Purification and partial amino acid sequence of plantaricin S, a bacteriocin produced by Lactobacillus plantarum LPCO10, the activity of which depends on the complementary action of two peptides.

Plantaricin S, one of the two bacteriocins produced by Lactobacillus plantarum LPCO10, which was isolated from a green-olive fermentation (R. Jiménez-Díaz, R.M. Ríos-Sánchez, M. Desmazeaud, J.L.Ruiz-Barba, and J.-C. Piard, Appl. Environ. Microbiol. 59:1416-1424, 1993), has been purified to homogeneity by ammonium sulfate precipitation, by binding to SP-Sepharose fast-flow, phenyl-Sepharose CL-4B, and C2/C18 reverse-phase chromatographies. The purification resulted in a final yield of 91.6% and a 352,617-fold increase in the specific activity. The bacteriocin activity was associated with two distinct peptides, termed alpha and beta, which were separated by C2/C18 reverse-phase chromatography. Although beta alone appeared to retain a trace of inhibitory activity, the complementary action of both the alpha and beta peptides was required for full bacteriocin activity, as judged by both the agar well diffusion and the microtiter plate assays. From the N-terminal end, 26 and 24 amino acids residues of alpha and beta, respectively, were sequenced. Further attempts at sequencing revealed no additional amino acids residues, suggesting that either modifications in the next amino acid residue blocked the sequencing region or that the C-terminal end had been reached. The amino acid sequences of alpha and beta show no apparent homology to each or to other bacteriocins purified from lactic acid bacteria.     

Biological and molecular characterization of a two-peptide lantibiotic produced by Lactococcus lactis IFPL105

The lactic acid bacterium Lactococcus lactis IFPL105 secretes a broad spectrum bacteriocin produced from the 46 kb plasmid pBAC105. The bacteriocin was purified to homogeneity by ionic and hydrophobic exchange and reverse-phase chromatography. Bacteriocin activity required the complementary action of two distinct peptides (alpha and beta) with average molecular masses of 3322 and 2848 Da, respectively. The genes encoding the two peptides were cloned and sequenced and were found to be identical to the ltnAB genes from plasmid pMRC01 of L. lactis DPC3147. LtnA and LtnB contain putative leader peptide sequences similar to the known 'double glycine' type. The predicted amino acid sequence of mature LtnA and LtnB differed from the amino acid content determined for the purified alpha and beta peptides in the residues serine, threonine, cysteine and alanine. Post-translational modification, and the formation of lanthionine or methyllanthionine rings, could partly explain the difference. Hybridization experiments showed that the organization of the gene cluster in pBAC105 responsible for the production of the bacteriocin is similar to that in pMRC01, which involves genes encoding modifying enzymes for lantibiotic biosynthesis and dual-function transporters. In both cases, the gene clusters are flanked by IS946 elements, suggesting an en bloc transposition. The findings from the isolation and molecular characterization of the bacteriocin provide evidence for the lantibiotic nature of the two peptides.     

Lactococcin Q, a novel two-peptide bacteriocin produced by Lactococcus lactis QU 4

A bacteriocin-producing strain, Lactococcus lactis QU 4, was isolated from corn. The bacteriocin, termed lactococcin Q, showed antibacterial activity only against L. lactis strains among a wide range of gram-positive indicator strains tested. Lactococcin Q was purified by acetone precipitation, cation exchange chromatography, and reverse-phase chromatography. Lactococcin Q consisted of two peptides, alpha and beta, whose molecular masses were determined to be 4,260.43 Da and 4,018.36 Da, respectively. Amino acid and DNA sequencing analyses revealed that lactococcin Q was a novel two-peptide bacteriocin, homologous to lactococcin G. Comparative study using chemically synthesized lactococcin Q (Qalpha plus Qbeta) and lactococcin G (Galpha plus Gbeta) clarified that hybrid combinations (Qalpha plus Gbeta and Galpha plus Qbeta) as well as original combinations showed antibacterial activity, although each single peptide showed no significant activity. These four pairs of lactococcin peptides acted synergistically at a 1:1 molar ratio and exhibited identical antibacterial spectra but differed in MIC. The MIC of Qalpha plus Gbeta was 32 times higher than that of Qalpha plus Qbeta, suggesting that the difference in beta peptides was important for the intensity of antibacterial activity.     

Purification and genetic characterization of plantaricin NC8, a novel coculture-inducible two-peptide bacteriocin from Lactobacillus plantarum NC8

A new, coculture-inducible two-peptide bacteriocin named plantaricin NC8 (PLNC8) was isolated from Lactobacillus plantarum NC8 cultures which had been induced with Lactococcus lactis MG1363 or Pediococcus pentosaceus FBB63. This bacteriocin consists of two distinct peptides, named alpha and beta, which were separated by C(2)-C(18) reverse-phase chromatography and whose complementary action is necessary for full plantaricin NC8 activity. N-terminal sequencing of both purified peptides showed 28 and 34 amino acids residues for PLNC8 alpha and PLNC8 beta, respectively, which showed no sequence similarity to other known bacteriocins. Mass spectrometry analysis showed molecular masses of 3,587 Da (alpha) and 4,000 Da (beta). The corresponding genes, designated plNC8A and plNC8B, were sequenced, and their nucleotide sequences revealed that both peptides are produced as bacteriocin precursors of 47 and 55 amino acids, respectively, which include N-terminal leader sequences of the double-glycine type. The mature alpha and beta peptides contain 29 and 34 amino acids, respectively. An open reading frame, orfC, which encodes a putative immunity protein was found downstream of plNC8B and overlapping plNC8A. Upstream of the putative -35 region of plNC8B, two direct repeats of 9 bp were identified, which agrees with the consensus sequence and structure of promoters of class II bacteriocin operons whose expression is dependent on an autoinduction mechanism.     

Chemical characterization and purification of the beta subunit of the DNA polymerase III holoenzyme from an overproducing strain

We have purified the beta subunit of the DNA polymerase III holoenzyme to homogeneity from an overproducing strain (Blanar, M., Sandler, S., Armengod, M., Ream, L., and Clark, A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4622-4626). From this procedure we can obtain 100 mg quantities of protein. The beta isolated from the overproducer is indistinguishable from that isolated from wild-type cells in terms of its activity and molecular weight. Partial amino acid sequence analysis has confirmed the DNA sequence of the dnaN gene (Ohmori, H., Kimura, M., Nagata, T., and Sakakibara, Y. (1984) Gene (Amst.) 28, 159-170) and established the sites for initiation and termination of translation. No processing that removes amino acid residues from beta occurs since the active protein begins with the initiating methionine and terminates at the position predicted from the DNA sequence. Our knowledge of the precise amino acid composition has been used to determine the extinction coefficient of beta to be 17,900 and 18,700 cm-1 M-1 at 280 and 277 nm, respectively. The extinction coefficient at 280 nm is reduced to 14,700 cm-1 M-1 under denaturing conditions in guanidine HCl. Conditions have been optimized so that 1 N-ethylmaleimide residue can be incorporated per beta monomer with full preservation of activity.     

Purification and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici.

A bacteriocin produced by Pediococcus acidilactici has been purified to homogeneity by a rapid and simple four-step purification procedure which includes ammonium sulphate precipitation, chromatography with a cation-exchanger and Octyl Sepharose, and reverse-phase chromatography. The purification resulted in an approximately 80,000-fold increase in the specific activity and about a 6-fold increase in the total activity. The amino acid composition and sequencing data indicated that the bacteriocin contained 43-44 amino acid residues. The predicted M(r) and isolectric point of the bacteriocin are about 4600 and 8.6, respectively. Comparing the amino acid sequence of this bacteriocin with the sequences of leucocin A-UAL 187, sakacin P and curvacin A (bacteriocins produced by Leuconostoc gelidum, Lactobacillus sake and Lactobacillus curvatus, respectively) revealed that all four bacteriocins had in their N-terminal region the sequence Tyr-Gly-Asn-Gly-Val-Xaa-Cys, indicating that this concensus sequence is of fundamental importance for this group of bacteriocins. The bacteriocin from P. acidilactici and sakacin P were very similar, having at least 25 common amino acid residues. The sequence similarity was greatest in the N-terminal half of the molecules--17 of the first 19 residues were common--indicating the fundamental importance of this region. Leucocin A-UAL 187 and curvacin A had, respectively, at least 16 and 13 amino acid residues in common with the bacteriocin from P. acidilactici.     

Identification and nucleotide sequence of genes involved in the synthesis of lactocin 705, a two-peptide bacteriocin from Lactobacillus casei CRL 705

The structural gene determinants of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705, have been amplified from a plasmid of approximately 35 kb and sequenced. Lactocin 705 is a class IIb bacteriocin, whose activity depends upon the complementation of two peptides (705alpha and 705beta) of 33 amino acid residues each. These peptides are synthesized as precursors with signal sequences of the double-glycine type, which exhibited high identities with the leader peptides of plantaricin S and J from Lactobacillus plantarum, brochocin C from Brochotrix campestris, sakacin P from Lactobacillus sake, and the competence stimulating peptides from Streptococcus gordonii and Streptococcus mitis. However, the two mature bacteriocins 705alpha and 705beta do not show significant similarity to other sequences in the databases.     

Purification, characterization, and sequencing of novel antimicrobial peptides, Tu-AMP 1 and Tu-AMP 2, from bulbs of tulip (Tulipa gesneriana L.)

Novel antimicrobial peptides (AMP), designated Tu-AMP 1 and Tu-AMP 2, were purified from the bulbs of tulip (Tulipa gesneriana L.) by chitin affinity chromatography and reverse-phase high-performance liquid chromatography (HPLC). They bind to chitin in a reversible way. They were basic peptides having isoelectric points of over 12. Tu-AMP 1 and Tu-AMP 2 had molecular masses of 4,988 Da and 5,006 Da on MALDI-TOF MS analysis, and their extinction coefficients of 1% aqueous solutions at 280 nm were 3.3 and 3.4, respectively. Half of all amino acid residues of Tu-AMP 1 and Tu-AMP 2 were occupied by cysteine, arginine, lysine, and proline. The concentrations of peptides required for 50% inhibition (IC(50)) of the growth of plant pathogenic bacteria and fungi were 2 to 20 microg/ml. The structural characteristics of Tu-AMP 1 and Tu-AMP 2 indicated that they were novel thionin-like antimicrobial peptides, though Tu-AMP 2 was a heterodimer composes of two short peptides joined with disulfide bonds.     

Purification and partial amino acid sequence of plantaricin 1.25ααα and 1.25βββ, two bacteriocins produced by Lactobacillus plantarum TMW1.25

Two bacteriocins produced by Lactobacillus plantarum TMW1.25 have been purified by a four-step purification procedure, including ammonium sulphate precipitation and cation-exchange chromatography followed by hydrophobic-interaction chromatography on octyl sepharose. The final purification was performed by repeated reversed-phase chromatography steps which yielded two bacteriocin fractions designated plantaricin 1.25 alpha and plantaricin 1.25 beta. The molecular masses of the peptides in these fractions were 5979 and 5203 Da, respectively. Combination of the fractions did not have any synergistic effects on bacteriocin activity, indicating that they each contain a one-peptide bacteriocin. The major peptide in the alpha fraction was blocked at its N-terminus, and a partial sequence (25 residues) could only be obtained after cleavage with CNBr. This sequence did not show clear homologies with known bacteriocins. The beta peptide has been sequenced almost completely and consists, presumably, of 53 residues. This peptide displayed strong homology to the known N-terminal part of brevicin 27 produced by Lactobacillus brevis SB27. The results showed that the beta peptide contains as many as six consecutive lysine residues at the N-terminus.     

A nicked beta-subunit of human chorionic gonadotropin purified from pregnancy urine.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone composed of two dissimilar subunits (alpha and beta) and normally excreted in urine of pregnant women. An uncommon beta-subunit of hCG was purified from fresh early normal pregnancy urine by Sepralyte C8, resin adsorption. Sephadex G-100 column chromatography, and reverse-phase HPLC. SDS-PAGE under non-reducing conditions showed that the apparent molecular weight (39,000) of this beta-subunit was extremely similar to that of the native beta-subunit, which is known to consist of 145 amino acid residues and carbohydrates. However, SDS-PAGE, under reducing conditions, resulted in two bands with apparent molecular weights of 22,000 and 18,000, indicating that it consisted of two peptide fragments connected with disulfide bridge(s). These two peptide fragments, separated and purified from the reduced and carboxymethylated protein, were subjected to amino acid and N-terminal sequence analyses. It was found that this beta-subunit consisted of two polypeptide chains composed of residues 1-47 disulfide-bridged to residues 48-145 of the beta-subunit, which may be produced by nicking of the beta-subunit at the one site (Gly47-Val48). This beta-subunit was termed a nicked beta-subunit of hCG (N-hCG beta). It was also found that N-hCG beta was present in urine as an alpha beta dimer, indicating that an intrachain nicking of this site in the beta-subunit does not inhibit alpha beta dimer formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a new bacteriocin isolated from a Carnobacterium sp.

A bacteriocin-producing Carnobacterium sp. was isolated from fish. The bacteriocin, termed carnocin UI49, was purified to homogeneity by a four-step purification procedure, including hydrophobic interaction chromatography and reverse-phase chromatography. Carnocin UI49 has a bactericidal mode of action. It was shown to be heat tolerant and stable between pH 2 and 8. At pH above 8, carnocin UI49 was rapidly inactivated. Amino acid analysis revealed a composition of about 35 to 37 amino acids in addition to an unidentified peak which migrates at the position of lanthionine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggests a molecular weight of about 4,500 to 5,000. Mass spectrometry gave a molecular weight of 4,635, which is about 1,000 larger than that calculated from the amino acid analysis data. Performic acid oxidation of carnocin UI49, followed by amino acid hydrolysis, revealed the presence of cysteic acid. The sequence of the first seven amino acid residues was determined to be N-Gly-Ser-Glu-Ile-Gln-Pro-Arg. After the seventh amino acid, carnocin UI49 was not available for further Edman degradation. The results suggest that carnocin UI49 belongs to the class of bacteriocins termed lantibiotics.     

Characterization of the bilin attachment sites in R-phycoerythrin

The amino acid sequence around the sites of attachment of all the bilin prosthetic groups of Gastroclonium coulteri R-phycoerythrin, (alpha beta)6 gamma, have been determined. The sequences of tryptic peptides derived from the alpha and beta subunits are (Formula: see text) where the designations alpha and beta refer to the subunits from which the peptides derived. Cysteinyl residues involved in bilin attachment are indicated with an asterisk. Each peptide carries a single bilin, either phycoerythrobilin (PEB) or phycourobilin (PUB). Spectroscopic studies on the gamma subunit indicate the presence of one PEB and three PUB groups. However, five unique tryptic peptides, gamma-A through gamma-E, were characterized, indicating that Gastroclonium R-phycoerythrin is a mixture of at least two species, (alpha beta)6 gamma and (alpha beta)6 gamma', with gamma subunits differing in amino acid sequence. The sequences of the gamma subunit bilin peptides (see below) were not homologous to those from alpha and beta subunits of any biliprotein. (Formula: see text) The bilins in all these peptides are attached through single linkages to a cysteinyl residue, except for the phycourobilin on peptide beta-3 which is attached through two thioether linkages to cysteinyl residues 10 amino acids apart. The availability of small bilin peptides was exploited to obtain more accurate molar extinction coefficients for peptide-linked PEB and PUB groups. Application of these extinction coefficients in the calculation of the bilin content of R-, B-, and C-phycoerythrins shows that there are 5 bilins/alpha beta in each of these three biliprotein types.     

Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins.

Spectroscopic measurement of protein concentration requires knowledge of the value of the relevant extinction coefficient. If the amino acid composition of a protein is known, however, extinction coefficients can be calculated approximately, provided that the values of the molar absorptivities for tryptophan and tyrosine residues in the protein are known. We have applied a matrix linear regression procedure and a mapping of average absolute deviations between experimental and calculated values to find molar extinction coefficients (epsilon M, 1 cm, 280 nm) of 5540 M-1 cm-1 for tryptophan and 1480 M-1 cm-1 for tyrosine residues in an "average" protein, as defined by a set of experimentally determined extinction coefficients for more than 30 proteins. Use of these values provides a significant improvement in extinction coefficient estimation over that obtained with the commonly used values obtained from solutions of model compounds in guanidine-HCl. The consistency of these results when compared to the large deviations often observed between experimentally determined extinction coefficients suggest that this method may offer acceptable accuracy in the initial estimation of molar absorptivities of globular proteins.     

Simple method of purification and sequencing of a bacteriocin produced by Pediococcus acidilactici UL5

H. DABA, C. LACROIX, J. HUANG, R.E. SIMARD AND L. LEMIEUX. 1994. A bacteriocin produced by a strain of Pediococcus acidilactici was successfully purified sequentially by acid extraction (at pH 2) and reverse-phase high-performance liquid chromatography (HPLC). Cell extracts of derivative strains deficient in bacteriocin production exhibited a similar HPLC elution profile to the active extracts except for the two peaks containing bacteriocin activity. The sequence of the antibacterial peptide consisted of 44 amino acid residues of which 42 were identified, and its molecular weight was 4624 Da, as determined by mass spectrometry. Moreover, according to the molecular weight of the peptide, the unidentified residues in the bacteriocin sequence must correspond to two tryptophan residues, confirming that the peptide isolated from Ped. acidilactici UL5 is pediocin PA-1. However, oxidized forms of the bacteriocin produced during storage also showed bacteriocin activity and resulted in a second peak with activity in the chromatograms. HPLC chromatograms of cell surface preparations from the active and from the deficient strains were confirmed by capillary electrophoresis. The purification method used is simple and effective in comparison with traditional methods, permitting a selective recovery of cell-associated bacteriocin at low pH, and its isolation in pure form for sequencing.     

Isolation, partial characterization, and mode of action of Acidocin J1132, a two-component bacteriocin produced by Lactobacillus acidophilus JCM 1132.

Lactobacillus acidophilus JCM 1132 produces a heat-stable, two-component bacteriocin designated acidocin J1132 that has a narrow inhibitory spectrum. Maximum production of acidocin J1132 in MRS broth was detected at pH 5.0. Acidocin J1132 was purified by ammonium sulfate precipitation and sequential cation exchange and reversed-phase chromatographies. Acidocin J1132 activity was associated with two components, termed alpha and beta. On the basis of N-terminal amino acid sequencing and the molecular masses of the alpha and beta components, it is interpreted that the compounds differ by an additional glycine residue in the beta component. Both alpha and beta had inhibitory activity, and an increase in activity by the complementary action of the two components was observed. Acidocin J1132 is bactericidal and dissipates the membrane potential and the pH gradient in sensitive cells, which affect such proton motive force-dependent processes as amino acid transport. Acidocin J1132 also caused efflux of preaccumulated amino acid taken up via a unidirectional ATP-driven transport system. Secondary structure prediction revealed the presence of an amphiphilic alpha-helix region that could form hydrophilic pores. These results suggest that acidocin J1132 is a pore-forming bacteriocin that creates cell membrane channels through the "barrel-stave" mechanism.     

Characterization of cytochrome c3 from the thermophilic sulfate reducer Thermodesulfobacterium commune

A c3 type cytochrome has been purified from the thermophilic, non-spore-forming, sulfate-reducing bacterium Thermodesulfobacterium commune. The purified protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and isoelectric focusing. A pI of 6.83 was observed. The molecular weight of the cytochrome was estimated to be ca. 13,000 from both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hemoprotein exhibited absorption maxima at 530, 408.5, and 351 nm in the oxidized form and 551.5 (alpha band), 522.5 (beta band), and 418.5 nm (gamma band) in the reduced form. The extinction coefficients of T. commune cytochrome c3 were 130,000, 74,120, and 975,000 M-1 cm-1 at 551.5, 522.5, and 418.5 nm, respectively. It contains four hemes per molecule, on the basis of both the iron estimation and the extinction coefficient value of its pyridine hemochrome. The amino acid composition showed the presence of eight cysteine residues involved in heme binding. T. commune cytochrome c3 had low threonine, serine, and glycine contents and high glutamic acid and hydrophobic residue contents. The electrochemical study of T. commune cytochrome c3 by cyclic voltammetry and differential pulse polarography has shown that the cytochrome system behaves like a reversible system. Four redox potential values at Eh1 = -0.140 +/- 0.010 V, Eh2 = Eh3 = Eh4 = -0.280 +/- 0.010 V have been determined. T. commune cytochrome c3, which acts as the physiological electron carrier of hydrogenase, is similar in most respects to the multiheme low-potential cytochrome c3 which is characteristic of the genus Desulfovibrio.     

Extensive post-translational modification, including serine to D-alanine conversion, in the two-component lantibiotic, lacticin 3147

Lacticin 3147 is a two-component bacteriocin produced by Lactococcus lactis subspecies lactis DPC3147. In order to further characterize the biochemical nature of the bacteriocin, both peptides were isolated which together are responsible for the antimicrobial activity. The first, LtnA1, is a 3,322 Da 30-amino acid peptide and the second component, LtnA2, is a 29-amino acid peptide with a mass of 2,847 Da. Conventional amino acid analysis revealed that both peptides contain the thioether amino acid, lanthionine, as well as an excess of alanine to that predicted from the genetic sequence of the peptides. Chiral phase gas chromatography coupled with mass spectrometry of amino acid composition indicated that both LtnA1 and LtnA2 contain D-alanine residues and amino acid sequence analysis of LtnA1 confirmed that the D-alanine results from post-translational modification of a serine residue in the primary translation product. Taken together, these results demonstrate that lacticin 3147 is a novel, two-component, D-alanine containing lantibiotic that undergoes extensive post-translational modification which may account for its potent antimicrobial activity against a wide range of Gram-positive bacteria.     

The primary structure of D-amino acid oxidase from pig kidney. I. Isolation and sequence of the tryptic peptides

D-Amino acid oxidase from pig kidney cortex was digested with trypsin. Thirty-two tryptic peptides were isolated by ion exchange chromatography, high voltage paper electrophoresis, descending paper chromatography, and reverse-phase high performance liquid chromatography. The last method permitted the isolation of 29 tryptic peptides, many in a single step, in yields usually greater than 75%. The purified peptides were characterized by amino acid analysis and their sequences determined by the manual 5-dimethylaminonaphthalene-1-sulfonyl-Edman degradation procedure or by the automated Edman-Begg degradation method. These peptides accounted for all 12 lysine and 21 arginine residues observed by amino acid analysis of the intact protein and for 347 amino acid residues of the 345 predicted by the analysis.     

Molecular view by fourier transform infrared spectroscopy of the relationship between lactocin 705 and membranes: speculations on antimicrobial mechanism

Lactocin 705 is a bacteriocin whose activity depends upon the complementation of two peptides, termed Lac705alpha and Lac705beta. Neither Lac705alpha nor Lac705beta displayed bacteriocin activity by itself when the growth of sensitive cells was monitored. To obtain molecular insights into the lactocin 705 mechanism of action, Fourier transform infrared spectroscopy was used to investigate the interactions of each peptide (Lac705alpha and Lac705beta) with dipalmitoylphosphatidylcholine liposomal membranes. Both peptides show the ability to interact with the zwitterionic membrane but at different bilayer levels. While Lac705alpha interacts with the interfacial region inducing dehydration, Lac705beta peptide interacts with only the hydrophobic core. This paper presents the first experimental evidence that supports the hypothesis that Lac705alpha and Lac705beta peptides could form a transmembrane oligomer. From the obtained results, a mechanism of action of lactocin 705 on membrane systems is proposed. The component Lac705alpha could induce the dehydration of the bilayer interfacial region, and the Lac705beta peptide could insert in the hydrophobic region of the membrane where the peptide has adequate conditions to achieve the oligomerization.     

Purification and characterization of human recombinant interleukin-1 beta

A human interleukin-1 (IL-1) beta cDNA was cloned, and the region coding for the mature protein was expressed in Escherichia coli. The 17-kDa biologically active product was purified in 40% yield to apparent homogeneity, without chaotropes, from the soluble fraction of sonicated cell lysates. The recombinant IL-1 beta was characterized by amino acid analysis, NH2- and COOH-terminal sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, and biological assay. Specific biological activity was 4.6 X 10(8) units/mg in a co-mitogenic IL-2 induction assay using cultured EL-4 T-lymphocytes. The molar extinction coefficient was determined to be 10,300 cm-1 M-1 at 280 nm. NH2-terminal sequence analysis revealed that 70% of the product begins with the Ala corresponding to the NH2 terminus of the natural protein, while 30% begins with the following Pro. No initiator Met was observed. Both of the sulfhydryl groups are reactive to Ellman's reagent and to iodoacetamide under nonreducing conditions, indicating that the Cys residues do not form disulfide bonds. S-Carboxamidomethyl-Cys-rIL-1 beta retained biological activity in the IL-2 induction assay. Circular dichroism suggested an extensive beta sheet structure for rIL-1 beta.