAGE and Western blotting from fertilization onward. After hatching, the main 110 kDa band of YP1 was degraded into smaller peptides during development, while YP2 hardly showed any such structural changes. The amino acid compositions of purified yolk proteins indicated that YP1, YP2 and YP3 were bester lipovitellin, β′-component, and phosvitin, respectively.

Vitellogenesis in Aquatic Animals

Vitellogenin (Vg) is the main precursor to egg yolk proteins (YPs) accumulated as nutrients for developing embryos of oviparous aquatic species. Recent gene cloning and immunobiochemical analyses verified the presence of multiple Vgs in teleost fishes, similar to the case in chickens and Xenopus. These findings lead us to abandon the classical “single Vg model” and explore different functions of individual Vgs and their YP derivatives during teleost oocyte maturation and embryogenesis. The course of proteolysis of Vgs and their YP products appears to differ among species. Detailed characterization of the relevant proteolytic enzymes has been partly accomplished only for salmonid fishes. Investigations of the endocrine regulation of teleost Vg and YP proteolysis have only just begun. Over the past decade, much attention has been paid to Vg due to its promise as a biomarker of contaminants that mimic estrogen, which are present in the aquatic environment. Sensitive and specific assays for measuring Vg in male and juvenile fish have been and will be valuable tools for identifying environmental estrogens to which humans and wildlife are potentially exposed.

Restoration of phosphorylation capacity to the dormant half of the α-subunits of Na +, K +-ATPase

Purified kidney Na +,K +-ATPase whose α-subunit is cleaved by chymotrypsin at Leu 266-Ala 267, loses ATPase activity but forms the phosphoenzyme intermediate (EP) from ATP. When EP formation was correlated with extent of α-cleavage in the course of proteolysis, total EP increased with time before it declined. The magnitude of this rise indicated doubling of the number of phosphorylation sites after cleavage. Together with previous findings, these data establish that half of the α-subunits of oligomeric membrane-bound enzyme are dormant and that interaction of the N-terminal domain of α-subunit with its phosphorylation domain causes this half-site reactivity. Evidently, disruption of this interaction by proteolysis abolishes overall activity while it opens access to phosphorylation sites of all α-subunits.

Disulfide bonds, N-glycosylation and transmembrane topology of skeletal muscle triadin.

Native triadin is a disulfide linked homopolymer of variable subunit number. Two monoclonal antibodies (mAbs), AE8.91 and GE4.90, recognize cytoplasmic regions of triadin between amino acids 110 and 163 and at the C-terminal 34 amino acids, respectively. Triadin in intact triads is largely unaffected by trypsin, while triads whose membrane has been disrupted by hypotonicity or by treatment with the detergent Triton X-100 yield both soluble and membrane bound fragments. Soluble fragments monitored by mAb GE4.90 appear to be formed sequentially during the course of proteolysis at 28, 16, 10 and 7 kDa in the presence of mercaptoethanol. Higher molecular weight bands are observed under nonreducing conditions. A two-dimensional electrophoresis immunoblot (first nonreducing; second reducing) of the soluble fragments developed with mAb GE4.90 shows the presence of several bands which can be interpreted as containing a dimer formed by a combination of any two of the fragments of 16, 10, or 7 kDa present in the digest. MAb AE8.91 does not detect these fragments. This observation indicates that one of the intermolecular disulfide bonds is formed between the identical domains of two triadin molecules at cysteine 671. Immunoblots performed with and without mercaptoethanol of the insoluble fragments using mAb AE8.91 indicate the presence of a dimer formed between identical domains of two triadin intermolecular disulfide linkage at cysteine 270. The glycosidase endo F/N-glycosidase F changed the mobility of intact triadin in TC/triads and its proteolytic fragments detected by mAb GE4.90.(ABSTRACT TRUNCATED AT 250 WORDS)

The tryptophan synthase alpha 2 beta 2 complex. Cleavage of a flexible loop in the alpha subunit alters allosteric properties

This study explores the catalytic and allosteric roles of a flexible loop in tryptophan synthase. Trypsin is known to cleave the tryptophan synthase alpha 2 beta 2 complex in an alpha subunit loop at Arg-188. Cleavage yields an active "nicked" alpha 2 beta 2 derivative. The new results provide evidence that the alpha subunit loop serves two important roles: substrate binding and communicating the effects of substrate binding to the beta subunit. A role for the loop in substrate binding is supported by our finding that addition of a substrate analogue of the alpha subunit, alpha-glycerol 3-phosphate, decreases the rate of cleavage by trypsin. An allosteric role for the loop is supported by the finding although the native alpha 2 beta 2 complex is strongly inhibited by alpha-glycerol 3-phosphate, the nicked alpha 2 beta 2 complex is desensitized to this inhibition. The time course of proteolysis in the presence and absence of alpha-glycerol 3-phosphate is followed by sodium dodecyl sulfate-gel electrophoresis and by assays of activity in the presence and absence of alpha-glycerol 3-phosphate. We use spectroscopic measurements of the pyridoxal phosphate-L-tryptophan intermediates at the active site of the beta subunit to determine the affinity of the native and nicked enzymes for L-tryptophan and alpha-glycerol 3-phosphate. Although cleavage alters the equilibrium distribution of intermediates and reduces the affinity for alpha-glycerol 3-phosphate, it has little effect on the affinity for amino acids bound to the beta subunit. We conclude that the loop in the alpha subunit is important for ligand binding and for communicating the effects of ligand binding from the alpha subunit to the beta subunit in the alpha 2 beta 2 complex.

The proline‐activating activity of the multienzyme gramicidin S synthetase 2 can be recovered on a 115‐kDa tryptic fragment

The multienzyme gramicidin S synthetase 2 was treated with trypsin to obtain fragments capable of activating proline. Three different active fragments were detected. The course of proteolysis was simulated by using a concentration range of trypsin; the cleavage pattern indicated that one of the fragments was particularly stable. This fragment was purified and shown to have a molecular mass of 115 kDa. It was compared chromatographically, by SDS/PAGE, and enzymatically to a Pro-activating fragment produced by a gramicidin-S-negative mutant. It can be concluded that the proteolytic fragment represents a structure which is contained on a continuous part of the polypeptide chain of gramicidin S synthetase 2 and has a relatively compact structure. This provides evidence that the multienzyme gramicidin S synthetase 2 is, at least in part, constructed from functional domains. An approach towards extending these studies to other parts of the gramicidin S synthetase 2 molecule has also been devised. This work complements recombinant DNA studies in the area, providing stable functional fragments.

Phosphofructokinase from baker's yeast: properties of a proteolytically modified active enzyme form

The structural and functional stability of an active fragment of yeast phosphofructokinase (called 12 S-enzyme) obtained by limited proteolysis was investigated. In the course of proteolysis the subunits of the native enzyme (α and β) are partially degraded to forms of lower molecular weight (designated as α' and β') which is accompanied by a dissociation of the native octamer to tetramers. Upon dilution the tetramic 12 S-enzyme becomes inactivated as a consequence of its dissociation. Hydrodynamic studies suggest a two-step dissociation mechanism with the formation of dimers and monomers. Inactivation of the enzyme fails or is retarded at millimolar concentrations of either fructose 6-phrosphate or ATP. Sulphate and phosphate have a similar effect but at molar concentrations. The latter two anions are also able to reactivate the inactivated enzyme. This reactivation is not due to an increase of the ionic strength but seems to be rather specifically caused by the lyotropic properties of sulphate and phosphate. The reactivation is accompanied by a partial reassociation of the enzyme. An association-dissociation model in the presence of sulphate providing only dimers as smallest species was calculated from the hydrodynamic date. The conclusion can be drawn that the tetramic form of the enzyme is the smallest active fragment of the 12 S-phosphofructokinase.

Identification of three sites of proteolytic cleavage in the hinge region between the two domains of the beta 2 subunit of tryptophan synthase of Escherichia coli or Salmonella typhimurium.

The beta 2 subunit of tryptophan synthase is composed of two independently folding domains connected by a hinge segment of the polypeptide that is particularly susceptible to limited proteolysis by trypsin [Högberg-Raibaud, A., & Goldberg, M. (1977) Biochemistry 16, 4014-4019]. Since tryptic cleavage in the hinge region inactivates the beta 2 subunit, the spatial relationship between the two domains is important for enzyme activity. However, it was not previously known whether inactivation results from cleavage of the chain or from the loss of internal fragment(s) subsequent to cleavage at two or more sites. We now report comparative studies of limited proteolysis by three proteinases: trypsin and endoproteinases Lys-C and Arg-C. Our key finding that endoproteinase Arg-C inactivates the beta 2 subunit by cleavage at a single site (Arg-275) demonstrates the important role of the hinge peptide for enzymatic activity. We have also identified the sites of cleavage and the time course of proteolysis by trypsin at Arg-275, Lys-283, and Lys-272 and by endoproteinase Lys-C at Lys-283 and Lys-272. Sodium dodecyl sulfate gel electrophoresis, Edman degradation, and carboxypeptidases B and Y have been used to identify the several fragments and peptides produced. Our finding that the beta 2 subunit and F1 fragments have a heterogeneous amino terminus (Met-1 or Thr-2) indicates that the amino-terminal methionine is incompletely removed during posttranslational modification. Our results show that Edman degradation can be effectively used with a protein of known sequence to analyze proteolytic digests that have at least four different amino-terminal sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Limited proteolysis at the carboxy end modifies interactions between the subunits of Escherichia coli phosphofructokinase.

The limited proteolysis of Escherichia coli phosphofructokinase by subtilisin involves the removal of a segment of 40-50 residues at the C-terminal end of each polypeptide chain [Le Bras, G., & Garel, J.R. (1985) J. Biol. Chem. 260, 13450-13453]. The time course of proteolysis has been followed by the appearance of shorter chains, the loss of allosteric inhibition by phosphoenolpyruvate, and the weakening of the tetrameric structure in the absence of fructose 6-phosphate. It is found that with only one shorter chain out of four the stability of the tetramer is altered so that it is no longer stable in the absence of fructose 6-phosphate. Also, the reduction in size of only two chains is sufficient to render the enzyme insensitive to allosteric effectors, albeit the protein still possesses the ability to bind such an effector (at least partially); the cleavage of all four chains is needed to lose all the effector binding ability. The C-terminal segment therefore plays an important role in subunit interactions as seen from the gradual changes in structural and functional properties which follow its removal from one, two, or four chains.

Localization of the active site and phosphorylation site of Acanthamoeba myosins IA and IB.

The 130- and 125-kDa heavy chains of Acanthamoeba myosins IA and IB were radioactively labeled at either the regulatory phosphorylation site or the catalytic site and then subjected to controlled proteolysis by either trypsin or chymotrypsin. The labeled and unlabeled peptides generated during the course of proteolysis were identified by autoradiography and Coomassie Blue staining after separation by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The relative positions of the phosphorylation and active sites could be deduced. The catalytic site of myosin IA is most probably within 38 kDa of one end of the 130-kDa heavy chain, and the phosphorylation site, which can be no more than 40 kDa away from the catalytic site, would then be between 38 and 78 kDa of that same end of the heavy chain. Possibly, the phosphorylation site is further restricted to the region between 38 and 64 kDa from the end of the heavy chain. The catalytic and phosphorylation sites of myosin IB are both contained within a segment of 62 kDa at one end of the 125-kDa heavy chain and are within 40 kDa of each other. The phosphorylation site may be restricted to a small segment between 60 and 62 kDa from one end of the heavy chain which would limit the possible position of the catalytic site to the region between 20 and 60 kDa of that end.

Proteolysis of caseins and the proteose-peptone fraction of bovine milk

SummaryThe proteolysis of highly purified samples of αs1-, αs2-, β-and κ-caseins by porcine plasmin and by bovine plasminogen with urokinase has been examined principally by gel electrophoresis. The resulting peptide band patterns were compared with those of total proteose-peptone (TPP) samples prepared from fresh and stored raw and pasteurized milk, and also with those obtained during the natural course of proteolysis by indigenous enzymes in milk during storage. TPP was found to contain at least 38 components detectable by a single electrophoresis run. Apart from residual traces of whey proteins and intact caseins nearly all of these components were fragments of caseins produced by indigenous plasmin, with products from the breakdown of αs1- and β-casein predominating. Over 90 % of TPP has been accounted for in this way. A fragment consisting of residues 29–105 of β-casein was isolated and characterized from both stored milk and from plasmin digests of β-casein. This fragment was a relatively major product of the natural proteolysis occurring during storage of milk, but contrary to a report in the literature it was not the same as proteose-peptone component 8-slow. Since many of the components of TPP resulted from proteolysis, the composition of TPP was found to vary according to the time and temperature of storage of the milk from which it was prepared. Thus, while the proteose-peptone fraction of milk can easily be defined operationally it cannot be rigorously defined in terms of its composition unless the history of the milk is also defined.