Search

1 A new diagonal electrophoretic technique for determining the order of the lysine-containing tryptic peptides of a protein is described. The protein is converted into its trifluoracetyl derivative, digested enzymatically (or chemically), and the resulting peptides separated by paper electrophoresis. The paper is then treated with ammonia vapour, which re-exposes the ɛ-amino groups of the lysine residues, and submitted to a second electrophoresis at right angles to the first direction. Peptides containing lysine residues, together with the N-terminal peptide of the protein, are found to lie off a diagonal formed by all other peptides, whence they may be readily purified. A study of these peptides enables the order of the lysine-containing tryptic peptides in the protein to be deduced. 2 The technique has been successfully tested with insulin. 3 When the method was applied to porcine pepsin, the four tryptic peptides isolated were easily ordered and the carboxyl-terminal sequence of the protein shown to be Arg-Gln-Tyr-Tyr-Thr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ala-Pro-Val-Ala. The three basic amino acid residues in the molecule are thus found clustering towards the C-terminus of the polypeptide chain. 4 A common ancestral gene for porcine pepsin and bovine (calf) rennin is suggested by the close homology between the C-terminal sequences of the two proteins.

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 amino acid sequence of the porcine vasoactive intestinal octacosapeptide is His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-L ys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2. Its amino acid residues 1, 2, 6 and 7, counted from the N-terminus, are identical to those in the corresponding positions in both porcine glucagon and secretin. The residues in positions 3, 12, 13, 14 and 23 are identical to those in secretin, but not in glucagon, and position 10 is occupied by a tyrosyl and position 28 by an asparaginyl residue, like in glucagon but not in secretin. If in addition to identical amino acid residues also chemically similar residues, such as isoleucine in position 26 of the octacosapeptide, as compared to leucine in secretin and glucagon, are taken into consideration then the similarity between these three polypeptides is still more evident. At a more remote level there is some structural resemblance also between these peptides and the other four peptides from the intestinal wall, cholecystokinin-pancreozymin, motilin, the gastric inhibitory peptide and substance P, the structures of which are known. The elucidation of the structure of the octacosapeptide was facilitated by the finding that pancreatic kallikrein preferentially cleaved only one of the three bonds in its N-terminal cyanogen bromide heptadecapeptide that are susceptible to cleavage with trypsin.

Cleavage with cyanogen bromide was employed to investigate the N-terminal amino acid sequence of the λ-chains of normal porcine immunoglobulin. The N-terminal homoserine-containing nonapeptide was isolated from all fractions of the λ-chains in a yield of 60% of theory. Its amino acid sequence was determined by mass spectrometry and was found to be uniform. Other homoserine-containing fragments were obtained from the cyanogen bromide hydrolyzate of the λ-chains with split disulfide bonds. Partial amino acid sequence of these fragments provided evidence that methionine in position 9 is present in more than 60% of variants and obviously has an invariant character. Variants of the λ-chains exist (by estimate not more than 15% of the total λ-chains) which, in addition to the invariant methionine residue in position 9, possess a variable methionine residue in the section between positions 46 and 51. The data obtained indicate that the sequence of 23 amino acid residues from the N-terminus of porcine λ-chains is uniform with the exception of a single replacement (in position 13). Moreover, in comparison with the sequence of λ-chains of man, mouse and birds, the sequence displays several species-specific replacements and a deletion of threonine which occupies position 5 in the chains of the other species. In the pig, the λ-chains represent the predominant type of light chains (some 70%). Hence it follows that some 50% of the light chains in this species possess a uniform amino acid sequence in the N-terminal section. These facts are difficult to reconcile with the germline theories of genetic control so that mechanisms of somatic diversification of immunoglobulins must be envisaged.

The peptide side chains of the peptidoglycan of the Pseudomonas aeruginosa cell envelope have the sequence H2N-l-Ala-d-Glu-A2pm-d-Ala-COOH. The d-glutamate is linked to the meso-2, 5-diaminopimelate (A2pm) by a peptide bond formed by the C-5 carboxyl group of d-glutamate. The C-1 carboxyl group of the glutamate does not occur in the amidated state in the cell wall. The configurations of alanine, glutamate, and 2, 5-diaminopimelate have been confirmed.

Most rabbit antibodies contain a methionine residue about 240 residues from the NH2-terminus of the heavy chain sequence. Cleavage of heavy chains by CNBr at this methionine leads to the formation of a fragment, C1, which contains the variable portion of the H-chain but is slightly longer than the Fd fragment. Published amino acid compositions of fractions from different antibodies, all taken to represent fragment C1, showed marked similarity. In this work it is shown that the primary reason for this similarity is that previous methods of isolating C1, by gel filtration using 6 M urea or 1 M acetic acid as solvent, led to the isolation of aggregates contaminated with an extensive section of the constant region. Six M guanidine · HCl prevented the formation of these aggregates and gel filtration in this solvent yielded a C1 fragment with an amino acid composition quite different from that of the contaminated aggregate. The amino acid compositions of the uncontaminated C1 fragments from antihapten antibodies from individual rabbits showed large differences. The differences were apparent because of the restricted heterogeneity of the antibodies being compared. Amino acid compositions of C1 fragments from γ-globulins from individual uninjected rabbits showed appreciably less variation because, unlike the antibodies, the γ-globulins were complex mixtures.

Cell-free protein synthesis has been studied in heterologous systems using rabbit reticulocyte postribosomal supernatant and ribosomes obtained from HeLa or mouse myeloma cells. These cell-free systems are quite active and incorporate labelled amino acids linearly for more than 30 min. The proteins synthesized have been analyzed by acrylamide gel electrophoresis, column chromatography and paper electrophoresis of the tryptic peptides. More than 70% of the protein synthesized with either type of ribosomes is α chain of rabbit hemoglobin; a small amount of β chain is also synthesized. This result can only be explained by the presence in the postribosomal supernatant of reticulocytes of messenger RNA for hemoglobin. This mRNA is translated more efficiently than the endogenous mRNA present on HeLa or myeloma ribosomes; previous measurements of the mRNA of reticulocyte postribosomal supernatant indicate that it is less on a weight basis than 1/20 of the mRNA associated with the corresponding amount of ribosomes used in an incubation. The preferential translation of mRNA present in the supernatant, presumably in a ribonucleoprotein particle, is discussed.

1 Adenylate kinase (ATP:AMP phosphotransferase) has been purified 490-fold from porcine muscle with a final yield of 60 mg/kg muscle. 2 The amino-acid composition is Asp11, Asn2, Thr14, Ser11, Glu19, Gln6, Pro6, Gly19, Ala8, Cys2, Val17, Met6, Ile9, Leu18. Tyr7, Phe5, Lys21, His2, Arg11. 3 The protein molecule is a single polypeptide chain of 194 amino-acid residues with an acetyl-methionine at the N-terminus and a lysine residue at the C-terminus. 4 Cyanogen bromide cleavage of carboxymethylated adenylate kinase yielded six fragments which were further degraded by using trypsin, chymotrypsin, thermolysin, subtilisin or α-protease. Sequence data on the resulting peptides are summarized in the present report, full details are given in a supplementary paper which has been deposited at CNRS from where copies can be obtained. 5 The primary structure of porcine adenylate kinase is: Ac-Met-Glu-Glu-Lys-Leu-Lys-Lys-Ser-Lys-Ile10-Ile-Phe-Val-Val-Gly-Gly-Pro-Gly - Ser-Gly20-Lys-Gly-Thr-Gln-Cys-Glu-Lys-Ile-Val-Gln30-Lys-Tyr-Gly-Tyr-Thr-His-Leu-Ser-Thr-Gly40-Asp-Leu-Leu-Arg-Ala-Glu-Val-Ser-Ser-Gly50-Ser-Ala-Arg-Gly-Lys-Met-Leu-Ser-Glu-Ile60-Met-Glu-Lys-Gly-Gln-Leu-Val-Pro-Leu-Glu70-Thr-Val-Leu-Asp-Met-Leu-Arg-Asp-Ala-Met80-Val-Ala-Lys-Val-Asp-Thr-Ser-Lys-Gly-Phe90-Leu-Ile-Asp-Gly-Tyr-Pro-Arg-Glu-Val-Lys100-Gln-Gly-Glu-Glu-Phe-Glu-Arg-Lys-Ile-Gly110-Gln-Pro-Thr-Leu-Leu-Leu-Tyr-Val-Asp120-Ala-Gly-Pro-Glu-Thr-Met-Thr-Lys-Arg-Leu-Leu130-Lys-Arg-Gly-Glu-Thr-Ser-Gly-Arg-Val-Asp140-Asp-Asn-Glu-Glu-Thr-Ile-Lys-Lys-Arg-Leu150-Glu-Thr-Tyr-Tyr-Lys-Ala-Thr-Glu-Pro-Val160-Ile-Ala-Phe-Tyr-Glu-Lys-Arg-Gly-Ile-Val170-Arg-Lys-Val-Asn-Ala-Glu-Gly-Ser-Val-Asp180-Asp-Val-Phe-Ser-Gln-Val-Cys-Thr-His-Leu190-Asp-Thr-Leu-Lys.

The primary structure of a non-immunoglobulin amyloid protein AS has been determined. The protein was found to consist of 76 amino acid residues corresponding to a molecular weight of 9145. The sequence analysis showed clearly that the protein was homogeneous. A characteristic distribution of hydrophobic amino acids was observed and suggested as being of importance for the ability of this protein to form fibrils. A comparison of the protein with other amyloid protein AS showed a high degree of variability, particularly in the carboxyl-terminal region.

The venom of Dendroaspis viridis has been fractionated by chromatography on Sephadex G-50 and SE-Sephadex. Thirteen polypetides containing either 59–63 amino acid residues and four disulphide bridges or 71–73 amino acid residues and five disulphide bridges have been isolated and characterized by amino-acid analysis and N-terminal residue determination. The compositions of the polypeptides show many similarities but the toxicities vary in excess of 100-fold, upwards from an LD50 of 0.08 mg/kg in mice. Enzymic and nerve growth factor activities have been located in protein fractions associated with higher molecular-weight material.

Tryptic and chymotryptic digestion of peptide B, the N-terminal fragment obtained by cyanogen bromide cleavage of thioredoxin, provided the information required to determine the contiguity of the peptic fragments studied previously. The complete sequence of the 37 residues of peptide B could thus be established. Peptide B contains the active center of thioredoxin. Its amino acid sequence was found to be: . This sequence supports earlier conclusions drawn from fluorescence studies of thioredoxin, which indicated a close relationship between one or both tryptophan residues and the disulfide bridge of the active site. Peptide maps of the tryptic digests of carboxymethylated thioredoxin and of the two cyanogen bromide fragments are described. Amide nitrogen analyses of thioredoxin and the two cyanogen bromide fragments demonstrate that all seven amide groups of thioredoxin are localized in the C-terminal fragment (peptide A).

The size and number of subunits of the sucrase · isomaltase complex (mol. wt approximately 220000) from rabbit small intestine were determined by dodecylsulfate-polyacrylamide gel electrophoresis, gel filtration and fingerprint analysis of tryptic hydrolysates. It was found that two subunits of similar size (estimated mol. wt 110000 to 120000 each) build up the sucrase · isomaltase complex, the one carrying sucrase, the other isomaltase activity. The isolation of the active isomaltase moiety, along with small amounts of active sucrase as a byproduct, has been achieved. Amino acid and carbohydrate analysis of the two subunits are reported.

The composition and the properties of a protein fraction isolated from the purified spore coats of Bacillus thuringiensis have been studied. This fraction, obtained by action of mercaptoethanol and urea or guanidine on intact coats, shows a strong similarity with crystal protein, with regard to amino acid composition, electrophoretical behaviour and end group analysis (an N-terminal phenylalanine and a C-terminal lysine have been found). Immunological studies indicate a partial homology with parasporal inclusion, but not identity. A similar fraction has also been extracted from the spore coats of an acrystalliferous mutant strain. It is concluded that a crystal-like protein, highly insoluble in character, is one of the components of the spore coats and might be related to other structural proteins associated with the morphogenesis of the spore.

A new Lepore-type hemoglobin in a person of Negro descent has been identified. In this person a minor hemoglobin (Hb-LeporeBaltimore) was detected. An abnormal δβ-chain was isolated and fingerprinted. This hybrid-type protein presumably arose as a consequence of unequal crossing over between the δ and β genes, as was concluded for Hb-LeporeBoston and Hb-LeporeHollandia. In the new Lepore-type hemoglobin described here the crossover could be placed between residues δ50 and β86. This type of crossover should occur with the highest frequency if crossing over is equally frequent between any nucleotide and if selection favours none of the crossover types.

The hemoglobins of a child homozygous for the Lepore Boston gene have been analyzed. Only hemoglobin F and Lepore were observed. Hemoglobin Lepore was isolated and the peptide chains of this hemoglobin were separated. The amino acid composition of the Lepore chain and of the tryptic peptides obtained from this chain was determined. The peptides isolated accounted for the amino acid composition of the Lepore chain and indicated that this chain is 146 amino acid long. The switch region from the δ-like sequence of the Lepore chain to the β-like sequence has been localized between residue 87 and residue 116.

The parasporal inclusion of Bacillus thuringiensis var. Berliner was solubilized by several processes. Action of denaturing and reducing agents on the crystal protein was studied and the dissolution products analyzed in three cases: (a) after treatment with mercaptoethanol and guanidine hydrochloride at pH 7,5, (b) after treatment with a reducing agent at pH 9.5 and (c) after treatment with 0.1 N and 1 N sodium hydroxide. By the first treatment, one N-terminal and one C-terminal amino acid were characterized whereas four N-terminal residues appeared by the other processes performed at pH 9.5 and above pH 12. It is suggested that peptide bonds were disrupted in these latter cases. Ultracentrifugation experiments revealed the existence of only one repetitive subunit of molecular weight 80000 in the solution obtained after treatment by mercaptoethanol and guanidine hydrochloride at pH 7.5. Furthermore reassociated particles obtained from this solution were observed under phase-contrast microscope.

By peptic or tryptic proteolysis of different fragments of bovine encephalitogenic protein, a set of small fragments covering the following regions of the native protein have been obtained: 44–68, 44–89, 75–91, 93–116, 114–130, 134–150, and 154–170 (the amino acid residues are numbered according to [1]). These fragments were used in the macrophage migration inhibition technique in guinea pigs (an assay which is vividly considered as a correlate in vitro to delayed type hypersensitivity) to study the localization of antigenic determinants in the protein. The results indicate the presence of at least one determinant in each of regions 44–68, 134–140, and 154–170. Furthermore, the rather strong determinant, which has previously been localized around the single tryptophan residue in the protein does not seem to be contained within peptide 114–130, as this peptide is considered unreactive in the assay. On the other hand, this peptide shows full encephalitogenic activity.

Amino acid analyses on thioredoxin reductase indicate the following amino acid composition: Asp 65Thr43Ser26Glu60Pro17Gly3Ala58Val32Met12Ileu39Leu53Tyr16Phe18Try2Lys23His18Arg29 (total half-cys)8 (CONH2)59. The enzyme consists of two identical or very similar polypeptide chains joined by noncovalent bonds. This is indicated from experiments employing ultracentrifugation in urea or guanidine hydrochloride, from the analysis of tryptic peptide maps and from quantitative determinations of NH2-terminal amino acids. Carboxymethylation of the enzyme with iodoacetic acid under different conditions showed that each polypeptide chain of the native enzyme contains one disulfide and two free sulfhydryl groups, the latter buried in the enzyme structure. Titration of the enzyme with NADPH under anaerobic conditions demonstrated that the complete reduction of one molecule of enzyme requires four molecules of NADPH. The results indicate that the enzyme contains two redox acceptors in addition to the two molecules of flavin adenine dinucleotide. Evidence is presented that these redox acceptors are identical with the two disulfides of the enzyme. Spectral studies of thioredoxin reductase employing anaerobic titration with substrate did not reveal the formation of a stable red 2-electron intermediate similar to that observed for two other flavoproteins, lipoly dehydrogenase and glutathione reductase. This finding suggests that the catalytic mechanism for thioredoxin reductase is different from that of lipoyl dehydrogenase and glutathione reductase.

A purified glycoprotein with blood-group-A specificity, was digested with insoluble pronase to give a serologically active product enriched in peptide-bonded serine and threonine residues; this was degraded with 0.25 M sulphuric in glacial acetic acid for 24 h. The resultant N-acetylgalactosaminyl-peptides were hydrolysed with 0.2 M hydrochloric acid for 24 h at 110°C. Fractionation of the products on Sephadex G-10, followed by preparative separation on an amino-acid analyser and paper electrophoresis at pH 1.9 yielded the following homogeneous oligopeptides; one tripeptide with the N-terminal sequence Thr-Thr-Ser, three tetrapeptides with the sequences, Thr-Ser-Thr-Ser, Thr-Pro-Thr-Ser and Pro-Thr-Thr-Ser, one pentapeptide with the sequence Thr-Pro-Thr-Ser-Ser, one hexapeptide with the sequence Pro-Thr-Thr-Thr-Pro-Ser and one heptapeptide with the sequence Ala-Pro-Thr-Thr-Ser-Gly-Ser. Galactosamine was present in several of these fragments and the hexapeptide and heptapeptide both contained sufficient of this sugar for all the serine and threonine residues to be glycosylated. The following five tetrapeptides sequences were deduced from mixtures of two peptides which differed by the amino-acid substituent at only one position in the chain; Pro-Thr-Ala-Ser, Thr-Thr-Pro-Ser, Thr-Thr-Ala-Ser, Ser-Pro-Thr-Ser, and Ser-Ala-Thr-Ser. A tripeptide mixture which differed only in the nature of the third amino acid yielded the following three tripeptide sequences: Pro-Thr-Ser, Pro-Thr-Gly and Pro-Thr-Ala. These peptides do not indicate a clearly defined amino-acid sequence surrounding the glycosylated hydroxy amino acids but establish that in the human blood-group-specific glycoproteins there is close packing of the serine and threonine residues in the region of the peptide moiety carrying the carbohydrate chains.

Histidine decarboxylase from Micrococcus sp.n. was carboxymethylated and two types of subunits, I and II, were separated in excellent yield by exclusion chromatography on Sephadex G-100 in 8 M urea. Total compositions, number of tryptic peptides and N-terminal groups of the subunits were analyzed. The larger subunit I is composed of all amino acids commonly found in proteins and has a blocked N-terminal residue, whereas the smaller subunit II does not contain proline, histidine or cysteine and has N-terminal methionine. The number of tryptic peptides detected in peptide mapping experiments of subunit I is about 27 and that of subunit II is about 15. These figures are in good agreement with the total number of lysine and arginine residues determined by amino acid analysis, if subunit I contains about 270 residues, corresponding to a molecular weight of 29000, and subunit II about 70 residues, corresponding to a molecular weight of 7000. It is concluded that histidine decarboxylase contains two apparently homogeneous but completely different polypeptide chains and that the native enzyme of molecular weight 110000 is a hexamer with three subunits of each type.

Highly purified calf thymus ALK-histone (histone rich in alanine, lecine and lysine) was isolated from F2a2 histone fraction by ion-exchange chromatography on Biorex 70 followed by gel filtration chromatography on Sephadex G-100. Peptides obtained by enzymatic hydrolyses (trypsin, chymotrypsin, thermolysin) of native or maleylated protein were fractionated by ion-exchange chromatography on Chromobeads P. Thermolysin peptides which account for 127 of the 129 residues of the protein provide overlaps of tryptic and chymotryptic peptides and led us to establish the complete amino-acid sequence of ALK-histone as follows: Ser1Gly-Arg-Gly-Lys-Gln-Gly-Gly-Lys-Ala-10Arg-Ala-Lys-Ala- Lys-Thr-Arg-Ser-Ser-Arg20-Ala-Gly-Leu-Gln-Phe-Pro-Val-Gly-Arg-Val30-His-Arg-Leu-Leu-Arg-Lys-Gly-Asn-Tyr-Ala40-Glu-Arg-Val-Gly-Ala-Gly-Ala-Pro-Val-Tyr50-Leu-Ala-Ala-Val-Leu-Glu-Tyr-Leu-Thr-Al60-Glu-Ile-Leu-Glu-Leu-Ala-Gly-Asn-Ala-Ala70-Arg-Asp-Asn-Lys-Lys-Thr-Arg-Ile-Ile-Pro80-Arg-His-Leu-Gln-Leu-Ala-Ile-Arg-Asn-Asp90-Glu-Glu-Leu-Asn-Lys-Leu-Leu-Gly-Lys-Val100-Thr-Ile-Ala-Gln-Gly-Gly-Val-Leu-Pro-Asn110-Ile-Gln-Ala-Val-Leu-Leu-Pro-Lys-Lys-Thr120Glu-Ser-His-His-Lys-Ala-Lys-Gly-Lys129. The amino end is acetylated. The sequence of ALK-histone is characterized by two hydro-phobic regions, the first from Val-43 to Ala-70 and the second from Val-100 to Pro-117 and presents many repetitive or analogous sequences. The NH2-terminal sequence of ALK-histone is highly basic and shows a remarkable analogy with that of GRK-histone. A cluster of five basic residues His-His-Lys-Ala-Lys-Gly-Lys is present at the COOH-terminal end.

Peptide B, the N-terminal fragment of thioredoxin obtained by cyanogen bromide treatment was aminoethylated and digested with pepsin. Eight peptides were separated by high voltage paper electrophoresis and chromatography and characterized with respect to amino acid sequence. The results account for all 37 residues of peptide B. The N-terminal sequence of peptide B was determined as: Ser-Asp-Lys-Ile-Ile-His-Leu-Thr-Asp-Asp-Ser-Phe.

The two rat proinsulins have been separated and the amino acid compositions determined. Using a method previously described for preparing C-peptides from ox and pork pancreases, three peptides were isolated from rat pancreases and the amino acid compositions of these peptides determined. Data are presented which strongly indicate that two of these peptides constitute the C-peptide part of the connecting peptide of the two rat proinsulins.

Low molecular weight solutes released during mild acid hydrolysis of the lipopolysaccharide of Pseudomonas aeruginosa were isolated from the fraction containing the partially degraded polysaccharide, by successive chromatography on columns of Sephadex G-75 and G-10, followed (as necessary) by preparative high voltage paper electrophoresis. The major components identified were 2-keto-3-deoxyoctonic acid, basic amino acids (free and bound), inorganic orthophosphate, ethanolamine phosphate and ethanolamine pyrophosphate. Ethanolamine pyrophosphate has not previously been found in acid hydrolysates of lipopolysaccharides. Ethanolamine phosphate and some of the orthophosphate were apparently produced by breakdown of ethanolamine pyrophosphate.

The glycine-rich, arginine-rich histone from calf thymus contains 102 residues of amino acids, distributed as follows: Asp5, Thr7, Ser2, Glu6, Pro1, Gly17, Ala7, Val9, Met1, Ile6, Leu8, Tyr4, Phe2, Lys10, Lys(Me)1, His2, Arg14. From a tryptic hydrolysate of Gly- and Arg-rich histone, 18 peptides were isolated by fractionation on a column of Chromobeads P followed by purification by paper electrophoresis and chromatography. The amino acid compositions of these peptides are reported. The sequence of 12 of these tryptic peptides has been established. The amino-terminal tryptic peptide is Ac-Ser-Gly-Arg. The carboxylterminal tryptic peptide is Thr-Leu-Tyr-Gly-Phe-Gly-GlyCOOH.

The amino-acid composition of a polypeptide fraction that contains a few closely related polypeptides was determined. these polypeptides are obtained in high yield from red kidney beans and stimulate RNA synthesis in a bacterial system and in mouse spleen lymphocytes. Peptide maps of the carboxymethylated fraction were prepared and tryptic peptides purified and analysed for total composition and amino acid sequence. The results revealed that the differences between the polypeptides are due to a few microheterogeneities in the form of amino acid exchanges, but no evidence for the existence of polypeptides of different size or overall structure were obtained. It is concluded that the purification method yields different types of highly similar polypeptides with a molecular weight in the range of 10000, corresponding to about 80 residues, and an unusually high cysteine content of 19 mol/100 mol. These facts show that the present polypeptides are different from all previously analysed factors stimulating RNA synthesis.

Bovine submaxillary mucin, after removal of its sialic acid component, is cleaved by trypsin into two principal glycopeptide fractions; their final yield corresponds to 70% by weight of the original mucin and 85% of the original amount of hexosamine in the mucin. These glycopeptides have amino acid and carbohydrate compositions that are very similar to those of the original and desialyzed mucin. The main glycopeptide contains about 28 amino acids; the second one seems to be a covalently linked trimer of the former and is resistant to further action by trypsin. The protein core of bovine submaxillary mucin is composed mainly of several hundred covalently bound sequences of these glycopeptides. These results are compatible with those reported by Downs and Pigman (1969) for the products obtained by chemical cleavage of this mucin.

The purification and chemical characterization of three Bence-Jones proteins and one light chain of an IgG myeloma protein of the rat strain LOU/Wsl are described. The amino acid composition of the four chains is very similar but not identical. Fingerprint analyses show a pattern of peptides common to all four proteins as well as unique features. The compositions of the common peptides are given. The data indicate that the four light chains are of the kappa type.

The reaction of phosphorothioate with bovine pancreatic ribonuclease at pH 9.0 in the absence of urea led to a derivative, denoted 4PS-ribonuclease, in which two of the four disulphide bonds in the molecule were opened. 4PS-ribonuclease is a unique molecular species, and not a mixture of native and open-chain ribonuclease; this is apparent from electrophoretic studies. By the use of the “diagonal map” technique it has been established unequivocally that the disulphide bonds opened were between half-cystines 3–8 and 4–5, counting from the amino terminus along the ribonuclease chain. 4PS-ribonuclease is fully active enzymically toward RNA, and more active than the native enzyme toward cytidine 2′,3′-cyclic phosphate. It is indistinguishable from native ribonuclease in its immunological reaction with antiserum to ribonuclease, and in its spectrophotometric titration, which shows that three of the six tyrosine phenolic groups in 4PS-ribonuclease titrate abnormally. Unlike native ribonuclease, 4PS-ribonuclease is digested by trypsin, even though at a lower rate than open-chain ribonuclease.