Hydrolytic cleavage of purine ribonucleosides in Aspergillus phoenicis

Cell-free extracts of nitrate-grown Aspergillus phoenicis could catalyze the hydrolytic cleavage of the N-glycosidic bond of inosine, guanosine and adenosine to the corresponding base and ribose by the nucleoside hydrolase. No evidence was obtained concerning the hydrolytic degradation of N-glycosidic bond of pyrimidine ribonucleosides namely cytidine and uridine by the same extracts. Optimum pH and temperature for adenosine, guanosine and inosine hydrolysis were the same at pH 3.5 and 55 degrees C, respectively. Citrate buffer showed the highest hydrolase activity when compared to the analogous activity obtained with the other buffers used. The rate of hydrolysis of the three nucleosides was in the order inosine > guanosine > adenosine. Incubation of extracts at 55 degrees C for 15 minutes caused about 85%, 75% and 62% loss of activity with adenosine, guanosine and inosine respectively. Dialyzing the extract caused a decrease in enzyme activity. Addition of inorganic arsenate to the reaction mixture (containing adenosine, guanosine or inosine) did not affect the amount of ribose liberated. Addition of EDTA at a concentration of 5 x 10(-3) M caused an inhibition of about 50%, however a complete inhibition for enzyme activity was obtained at 10(-2) M EDTA. MgSO4, CoSO4 and ZnSO4 at a final concentration of 5 x 10(-3) M showed activation of ribonucleoside hydrolase.     

Cytidine deaminase activity in Penicillium politans NRC-510

Cell-free extracts of nitrate-grown Penicillium politans NRC-510 catalyzes the hydrolytic deamination of cytidine to uridine. Uridine was chromatographically identified in cell-free extracts. The enzyme exhibited optimum pH and temperature activities at 6.5 and 80 degrees C respectively. Thermal stability experiments indicated that the enzyme restored its activity at 80 degrees C for at least 60 minutes. When cell-free extracts were incubated at 90 degrees C for 5 minutes enzyme activity was inhibited by about 33%. The involvement of sulfhydryl group(s) in the catalytic site of the enzyme was shown. HgCl2 (5 x 10(-3) M) and CuSO4 (10(-2) M) caused a complete inhibition of enzyme activity. Ethylene diamine tetraacetate at a concentration of 5 x 10(-3) M and 10(-2) M inhibited the enzyme as well. Whereas, MgCl2, CoSO2 and MnCl2 had a remarkable activating effect. Dialysis of the cell-free extracts resulted to an increase in enzyme activity by about 30%. To our knowledge the thermophilic nature of the cytidine deaminase of P. politans NRC-510 is unique.     

Factors affecting D-galactonate degradation by extracts of Aspergillus niger

Biochemical studies on the degradation of D-galactonate by cell-free extracts of Aspergillus niger indicated that the pH value and temperature optima were 8.0 and 7.5 and 40 degrees C and 50 degrees C for the two enzymes responsible for this degradation namely D-galactonate dehydratase and 2-keto-3-deoxy-D-galactonate (KDGal) aldolase respectively. The effects of the nature of the buffer substance, buffer molarity and enzyme concentration were also studied. Thermal stability behaviour studies show that D-galactonate dehydratase was stable at 40 degrees C for 60 minutes and about 41%, 80% and 90% of enzyme activity were lost by exposing the extracts to 50 degrees C, 60 degrees C and 70 degrees C, respectively, for the same period. However, exposing the extracts to 70 degrees C after 60 minutes caused a complete inhibition for KDGal aldolase activity and a gradual decrease in activity was noticed by incubation the extracts at 60 degrees C. The results of freezing and thawing treatment indicated that KDGal aldolase was more stable than D-galactonate dehydratase in this respect, as only 27% of enzyme activity was lost after 5 days of storage at -5 degrees C. Dialyzing the extracts significantly affects KDGal formation from D-galactonate. Results obtained also indicated the non-requirement of metal ions for activation of KDGal aldolase. On the other, hand D-galactonate dehydratase has a requirement for Mg++ and Mn++, however ZnSO4 and HgCl2 caused a complete inhibition of the enzymatic activity of this enzyme.     

Properties of adenosine deaminase from Candida albicans

Adenosine deaminase (ADA; adenosine aminohydrolase, E.C. 3.5.4.4), a purine catabolic enzyme, was studied in Candida albicans, an opportunistic yeast that causes diseases ranging from superficial infections to the deep systemic disease, candidiasis, in immunosuppressed humans. The fungus was grown as a yeast form in Lee 's synthetic medium, pH 4.5, at room temperature for various growth periods. Adenosine deaminase (ADA) activity was determined from the cell free extract by measuring the change in absorbance 265 nm resulting from the deamination of adenosine. In yeast form, maximum growth and ADA activity were found at 72 and 24 hours, respectively, whereas in the mycelial form both the growth and ADA activity were maximum after 48 hours. Among the three media tested, tryptic soy broth supported maximum growth and enzyme production, compared to Lee synthetic medium or Sabouraud dextrose broth. The enzyme was active over the pH range 4–8 and the optimum temperature for ADA activity was found to be 37 °C.     

Creatine phosphokinase activity in rabbit alveolar macrophages

An enzyme is present in extracts of rabbit alveolar macrophage which can catalyze the reaction of creatine phosphate with adenosine diphosphate to form adenosine triphosphate and creatine. The enzyme is moderately activated by reduced glutathione, has a pH optimum between pH 6.5 and 7.0, and shows an absolute requirement for Mg(2+). The K(m) for creatine phosphate is approximately 3.6 mm while the K(m) for adenosine diphosphate is about 1.1 mm. The enzyme may play a role in the energy balance of the cell by creating a reserve of energy in the form of creatine phosphate.     

Isolation and characterization of a cysteine proteinase from Fasciola hepatica adult worms

Adult Fasciola hepatica worms contain multiple proteinases capable of degrading hemoglobin, immunoglobulins and collagen. Here we report the isolation and biochemical characterization of a cysteine proteinase from acidic extracts of these worms. The enzyme was purified to homogeneity by cation exchange and molecular sieve high-performance liquid chromatography. It eluted at a native molecular weight of approximately 14,500 and migrated as a single band at approximately 14,500 Da upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Activity was assessed by employing synthetic peptide substrates, such as carbobenzoxy-phenylalanyl-arginyl-7-amino-4-trifluoro-methylcoumarin, commonly used to assay other cysteine proteinases. The proteinase was maximally active at pH 6.0, with 50% or more of the activity detected between pH 4.5 and 7.5. Inhibition of activity at pH 5.5 was seen only with compounds known to inhibit cysteine proteinases. No effect was seen with inhibitors of aspartic, serine, or metalloproteinases. The purified enzyme was stable at acidic pH at 4 degrees C, 25 degrees C, -20 degrees C, and in 1 M urea.     

Colorimetric determination of serum acid phosphatase activity using adenosine 3′-monophosphate as substrate

The hydrolysis of adenosine 3'-monophosphate by serum acid phosphatase has been coupled to the liberation of ammonia from the adenosine generated through the action of exogenous adenosine deaminase. The ammonia is measured at the end of the incubation by a modification of the phenol-hypochlorite reaction of Berthelot. Optimum conditions for the enzyme reaction have been defined. Inhibition of the Berthelot reaction by the serum used in the assay is small, and may be compensated by a correction factor. Although the value for the control is high in relation to the test over the normal range, this is largely outweighed by the good sensitivity and precision of the method.The substrate is not significantly hydrolysed by erythrocyte acid phosphatase within the limits encountered in haemolysed sera. Experience of the method in routine hospital diagnosis compared favorably with that of a standard method employing disodium phenyl phosphate as substrate. It is suggested that activities greater than 3.1 IU/l should be further investigated and those greater than 3.7 IU/l should be regarded as definitely raised. The stability of human serum AcPase when promptly separated and held at 4 degrees C or - 20 degrees C was confirmed. At room temperature, acidification to pH 6.0 greatly improved stability.     

Some properties of two aldolases in extracts of Aspergillus oryzae

Fructose 1,6-diphosphate (FDP) aldolase and 2-keto-3-deoxy-D-gluconate (KDG) aldolase the two key enzymes of Embden-Meyerhof-Parnas (EMP) and the nonphosphorolytic Entner-Doudoroff (ED) pathways respectively, were identified in cell-free extracts of four Aspergillus oryzae strains grown on D-glucose as sole source of carbon. A. oryzae NRRL 3435 gave the highest enzymatic activity for the two enzymes and selected for further studies. Studies on the properties of the two key enzymes indicated that the optimum conditions for the activities of FDP aldolase and KDG aldolases occurred at pH 8.5, 45 degrees C and pH 8.0, 55 degrees C, respectively. Tris-acetate buffer and phosphate buffer showed the highest enzymatic activity for these two enzymes respectively. KDG aldolase was stable at 55 degrees C for 60 minutes however FDP aldolase was found to be less stable above 45 degrees C. On the other hand the two aldolases showed a high degree of stability towards frequent freezing and thawing. Dialysis of the extracts caused a decrease in the enzymatic activity of KDG aldolase, and an increase in FDP aldolase activity. The addition of ethylene diamine tetraacetate to the crude extracts caused an inhibition of KDG aldolase, whileas FDP aldolase was not affected. Addition of MnCl(2), CoSO(4), MgCl(2) and ZnSO(4) to the dialyzed extracts increased the activity of KDG aldolase by 67%, 54%, 61% and 37%, respectively. On the other hand the addition of some metal salts caused an inhibition of FDP aldolase. The results obtained indicate the absence of evidence for the involvement of sulfhydryl groups in the catalytic sites of the two aldolases.     

The second component of human complement: detection of two hemolytic forms in plasma by pH variation

The second component of human complement (C2) in pseudoglobulin prepared from normal plasma eluted as a single peak at high conductivity (30 mS) and pH 4.5 from the cationic exchangers S-Sepharose or Mono S in the Fast Protein Liquid Chromatography (FPLC) System. The C2 was stable at pH 4.5 and 0 degrees C if enzyme inhibitors were used and the pH was raised to 6.0 after elution from the columns. After rechromatography on Mono S in the FPLC System at the median isoelectric point of 5.5 or pH 6.0, the C2 eluted as two distinct hemolytic forms: the first peaked at 16 mS, the second at 30 mS. The two forms of C2 did not correlate with the allotypic variant of C2 in individual, normal human plasmas. After elution at pH 4.5 from S-Sepharose and rechromatography at pH 5.5 or 6.0 on Mono S, the hemolytic activities of the two forms in individual plasmas eluted in 3 patterns: 1) high activity at 16 mS, low activity at 30 mS; 2) low activity at 16 mS, high activity at 30 mS; 3) high activity at 16 mS, high activity at 30 mS. The specific activities of both forms were approximately the same; both eluted the same after gel filtration at pH 5.5, and both had the same pattern on SDS-PAGE and immunoblots. The pattern of elution was characteristic for each individual plasma, and the first hemolytic form appeared to elute independent of the second form. At pH 4.5, C2 was completely separated from Factor B, a functionally and structurally similar protein of the alternative complement pathway, whereas at pH 5.5 or 6.0, the two proteins eluted together. From these results, the two forms of hemolytic C2 can be purified for structural and functional analyses.     

Amylase of the thermophilic actinomycete Thermomonospora vulgaris

α-Amylase of the thermophilic actinomycete Thermomonospora vulgaris was partially purified. Maximal enzyme activity was obtained at 60 °C and pH 6.0. K_M value was 1.4%. The effect of some metal salts on enzyme activity was studied. Enzyme activity was inhibited by KCN, EDTA, and iodoacetate. Inhibition by EDTA was completely nullified by CaCl₂, but the inhibition by iodoacetate was not overcome by 2-mercaptoethanol. Exposure of the enzyme to pH 7.0 and 9.0 for 2hr. did not affect the enzyme, but exposure to pH 3.0 for few minutes completely inactivated the enzyme. Exposure of the enzyme to 60 °C resulted in an appreciable inactivation and exposure to 80 °C completely inactivated the enzyme. Addition of CaCl₂, 2-mercaptoethanol, or enzyme substrate did not stabilize the 60 °C exposed enzyme. However, bovine serum albumin had a protective effect when the enzyme was exposed to 60 °C but not to 80 °C. The enzyme was stable in the presence of 8 M urea.     

Purification and characterization of a protease produced by an aerobic haloalkaliphilic species belonging to the Salinivibrio genus

An extracellular protease produced at the end of the exponential growth phase was purified to homogeneity and characterized from the new isolate haloalkaliphilic strain 18AG, phylogenetically related to Salinivibrio costicola subsp. costicola. The protease molecular mass was about 38 kDa. The enzyme was dependent on salt concentration for activity and stability, and it showed optimal activity at 60 degrees C in the presence of 2.0% NaCl and 2.0 mM CaCl2, while in the absence of CaCl2 the optimum temperature was 50 degrees C. The enzyme was stable for 24 h at 30 degrees C, whereas at 50 degrees C in the presence of CaCl2 the half life was about 5 h. The enzyme had an optimum pH of 8.0 with 80% of residual activity at pH 9.0. The protease was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), slightly activated by denaturing agents such as SDS and urea, and partially inhibited by thiol-containing reducing agents. The synthesis of the enzyme in culture media was influenced by the medium composition: it was specifically dependent upon the NaCl concentration and was induced by the presence of gelatin.     

On the nature of endogenous purines modulating cholinergic neurotransmission in the guinea-pig ileum

By means of agonist and enzyme experiments, the relative importance of endogenous adenosine, adenine nucleotides or other purines as modulators of cholinergic neuroeffector transmission in preparations of guinea-pig ileum muscle has been examined. Adenosine, 2-chloroadenosine, AMP, ADP, ATP and AMPPNP reversibly inhibited contractile responses to transmural stimulation of the guinea-pig ileum longitudinal muscle. 5'-adenylate deaminase dose-dependently antagonized the inhibitory effect of adenosine, AMP, ADP, ATP and AMPPNP, but not that of 2-chloroadenosine. 8-p-sulphophenyltheophylline, adenosine deaminase and 5'-adenylate deaminase enhanced contractile responses to transmural nerve stimulation. Adenosine deaminase and 5'-adenylate deaminase were virtually equiactive whereas 8-p-sulphophenyltheophylline was much more effective, and the theophylline derivative also enhanced contractile responses in preparations pretreated with adenosine deaminase or 5'-adenylate deaminase. Moreover, 8-p-sulphophenyltheophylline abolished the inhibition by dipyridamole, whereas adenosine deaminase and 5'-adenylate deaminase only partly antagonized the inhibitory effect of dipyridamole. Application of 5'-adenylate deaminase did not enhance the nerve-induced contractions in preparations pretreated with adenosine deaminase or a combination of dipyridamole and adenosine deaminase. In conclusion, adenosine deaminase and 5'-adenylate deaminase enhanced the nerve-induced contractions in the ileum, and, since 5'-adenylate deaminase was inactive after pretreatment with adenosine deaminase, this suggests that endogenous adenosine rather than 5'-adenine nucleotides modulated cholinergic neurotransmission in the ileum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vaccinia virus E3L interferon resistance protein inhibits the interferon-induced adenosine deaminase A-to-I editing activity.

The RNA-specific adenosine deaminase (ADAR1) is an interferon-inducible editing enzyme that converts adenosine to inosine. ADAR1 contains three distinct domains: a N-terminal Z-DNA binding domain that includes two Z-DNA binding motifs; a central double-stranded RNA binding domain that includes three dsRNA binding motifs (dsRBM); and a C-terminal catalytic domain responsible for A-to-I enzymatic activity. The E3L protein of vaccinia virus mediates interferon resistance. E3L, similar to ADAR1, also contains Z-DNA binding and dsRNA binding motifs. To assess the possible role of E3L in modulating RNA editing by ADAR1, we examined the effect of E3L on ADAR1 deaminase activity. Wild-type E3L protein was a potent inhibitor of ADAR1 deaminase enzymatic activity. Analysis of mutant E3L proteins indicated that the carboxy-proximal dsRBM of E3L was essential for antagonism of ADAR1. Surprisingly, disruption of the Z-DNA binding domain of E3L by double substitutions of two highly conserved residues also abolished its antagonistic activity, whereas deletion of the entire Z domain had little effect on the inhibition. With natural neurotransmitter pre-mRNA substrates, E3L weakly inhibited the site-selective editing activity by ADAR1 at the R/G site of the glutamate receptor B subunit (GluR-B) pre-mRNA and the A site of serotonin 2C receptor (5-HT2CR) pre-mRNA; editing of the intronic hotspot (+)60 site of GluR-B was not affected by E3L. These results demonstrate that the A-to-I RNA editing activity of the IFN-inducible adenosine deaminase is impaired by the product of the vaccinia virus E3L interferon resistance gene.     

Characterization of extracellular neuraminidase produced by Actinomyces pyogenes

Extracellular neuraminidase from Actinomyces pyogenes could be isolated by ammonium sulfate precipitation, ion exchange chromatography with DEAE cellulose and gel filtration on Ultrogel ACA 54. The purified enzyme had a molecular weight of approximately 50,000 Dalton, a pH optimum at pH 6.0, a temperature optimum at 55 degrees C and a Km value of 1.4 X 10(-4) mol/l with N-acetyl-neuraminlactose as substrate. Preparative isoelectric focussing of the culture supernatant revealed neuraminidase activity mainly at pH 6.5. The enzyme activity was not influenced by metalions or EDTA.     

Adenosine induced immunosuppression: the role of the adenosine receptor--adenylate cyclase interaction in the alteration of T-lymphocyte surface phenotype and immunoregulatory function

Adenosine (1 microM at 37 degrees C) rapidly modulates the expression of T-lymphocyte surface antigens (OKT4 and OKT8) and Fc gamma receptors, and increases T-suppressor activity for pokeweed mitogen driven in vitro immunoglobulin synthesis. The adenosine induced loss of OKT4 expressed was maximal within 5 min, while increased OKT8 expression developed more slowly; increased Fc gamma expression was maximal at 30 min after initiation of incubation with adenosine. Adenosine, 2-chloroadenosine and adenosine in the presence of nitrobenzylthioinosine (NBTI) induced the decrease in OKT4 expression. In contrast only adenosine induced the enhancement of OKT8 and Fc gamma receptor expression. Neither 2-chloroadenosine, a poorly transported analog, nor adenosine in the presence of the adenosine transport inhibitor NBTI were capable of enhancing OKT8 or Fc gamma receptor expression. Adenosine was shown to cause a rapid biphasic increase in cAMP, while 2-chloroadenosine and adenosine with NBTI induces a prolonged elevation in cAMP. Similarly isoproteranol which induces a sustained elevation in cAMP suppressed the adenosine induced increases in OKT8 and Fc gamma receptor expression. Incubation of T-helper/inducer lymphocytes with 1 microM adenosine for 30 min at 37 degrees C caused the loss of T-helper function; this loss of T-helper activity has previously been shown to result from the activation of T-suppressor cells. The loss of T-helper function was blocked by the simultaneous addition of isobutylmethylxanthine, an R site adenosine receptor antagonist, or NBTI, an adenosine transport inhibitor. Moreover, 2-chloroadenosine could not induce loss of T-helper function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characteristics of collagenolytic enzyme produced by Candida albicans.

In media containing collagen as the nitrogen source, the pathogenic yeast Candida albicans secreted a collagenolytic enzyme. Purification of the enzyme from a culture filtrate was achieved by DEAE-Sephacel chromatography at pH 6.7. The molecular weight was found to be 46,000 by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was at pH 4.2. The pH optimum lay between 3.5 and 4.0, and above pH 6.0, the enzyme underwent alkaline denaturation. The enzyme was heat labile, and a decrease in activity was found above 55 degrees C. The enzyme activity was inhibited by the addition of urea, cysteine, and pepstatin. No other inhibitor among those tested had any effect. The C. albicans enzyme degraded both the native acid-soluble collagen and the insoluble dentinal collagen.     

Degradation of the polysaccharide component of gonococcal lipopolysaccharide by gonococcal and meningococcal sonic extracts.

An extract made from the supernatant of Neisseria gonorrhoeae Gc2 strain 1291 degraded the Gc2 polysaccharide antigen. Chemical analysis of this polysaccharide indicated it contains glucose, galactose, glucosamine, galactosamine, glucosamine-6-phosphate, heptose, 2-keto-3-deoxyotonate, and ethanolamine and is the polysaccharide component of gonococcal lipopolysaccharide. Degradation of the polysaccharide by sonic extracts resulted either in complete loss of antigenicity and immunogenicity or in partial degradation to subunits that could inhibit the Gc2-specific hemagglutination inhibition. The factors responsible for degradation were destroyed by heating at 100 degrees C for 5 min or by Pronase digestion, but were unaffected by ribonuclease, deoxyribonuclease, Mg2+, Ca2+, or ethylenediaminetetraacetic acid. The process was pH dependent, with optimal activity occurring at pH 7. Sonic extract supernatants from group B and C meningococcal strains contained degrading properties, whereas similar extracts produced from Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Streptococcus pneumoniae type II failed to degrade the Gc2 polysaccharide.     

Purification and characterization of thermoalkalophilic xylanase isolated from the Enterobacter sp. MTCC 5112

Thermoalkalophilic Enterobacter sp. MTCC 5112 was isolated from a sediment sample collected from the Mandovi estuary on the west coast of India. This culture produced extracellular xylanase. The xylanase enzyme was isolated by ammonium sulfate (80%) fractionation and purified to homogeneity using size exclusion and ion exchange chromatography. The molecular mass of the xylanase was approximately 43 kDa. The optimal pH of the xylanase activity was 9, and at room temperature it showed 100% stability at pH 7, 8 and 9 for 3 h. The optimal temperature for the enzyme activity was 100 degrees C at pH 9.0. At 80 degrees C and pH 9, 90% of the enzyme activity was retained after 40 min. At 70 and 60 degrees C, the enzyme retained 64% and 85% of its activity after 18 h, respectively, while at 50 degrees C and pH 9 the enzyme remained stable for days. For xylan, the enzyme gave a K(m) value of 3.3 mg ml(-1) and a V(max) value of 5,000 micromol min(-1) mg(-1) when the reaction was carried out at 100 degrees C and pH 9. In the presence of metal ions such as Co(2+), Zn(2+), Fe(2+), Cu(2+), Mg(2+) and Ca(2+) the activity of the enzyme increased, whereas strong inhibition of enzyme activity was observed in the presence of Hg(2+) and EDTA. To the best of our knowledge, this is the first report on the production of xylanase by this bacterium.     

Isolation and properties of levanase from Streptococcus salivarius KTA-19

Fructan-hydrolyzing enzyme from Streptococcus salivarius KTA-19 isolated from human dental plaque was investigated. The enzyme was purified by ammonium sulfate precipitation, acetone fractionation, and column chromatography on Bio-Gel and DEAE-cellulose. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Its molecular weight was 100,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme exhibited an optimum pH of 6.5 and decreased its activity from pH 6.0 and especially below pH 5.5. The optimum temperature was 40 to 50 degrees C, and enzyme activity was reduced by 90% at 55 degrees C. Enzyme activity was markedly inhibited by Hg2+, Ag+, Cu2+, and p-chloromercuribenzoate at a concentration of 10(-3) M, but not by other metal ions or chemical effectors. Fructose was the only by-product of the enzyme action on levan. These results indicated that the levanase of S. salivarius KTA-19 is an exo-beta-(2,6)-fructofuranosidase.     

Loss of bactericidal capacity of long-chain quaternary ammonium compounds with protein at lowered temperature.

Amphiphilic betaine esters are quaternary ammonium compounds (QACs) with rapid microbicidal effect, which spontaneously hydrolyze into nontoxic products. thus being referred to as soft antimicrobial agents. The bactericidal effect of 1-decyl (B10), 1-dodecyl (B12), and 1-tetradecyl (B14) betaine esters on Salmonella typhimurium was strongly influenced by temperature, pH and length of hydrocarbon chain. At pH 6.0, presence of 1.5 mM (10% w/v) BSA raised the concentration of B14 for 99% killing (BC2) from 0.006 mM to 1.8 mM. There was a stoichiometric relationship between concentration of BSA and BC2 of B14, indicating that one molecule of B14 was bound per BSA molecule when 99% killing was achieved. When the temperature was lowered to 0 degrees C only minor killing was seen in 1.5 mM BSA at the highest concentration of B14 tested, 57 mM. With B10 at 30 degrees C and pH 6.0, the presence of 1.5 mM BSA raised the bactericidal concentration (BC2) from 0.69 mM to 4.1 mM, and at 0 degrees C and 1.5 mM BSA the BC2 was 11 mM. Thus, the impairment caused of the bactericidal effect of B10 by BSA and lower temperature was less than for B14, since B14 is much more active than B10 at 30 degrees C in the absence of BSA, somewhat more active than B10 at 30 degrees C in the presence of 1.5 mM BSA, and much less active than B10 at 0 degrees C in the presence of BSA. B12 showed properties intermediate between B10 and B14. Lowered pH reduced the bactericidal effect particularly when reduced from pH 5.0 to 4.0 with B10. In the presence of 1.5 mM BSA, the bactericidal effect of 1-dodecyl (DTAB) and 1-hexadecyl (CTAB) trimethylammonium bromide decreased in the same manner as for B10 and B14, respectively. Increasing the time of incubation at 0 degrees C to 50 min, a 99% killing effect was seen with 17 mM CTAB, whereas the same killing effect was reached in 8 min with 17 mM DTAB. Binding of [3H]CTAB to S. typhimurium was also reduced at 0 degrees C in the presence of BSA. Thus, in the presence of 1.5 mM BSA, QACs with the longer hydrocarbon chain were most efficient at 30 degrees C, whereas at 0 degrees C those with the shorter hydrocarbon chain were most active. Consequently, QACs with shorter tails should be used for disinfection in the presence of proteins at lower temperatures.