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Previous methods which have been used for the preparation of protein A, such as thermal release of protein A with Iysozyme from the bacteria, have given a heterogenous product in a low yield. The present publication reports the isolation procedure of protein A from Staphylococcus aareus digested by lysostaphin for 2 h at 37 "C. 450-500 mg of protein A was obtained from 300 g of wet bacteria. The preparation is homogenous as seen from its disc electrophoretic pattern.Amino acid composition was determined and based on a molecular weight of 42000. The number of amino acid residues found was 378 f 6. Less than 0.2O/, hexosamines was present.The ultraviolet spectrum and the absorption coefficient, = 1.65 of protein A is given. Less than 0.1 mol amino terminal amino acid per mol protein was detected which indicates that the protein has a blocked N-terminal amino acid. The protein was slowly digested with a mixture of carboxypeptidase A and B and 2 mol lysine/mol protein was released.Precipitation of protein A with normal human y-globulin gave an IgG: protein A molar ratio of 2.1 : 1 within the equivalence zone.The results are discussed with regard to the subunit structure of the protein and the type of attachment of protein A to the peptidoglycan part of the cell wall.

A novel effective procedure for the purification of cathepsin D inhibitor from potatoes (PDI) was developed. The amino acid sequence of PDI was determined by analysis of the cyanogen bromide digest and of the limited tryptic and chymotryptic digests of the protein. The inhibitor is a single polypeptide chain protein consisting of 188 residues with a simple sugar moiety attached to Asn-19. The tentative disulfide pairings are also suggested. The sequence data clearly indicate that PDI is homologous with the soybean trypsin inhibitor (STI) (Kunitz) family. The active center of PDI for trypsin inhibition was identified as Pro-Val-Arg-Phe in analogy to STI.

Thermitase, a thermostable alkaline proteinase, consists of a single polypeptide chain, containing 279 amino acid residues (MI = 28 369). The enzyme shows remarkable structural features of proteinases of the subtilisin type as shown by pronounced sequential homologies. The amino acid replacements, insertions and deletions observed when the amino acid sequence of the enzyme is compared with the sequences of several subtilisins are discussed with respect to substrate specificity and expected tertiary structure. The existence of a cysteinecontaining subgroup of subtilisin-like proteinases is postulated.Thermitase Subtilisin Amino acid sequence Homology Secondary structure prediction Cysteine-containing subtilisin-like proteinase

Nitration of pancreatic trypsin inhibitor with tetranitromethane affords a mono-and a disubstituted derivative which was shown by sequential analysis to contain nitrotyrosine in position 10 and in positions 10 and 21, respectively. Both retain full trypsin inhibitor activity. Measurements of optical rotatory dispersion in the 230 nm region indicate that the secondary structure of the protein is unaffected by nitration. The nitrated derivatives exhibit a conformation-dependent side-chain Cotton effect in the region of nitroaromatic absorption. No such Cotton effect is shown by a nitrotyrosine-containing pentadecapeptide isolated from the tryptic digest from the nitrated and oxidized inhibitor. We have earlier shown [a] that the optical rotatory dispersion curve of pancreatic trypsin inhibitor shows a Cotton effect at 278 nm due to the tyrosine residues and that this effect is strongly dependent on conformation. It therefore appeared of interest to modify the tyrosine residues and to study the effect of such modification on the inhibitory activity and optical rotatory dispersion behaviour of pancreatic trypsin inhibitor. The recently introduced tetranitromethane reagent [9-111 has accordingly been used to nitrate pancreatic trypsin inhibitor and the properties of the nitrated product have been investigated. MATERIAL AND METHODSPancreatic trypsin inhibitor was prepared and characterized as described in the preceding paper [4].Non-Standard Abbreviation. Dansyl, I-dimethylaminonaphthalene-5-sulfonyl. Enzyme. Trypsin (EC 3.4.4.4).Trypsin used for the digestion was a crystalline preparation obtained from LBFiva (Prague) and was recrystallized three times with magnesium sulfate. Its residual chymotryptic activity was inhibited by reaction with l-chloro-4-phenyl-3-tosylamino-2-bu-The activity of trypsin was determined according to Nagel et al.[13] from the increase of absorbance at 405 nm due to the p-nitraniline liberated from benzoyl-DL-arginine p-nitranilide.Pancreatic trypsin inhibitor activity was determined by a difference method. The solution to be assayed (50 pl) was incubated with 100 pl of 0.1 M Tris-HC1 buffer, pH 7.8, and 250 pl of 0.001 N HC1 containing 40 pg of trypsin at 25" for 1, 5, I0 or 15 minutes and the activity of the remaining trypsin was determined as above.Protein concentrations were measured spectrophotometrically using A&ilo = 17.24 for trypsin s o htions and AiAlo = 8.33 for solutions of the inhibitor.Ultraviolet absorption spectra and optical rotatory dispersion curves were measured on a Jasco ORD/UV-5 recording spectrophotometer and spectropolarimeter. The absorption spectra were recorded in 1 cm cells. Solutions in 0.05 M sodium acetate (pH 4.6) and in 0.1 M Tris-HC1 buffer (pH 7.8) were prepared from weighed amounts of the dry protein (about 0.04 g/100 ml). Optical rotatory dispersion measurements were performed with the same solutions in cells of 1.0, 0.1, and 0.01 dm light path. For some optical rotatory dispersion measurements anhydrous trifluoroacetic acid served as the solvent. The so...