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Thiolysis of an imidazolyl-dinitrophenyl-histidyl peptide at either pH 3.5 or 6.5 results in an increase in the net positive charge on the peptide. It is shown that this property can be used to form the basis of a diagonal paper electrophoretic purification of histidyl peptides from proteins. The amino groups of the protein are first reacted with citraconic anhydride and then the citraconyl protein is reacted with 1-fluoro-2,4-dinitrobenzene. The dinitrophenyl-citraconyl protein is digested with pepsin in 10% formic acid and, if necessary, with other proteolytic enzymes. The enzymatic digest is subjected to high-voltage paper electrophoresis at either pH 3.5 or 6.5. A guide strip is removed, thiolyzed with 2-mercaptoethanol, and subjected to electrophoresis at the same pH at right angles to the original direction of electrophoresis. The histidyl peptides are displaced off the diagonal toward the cathode. The off-diagonal peptides are isolated from the original electrophoretogram by thiolysis and electrophoresis using the diagonal electrophoretogram to locate the positions of the dinitrophenyl-histidyl peptides.

Various birch-pollen and dog-epithelium allergen extracts were chromatographed using Sephadex G-75 and Sephadex G-200 columns. The chromatographic tractions were then tested by an in vitro paper disc radioallergosorbent test, using sera from allergen-sensitive patients. Calibrated columns were used to approximate molecular weights of some birch-pollen and dog-epithelium allergenic active components.

1. A new method is described for f∈≥rpr∫∈g'cysteicaceptsderivedomthedisϕ––debrsofprote∈s.Cyst∈epeptsareseparatedbypapere≤ctrophoresisand⊗zedonpaperbyperformicacapour.E≤ctrophoresisatright∠s→thefirstdirection∏ucesparal≤lgroupsofcysteicaceptsly∈goffadiagonal.Thisf∈≥rpr∫∈g′cysteicaceptsderivedomthedisϕdebrsofprote∈s.Cyst∈epeptsareseparatedbypapere≤ctrophoresisand⊗zedonpaperbyperformicacapour.E≤ctrophoresisatright∠s→thefirstdirection∏ucesparal≤lgroupsofcysteicaceptsly∈goffadiagonal.Thisfingerprint' reveals the way in which the cysteic acid peptides were originally joined in the protein. 2. The method allows a very easy selective purification of cysteic acid peptides. 3. By applying this method to bovine chymotrypsinogen A, we found that the half-cystine residues were linked 1–122, 42–58, 136–201, 168–182 and 191–220.

IN the first report1,2, on ‘crossing paper electrophoresis’, a direct demonstration of enzyme-substrate complex has been described. The enzymes tested included crystallized pure trypsin, chymotrypsin and ribonuclease.

IT has been shown1 that, by the action of chymotrypsin, insulin is broken down into a number of peptide fragments of comparatively small molecular weight, and a more massive residue. The higher molecular weight material can be precipitated by means of trichloro-acetic acid. Attempts have been made to separate the lighter peptide mixture, and the most effective method so far found has been paper partition chromatography2.

Publisher Summary This chapter presents a study on mechanisms of enzymic catalysis. Enhancement of the polar interactions by restricting motions of polar groups in their local environment and by high polarizability but low permanent polarization of oily regions in local environments can rigidify the structure. The similarity of the imidazole-carboxylate geometry in subtilisin to that in the chymotrypsin and trypsin suggests that electronic tricks and specificity have developed independently in evolution, but are frequently linked. The sensitivity of the viscosity within membrane bilayers to the same factors that influence soluble proteins through changes in the properties of bulk phase water suggests that, compensation is not limited to soluble proteins. The study states that the analogy is not perfect, but it provides a rough description of the way the protein conformation can produce a special electronic situation.

The complete amino acid sequence of the kappa-chain of the mouse myeloma protein MOPC 21 was established. The protein was reduced and alkylated with iodo[2-(14)C]acetic acid, and 21 tryptic peptides were isolated, mainly by paper electrophoresis and paper chromatography. Three large tryptic peptides (of 35, 36 and 42 residues), which were difficult to isolate in this manner, were obtained pure and in excellent yields by a combination of Sephadex G-50 gel filtration in 1% (w/v) NH(4)HCO(3) and chromatography on a DEAE-cellulose column in ammonium acetate buffer, pH8.1. Peptides overlapping the tryptic peptides were isolated from a chymotryptic digest. The chain is 214 residues long. Microheterogeneity of two peptides was observed and is believed to be due to deamidation. It was not excluded that such deamidation could occur in serum from which the protein was isolated. The sequence is compared with the sequences of two other mouse kappa-chains, and with the human kappa-chain basic sequences.

The positions of the disulfide bridges in toxin II of Androctonus australis Hector were investigated using proteolytic enzymes. The resulting peptides were separated by column and paper chromatography and high-voltage electrophoresis. Determination of the amino acid compositions of the cystine-containing peptides or their oxidized derivatives and, when necessary, dansyl Edman degradation allowed to locate the disulfide bonds in the toxin. The four disulfide bridges were found to link the half-cystine residues number 12 and 63, 16 and 36, 22 and 46, 26 and 48. These positions are probably the same in all scorpion neurotoxins.

The primary structure of sheep para-xA-casein, the N-terminal portion of x-casein, was established. The reduced alkylated protein was subjected to digestion with trypsin. The resulting soluble peptides were purified by a combination of Dowex 1 X2 chromatography and electrophoresis and chromatography on paper; the core peptides were solubilized in 50% formic acid and filtered on Sephadex G-50. The amino-acid sequence of these peptides was determined chiefly with a Sequencer. Alignment of the tryptic peptides into a single chain containing 105 amino acids was determined from basic overlap peptides (tryptic core peptides with several basic amino acids; chymotryptic peptides). Some comparisons were achieved with cow para-xa-casein. %-Casein is the principal casein fraction affected by chymosin (or rennin) in the primary phase of the milk clotting process [ 11. The group of Jollbs established already in 1965 that during this enzymic reaction, a Phe -+ Met linkage was specifically split in cow as well as in sheep x-caseins [2-41. An insoluble part, para-x-casein (N-terminal moiety of x-casein), was from Merck or Prolabo except those employed for the Sequencer which were purchased from Socosi (94100Saint Maur, France) and 4-sulfophenylisothiocyanate from Pierce.

The antitumor protein neocarzinostatin has been purified by repeated CM-cellulose chromatography and Sephadex G-50 gel filtration. The protein possesses 109 amino acid residues in a single chain, crosslinked by two disulfide bonds. Reduction and S -alkylation could not be accomplished in aqueous solution and required the use of dithiothreitol in liquid ammonia, followed by treatment with alkyl chloride. Tryptic digestion of (tetra- S -carboxymethyl)neocarzinostatin afforded five tryptic fragments which were fractionated by preparative paper chromatography and high-voltage paper electrophoresis, purified by Sephadex gel filtration and characterized by amino acid and amino end-group analysis. The total number of amino acid residues of these fragments account for the 109 residues of neocarzinostatin.

Angiotensin II (ATII), angiotensin I (ATI), and Acpc analogues of ATII, 8-Achc-ATII, 8-D-Phe-ATII, and 8-Ala-ATII, were incubated in vitro with carboxypeptidase, chymotrypsin, and leucine-aminopeptidase in order to study the influence of unnatural amino acids (Acpc, Achc, and D-Phe) and of L-Ala on the activity of peptidases.Fragments occurring during the breakdown of peptides were demonstrated by paper chromatography in an ascending system.ATII and ATI are rapidly inactivated by carboxypeptidase and chymotrypsin, while the degradation by leucine-aminopeptidase is slower.Substitution of L-Phe with Acpc, Achc, D-Phe, or L-Ala in position 8 prevents the degradation by carboxypeptidase. Chymotrypsin degrades 3-Acpc-ATII and 5-Acpc-ATII but not 4-Acpc-ATII. The action of leucine-aminopeptidase is not influenced by substituting Acpc to each one of the first five amino acids composing the molecule of angiotensin.The possible implications of these findings for the peptide-receptor interaction is discussed.

Crystals of sperm whale ferrimyoglobin were treated with 0.2 m sodium bromoacetate in concentrated ammonium sulfate solution at pH 6.8 for 10 days. The product obtained after dissolving the crystals retained most of the physical properties of the unmodified protein, except for the development of a small number of electrophoretic components representing an increment of about six to eight net negative charges on the average, judged at pH 9.2. The most fully studied preparation contained 6.4, 2.7, 2.0, and 0.8 residue per molecule of histidine, 1,3-dicarboxymethylhistidine, 3-carboxymethylhistidine, and 1-carboxymethylhistidine, respectively. The amino-terminal valyl residue was largely alkylated. Between 1 and 2 residues of lysine were alkylated. No modification of methionine was detected. After removal of heme the modified protein was subjected to cleavage by trypsin and chymotrypsin and the peptides were isolated by chromatography on ion exchange resins and, as required, by paper chromatography or paper electrophoresis. The state of alkylation of 10 of the 12 histidyl residues was established by quantitative estimation of the yields of appropriate tryptic and chymotryptic peptides. The state of residue 12 was determined qualitatively, and that of residue 113 surmised by difference. The following residues were recovered as unmodified histidine: 24, 48, 82, 93, and 97. Residue 119 was recovered primarily in the unmodified form but partly as 1-carboxymethylhistidine. The following residues were recovered primarily or exclusively as 1,3-dicarboxymethylhistidine: 12, 81, 114, and 116. Residue 116 was recovered partly as 3-carboxymethylhistidine. Residue 36 was recovered in high yield exclusively as 3-carboxymethylhistidine. Without exception these results appear to conform to the implications of the crystalline structure reported by Kendrew and Watson when account is taken of the geometry of interactions between neighboring myoglobin molecules in the crystal lattice.

The protected heptapeptide derivative t -butyloxycarbonyl- l -threonyl-β-benzyl- l -aspartyl- l -asparaginyl- O -benzyl- l -tyrosyl- l -threonyl-nitro- l -arginyl- l - leucine methyl ester was prepared by stepwise chain lengthening. The protecting groups on the side chains of arginine, tyrosine, and aspartic acid residues were removed by hydrogenolysis and the partially deprotected heptapeptide ester converted to the hydrazide, an intermediate in the synthesis of the (porcine) vasoactive intestinal peptide (VIP). After the removal of the tert -butyloxycarbonyl group, the heptapeptide ester was exposed to the action of trypsin which split off its C-terminal residue, l -leucine methyl ester. The hexapeptide was then exposed to chymotrypsin, which cleaved it into an acidic, and a basic fragment. The former was, under the conditions used, indistinguishable on paper chromatography and paper electrophoresis from the tetrapeptide threonyl-aspartyl-asparaginyl-tyrosine which had previously been isolated from natural VIP, of which it comprises the sequence 7–10. Similarly, the basic fragment was indistinguishable from threonyl-arginine, the sequence 11–12 of VIP. This intestinal peptide increases visceral blood flow and reduces blood pressure in the dog, and also causes relaxation of different smooth muscle preparations, e.g., the trachea of guinea pigs. The principal aim of the present synthesis is to provide independent evidence for the sequence of (porcine) VIP.

Guinea-hen egg-white lysozyme was obtained in a chromatographically pure state by ion-exchange chromatography on Amberlite CG-50. The reduced alkylated enzyme was subjected to digestion with trypsin. The resulting peptides were purified by a combination of Dowex 1-X2 chromatography and electrophoresis and chromatography on paper. The amino acid sequence of these peptides was determined chiefly by the Edman dansylation procedure. Alignment of the tryptic peptides into a single chain containing 129 amino acids was determined from basic overlap peptides obtained by digestion of the enzyme with chymotrypsin. 10 replacements were noted between guinea hen and hen lysozymes. Further comparisons were achieved with 4 other bird egg-white lysozymes the structures of which were already known.

Tryptic and chymotryptic peptide fragments obtained from performic acid-oxidized porcine neurophysin-I were isolated and purified by gel filtration, ion exchange chromatography, paper electrophoresis, and paper chromatography. The larger tryptic peptides were further hydrolyzed by papain, chymotrypsin, pepsin, or HCl to yield shorter fragments amenable to Edman degradation. The sum of the compositions of the tryptic peptides, however, exceeded the total number of amino acid residues of porcine neurophysin-I reported earlier. Re-examination of the amino acid composition and further investigation of COOH-terminal peptides showed that the porcine neurophysin-I preparation was not homogeneous with respect to the COOH terminus. The preparation contained two species of neurophysin molecules, in approximately equal amounts, differing in length only by 1 amino acid residue at the COOH terminus. The complete amino acid sequence of both species of porcine neurophysin-I, containing 92 and 91 residues, was determined by conventional methods. The larger molecule contains the entire sequence of the shorter molecule plus an extra leucyl residue at its COOH terminus. Alanine is the NH2-terminal residue of both molecules. Phenylalanine is the COOH terminus of the shorter molecule. Single methionine and tyrosine residues occupy positions 2 and 49, respectively. The location of the seven disulfide linkages of porcine neurophysin-I remains to be determined.

Although the amino acid sequences of two subtilisins' do not resemble those of the pancreatic proteinases chymotrypsin2 and trypsin,3' 4 the two groups of enzymes are similar in that they are both inhibited by reaction with diisopropylfluorophosphate at a single specific serine residue.5 Further, a specific histidine residue has been identified in the active sites of trypsin and chymotrypsin by reaction with the specific alkylating agents TLCKI6 I and TPCK.8 Although these reagents do not inhibit the subtilisins,9 kinetic data do suggest involvement of an un-ionized histidine residue in the active sites of these enzymes.10 Shaw and Ruscicall have found that the increased reactivity of the bromomethyl ketone of carbobenzoxy-L-phenylalanine (ZPBK) is sufficient to inhibit the subtilisins. There is close agreement between the loss of activity, incorporation of tritium from 3H-ZPBK, and loss of histidine with subtilisin BPN'. The reagent is stereospecific with only the L-form being reactive with the enzyme. That the reactive histidine must be involved in the enzymic activity is demonstrated by the finding that subtilisin BPN' treated with ZPBK no longer reacts with diisopropylfluorophosphate. After performic acid oxidation, 3-carboxymethylhistidine was identified. We wish to report on the identification of the active-site histidine residue in the subtilisins with the use of ZPBK. Materials and Methods.-Chemicals: Subtilisin Carlsberg was obtained from Novo Pharmaceutical Company, Copenhagen, Denmark (batch 60420), and subtilisin BPN' (as Nagarse) from Biddle Sawyer Corp., New York. ZPBK and 3H-ZPBK were synthesized by the method of Shaw and Ruscica."1 Radioactivity measurements: Radioactivity of the tritium-labeled protein or peptides was measured in a Packard (model 3003) liquid scintillation spectrometer. Liquid samples of 20-100 ,ud (usually 50 ,ul) were counted in 10.0 ml of Bray's solution,'2 with corrections for background but not for differences in efficiencies with the different solvents or different sized aliquots. To locate radioactive peptides after preparative chromatography or electrophoresis, guide strips that had been sprayed with ninhydrin or the Pauly reagent'3 were cut into small pieces and counted as described above in 10.0 ml of a scintillation solvent that contained 6 gm of PPO and 0.3 gm of dimethyl POPOP in 1 liter of toluene. In later stages of the work, a Beckman solubilizer BBS-3 was added to the toluene scintillation solvent'4 to improve the counting efficiency. Peptide separation: Peptides were separated either by gel filtration on Sephadex G-75 or G-25 in formic acid-propanol-water (30:20:50 by volume) and detected with ninhydrin after alkaline hydrolysis.'5 Paper chromatography in n-butanol-acetic acid-water (200:30:75 by volume) (BAW) and paper electrophoresis at pH 1.9 were used preparatively. Peptides were eluted from paper with 35% acetic acid and, unless otherwise specified, were dried on a rotary evaporator. Peptide maps were run with electrophoresis at pH 1.9 and chromatography in BAW. Amino acid analysis: Samples were hydrolyzed at 1100 for 24 hr in evacuated glass tubes with 6 N HCI containing a drop of 5% phenol in water. Analyses were performed with the Spinco automatic amino acid analyzer model 120 B with an accelerated system.'6 17 Results.-Reaction of 3H-ZPBK wvith subtilisin Carlsberg: M\1ost of the work

Ascaris suis was extracted with 60% ethanolin dilute formic acid after previous removal of fat with organic solvents. After dialysis, the extract was further purified by ammonium sulphate precipitation, treatment with Amberlite IRC-50 (XE-64), chromatography on CM- cellulose and gel filtration on Sephadex G-25. The purified product with a molecular weight probably within the range 2000–3000, had basic character with an isoelectric point ca 12.3 in 0.05 M borate buffer. It behaved as a nearly homogeneous compound on paper electrophoresis and on gel filtration. Nitrogen content: 16.7%. Fifteen amino acids were identified by paper chromatography after acid hydrolysis: alanine, arginine, cystine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine and valine. The purified product was degradrd and inactivated by the proteolytic enzymes papain, pronase, trypsin, α-chymotrypsin and partially by pepsin. The polypeptide caused degranulation of rat mesentery mast cells, even such cells from germ free animals, indicating that the mast cell respome was the result of a direct action of the Ascaris principle on the mast cell and not due to an antigen antibody reaction.

The badger apomyoglobin was first submitted to tryptic digestion. The tryptic hydrolysate of whole protein was fractionated by resin chromatography and each tryptic peptide was sequenced. The apomyoglobin was also cleaved by cyanogen bromide and the resulting fragments were fractionated by gel filtration; three segments were obtained; the N-terminal segment (55 residues), the C-terminal segment (22 residues) and the median segment (76 residues). Each segment was submitted to a variety of enzymatic digestions and the complete amino acid sequence of resulting peptides was established. In addition, the whole protein and the median segment were analyzed by using a sequenator (Edman); the first 31 N-terminal residues of the intact protein and the first 28 N-terminal residues of the median segment of CNBr resulting peptide were confirmed. The complete sequence of the C-terminal segment (22 residues) was also established and confirmed by manual Edman's degradation. Between badger and harbor seal myoglobins, 19 differences out of 153 residues were found. Of these amino acid replacements, one corresponds to the exchange of two bases and 18 to the exchange of one base in the coding triplets.

The isolation and amino-acid and carbohydrate composition of three tryptic glycopeptides, containing 16,17 and 20 amino-acid residues, respectively, from the neuraminidase-treated major form of human ceruloplasmin have been described earlier. It has also been suggested that the peptides possibly represent all of the carbohydrate-binding sites in the protein. The present paper describes the amino-acid sequences of these peptides as obtained by stepwise Edman degradation and carboxypeptidase digestions of the whole peptides and their fragments after cleavage by chymotrypsin, pepsin and thermolysin. The results confirm that the carbohydrate in all cases is attached via an asparagine (or aspartic acid) residue, that is followed by an X-Thr/Ser sequence. No sequence homologies are found when the peptides are compared to each other or to other glycopeptides of known sequence.

Summary The primary structure of cytotoxin VII4, consisting of 60 amino acid residues cross-linked by four disulphide bridges, was determined. Cytotoxin VII4 differs in altogether 17 residue positions from the other cytotoxins of the same snake. Comparing the amino acid sequence of cytotoxin VII4 from Naja mossambica mossambica venom to the known sequences of cytotoxins from the genera Naja and Haemachatus , 33 residues, including 8 half cystines, are identical. The amino acid sequence of cytotoxins as a group has 11 residue positions in common with the amino acid sequence of neurotoxins and based on this observation, 8 residues in the primary structure of cytotoxins could be implicated as probably being functionally important.

“Soluble prekeratin”, a fibrous protein extractable from the non-keratinized layers of cows' nose epidermis with citrate buffer, pH 2.6, has been examined in order to determine the size of any repeating subunit. From the content of the least occurring amino acid residues, the number of peptides produced by tryptic digestion and the specific staining properties of peptides, the presence of a repeating subunit was established which was calculated to contain 252 amino acid residues and to have a subunit weight of 26 900.

The amino acid sequence of the light chain of the myeloma protein Dee was studied. The light chain is of the κ type and of subgroup I. The variable part contains some substitutions that are unique and also some that have been observed already in other κI chains (repeated variants). Based on these repeated variants a subdivision of the κI subgroup is proposed.

Cyanogen bromide treatment of reduced, aminoethylated enterotoxin B yielded seven major peptides plus homoserine and homoserine lactone, as was expected from the number of methionine residues present in the enterotoxin B molecule. The cyanogen bromide peptides were fractionated on Sephadex G-50 and further purified by rechromatography on Sephadex G-50 or paper electrophoresis at pH 1.9. They contained a total of 239 amino acid residues. Tryptic peptides of each of the cyanogen bromide peptides were purified and their amino acid composition was determined. The order of these peptides in each cyanogen bromide peptide and the order of the cyanogen bromide peptides in enterotoxin B were established. Based on the information from tryptic, chymotryptic, and cyanogen bromide peptides, the complete amino acid sequence of enterotoxin B was established. Staphylococcal enterotoxin B is a single polypeptide chain containing 239 amino acid residues.

The primary structure of the hormone specific β subunit of human pituitary thyrotropin has been deduced from the composition and complete or partial amino acid sequences of the tryptic and chymotryptic peptides. It consists of 112 amino acid residues with the single oligosaccharide moiety which is assumed to be linked to the asparagine residue at position 23.

1. Cysteic acid peptides from various digests of calf rennin were purified by diagonal paper electrophoresis. 2. The amino acid sequence of these peptides accounts for 38 amino acids around three unique disulphide bridges in rennin. 3. One bridge connects two acidic regions of the chain, one forms a loop of five residues and the other a loop of six residues. 4. These bridges are homologous with those of hog pepsin. 5. Tryptic peptides from the C-terminus of rennin account for 22 residues, 17 of which are homologous with the C-terminus of pepsin. 6. Altogether, sequences accounting for 94 of the 270 residues in rennin are described and the degree of homology with pepsin approximates to 70%.

The primary structures of the ribosomal proteins A1 (= L7) and A2 (= L12) have been elucidated. Comparison of the two amino acid sequences confirms earlier studies by us (1972) which indicated that the two proteins are identical except that A1 possesses an N-terminal acetyl group. Sequencing of tryptic and chymotryptic peptides was accomplished primarily by automatic solid-phase Edman degradation of 50- to 70-nanomole peptide samples. A large tryptic peptide T1Phe, which could not be sequenced by this method, was fragmented with elastase and its sequence mainly derived by conventional methods. The sequence of the first 51 residues of the nonacetylated A2-protein was obtained by Edman degradation in the Beckman sequencer. A-protein comprises three distinct regions: I (residues 1—55), which is negatively-charged and hydrophobic, II (56—81), which is positively charged, and III (82—120), which is negatively charged and hydrophilic. α-Helix promoting residues are located primarily in regions I and III. ɛ-N-Monomethyllysine, which occurs in 50% of the A1 and A2 chains is located in the more flexible region II at position 81. The distinctive clustering of hydrophobic and charged residues is quite remarkable and suggests that in situ certain of these regions may contain a binding site for components involved in peptide chain elongation.

The active thiol group of lombricine kinase from Lumbricus terrestris muscle was labelled with N-ethyl-[1-14C]maleimide. The resulting inactivated N-ethyl-[1-14C]succinimido enzyme was then subjected to tryptic hydrolysis. The peptide containing the labelled essential thiol group was isolated and found to contain: Leu-Gly-Tyr-Ile-Thr-[14C]Cys-Pro-Gly-Ser-Asn-Leu-Gly-Thr-Leu-Arg. The amino acid sequence around this thiol group was very similar with that of homologous ATP: guanidine phosphotransferases previously studied, arginine kinase from Homarus vulgaris muscle, creatine kinases from ox brain and ox muscle and from rabbit muscle. In addition among the other enzymes of this group, lombricine kinase is of special interest since it is the only dimeric enzyme of molecular weight ≃ 80000 which possesses only one essential thiol group and one nucleotide binding site per two subunits.

1. 1.|Extracellular alkaline proteinases from the molds Aspergillus flavus , Aspergillus oryzae , Aspergillus sojae and Aspergillus sulphureus , isolated and characterized by individual authors in four different laboratories, have been compared using uniform methods under identical conditions in one of our laboratories. 2. 2.|The enzymes studied were compared by gel filtration, disc electrophoresis, amino acid analysis, determination of the pH optima of their proteolytic activity, estimation of their specificity by cleavage of the B-chain of oxidized insulin, and by peptide maps of the tryptic, chymotryptic, and peptic digests of the inactivated proteinases. 3. 3.|The results of this comparison led to the following conclusion: Alkaline proteinases from A. flavus and A. oryzae are probably indentical (or very closely related), alkaline proteinase from A. sojae is closely related but not identical, alkaline proteinase from A. sulphureus is homologous yet less related. 4. 4.|The combination of methods used in this study requires only small quantities of enzymes. It is therefore suitable for preliminary comparison of proteinases and for studies on the degree of their homology.

The mitogens of a phytohemagglutinin from red kidney beans contain two types of subunits. These subunits were isolated in fairly large scale using a preparative gel electrophoresis in sodium dodecyl sulfate-urea. One type (α) was made up of a single subunit species of MW 35,000 (a dimer at pH 7), and this molecule kept its mitogenic activity after sodium dodecyl sulfate treatment. The other type (β) which was mitogenically inactive after sodium dodecyl sulfate treatment, appeared to consist of two kinds of subunits with molecular weights of 35,500 and 36,500. Analysis for amino acids and carbohydrates, mapping techniques for tryptic and chymotryptic peptides and molecular weight determination have established structural differences between the two subunits. However, terminal amino acids (NH 2 - and COOH-) of both subunits were identical, suggesting that they had closely related primary structures.

Various biological and chemical properties of colostrokinin were studied. Vasodilatation in dogs, stimulation of smooth muscles of rats and increasing capillary permeability in guinea pigs were observed as the pharmacological actions. Among these, the change in capillary permeability is the strongest when compared to the activity of bradykinin. Colostrokinin activity is destroyed by chymotrypsin, but only slightly by trypsin. It is more stable in acid than in alkaline solution. This substance is dialyzable, and is soluble in water, in 67% ethanol and in 75% acetone, but not in absolute acetone. The isoelectric point is close to pH 7.4. Phenylalanine and serine were detected as the N -terminal and the C-terminal amino acids respectively. The molecular weight, on the basis of amino acid analysis, was calculated to be about 2000. Colostrokinin absorbs light close to λ = 260 m μ .

The use of ion exchange chromatography, gel filtration, and paper electrophoresis for the separation of the peptides obtained by tryptic digestion of the cowpea strain of tobacco mosaic virus protein is described. The amino acid compositions of the 13 tryptic peptides obtained were determined and accounted for 159 residues compared with the 158 found for type tobacco mosaic virus protein. The only tryptic peptide that cowpea tobacco mosaic virus protein and type tobacco mosaic virus protein have in common is asparaginyl-arginine despite the fact that the former is very similar to type tobacco mosaic virus protein in a number of its properties. The amino terminal sequence was found to be acetyl alanyl-tyrosine and the carboxyl terminal sequence was confirmed as alanine preceded by threonine.

The amino acid sequence of cytochrome c553 from Desulfovibrio vulgaris (NCIB 8303) is presented. The protein molecule consists of a single polypeptide chain of 82 residues. This sequence is deduced from data obtained by enzymatic digestion of the apoprotein or by chemical cleavage with cyanogen bromide. Comparison of the sequence of cytochrome c553 of Desulfovibrio vulgaris with that of cytochrome c3 of the same organism revealed no homology. However, some similarities with Pseudomonas cytochrome c551 were found.

A concentration of 10 μg of fluorophenylalanine per ml added to a chemically defined medium reduced by 100-fold the number of bacteriophage MS2 produced on Escherichia coli C3000 and increased the latent period. Fluorophenylalanine was most effective when added concurrent with infection. Addition of a 10-fold greater concentration of phenylalanine reversed the inhibition caused by fluorophenylalanine. Radioactive fluorophenylalanine was incorporated into the coat protein. The four phenylalanine-containing chymotryptic peptides are not equally accessible to fluorophenylalanine. Only two of the peptides are highly labeled by fluorophenylalanine. Incorporation of fluorophenylalanine decreased the specific infectivity and the rate of adsorption but did not increase the sensitivity of the whole virus to ribonuclease. MS2 ribonucleic acid (RNA) functioned as messenger RNA for the incorporation of both phenylalanine and fluorophenylalanine in a cell-free incorporating system from E. coli.

A procedure for synthesizing 14C-labeled N-benzoyl-l-tyrosine ethyl ester with the label in the benzoyl group has been described. Using this substrate, which is specific for chymotrypsin, and employing trypsin activation of chymotrypsinogen in an incubation medium containing radiolabeled BTEE, we have presented a method which permits assay of nanogram quantities of chymotrypsin activity in organ-cultured tissue. The radiolabeled product of enzyme hydrolysis, N-benzoyltyrosine, is readily separated by paper chromatography from the unreacted labeled substrate and measured by radioactivity counting.

A technique for the analysis of certain amino acids in protein hydrolyzates is proposed which consists in one-dimensional paper chromatography followed by elution of the proper areas and colorimetric analysis by selective methods. Results are reported for the histidine, tyrosine, methionine, and arginine contents of a few crystalline protein. This method has been applied to the differentiation of some known and unknown strains of TMV. Application to DNP-derivatives of chymotrypsin and DFP-inhibited chymotrypsin has not yielded any evidence concerning the mechanism of the inhibition by the alkyl phosphates.

For the elucidation of the amino-acid sequence of the carboxyl-terminal region of soybean trypsin inhibitor (Kunitz), fragments C and D were digested with trypsin, and the resulting peptides were separated by ion-exchange chromatography on Dowex 50X2 or by gel nitration on Bio-Gel P-4. Further fractionation and purification of the peptides were performed by ion-exchange chromatography on Dowex 1X2, by gel filtration on Bio-Gel P-2 or by high-voltage paper electrophoresis at pH 1.9 and 3.6. Three peptides were obtained in pure form from fragment C and ten peptides from fragment D, and their amino-acid sequences were determined by the direct Edman method and by carboxy-peptidase digestion technique. Overlapping peptides necessary for the alignment of the tryptic peptides from fragment D were obtained from a chymotryptic hydrolysate of fragment D. Nine main peptides and nine minor peptides were obtained. The amino acid composition and the partial amino-acid sequence of the 18 chymotryptic peptides of fragment D made it possible to establish the amino-acid sequence of the carboxyl-terminal region of the inhibitor. The complete amino-acid sequence of soybean trypsin inhibitor (Kunitz) deduced here was compared with that of Bowman-Birk inhibitor, another well-known soybean proteinase inhibitor.

A short glycopeptide was isolated from bovine χ-casein by enzymic digestions, filtrations on Sephadex G-25 and was electrophoretically homogeneous. An O-glycosidic linkage was characterized between a residue of threonine and N-acetylgalactosamine. By chemical and enzymic proce- dures, the following formula was established: where NeuNAc =N-acetylneuraminic acid. The phosphoserine residue of χ-caseinoglycopeptide was characterized in the short glycopeptide which was localized in the C-terminal part of χ-casein. Thr-X-Pro might be suggested as “code sequence” for glycopeptides containing an O-glycosidic linkage.

Reduced S -carboxymethylated cobrotoxin was digested with trypsin and chymotrypsin. Nine peptides were isolated from the tryptic digest by a combination of electrophoresis and chromatography on paper. The amino acid sequence in these peptides was determined by applying the Edman degradation and by using proteolytic enzymes, leucine aminopeptidase and carboxypeptidases. The arrangement of nine peptides in a linear structure was determined by comparing the amino acid compositions of the overlapping peptides isolated from the chymotryptic digest of reduced S -carboxymethylated cobrotoxin. The striking distributional regularities of the basic and hydrophilic residues in the sequence and the importance of the positions of half cystinyl residues which form the disulfide bonds for maintaining the protein in its active configuration, are considered in connection with the structure-activity relationship of the protein.