Influence of organic solvents on papain kinetics.

Papain activity was studied in water-organic solvent mixtures using the fluorogenic substrate Dabcyl-Lys-Phe-Gly-Gly-Ala-Ala-Edans. The increase of organic solvent (MeOH, EtOH, iPrOH, TFE, MeCN, (MeO)2Et and DMF) concentration in the mixture caused a substantial decrease the initial rate of papain-catalyzed hydrolysis. Moreover, the number of papain active sites decreased with the increase of DMF and MeOH concentration.

Influence of Me2SO and incubation time on papain activity studied using fluorogenic substrates.

Papain activity in a buffer containing Me2SO was studied using fluorogenic substrates. It was found that the number of active sites of papain decreases with increasing Me2SO concentration whereas the incubation time, in a buffer containing 3% Me2SO does not affect the number of active sites. However, an increase of papain incubation time in the buffer with 3% Me2SO decreased the initial rate of hydrolysis of Z-Phe-Arg-Amc as well as Dabcyl-Lys-Phe-Gly-Gly-Ala-Ala-Edans. Moreover, an increase of Me2SO concentration in working buffer decreased the initial rate of papain-catalysed hydrolysis of both substrates. A rapid decrease of the initial rate (by up to 30%) was observed between 1 and 2% Me2SO. Application of the Michaelis-Menten equation revealed that at the higher Me2SO concentrations the apparent values of k(cat)/Km decreased as a result of Km increase and kcat decrease. However, Me2SO changed the substrate binding process more effectively (Km) than the rate of catalysis k(cat).

Properties of lactate dehydrogenase from the isopod, Saduria entomon.

Saduria entomon lactate dehydrogenase (LDH-A4*) from thorax muscle was purified about 89 fold to specific activity 510 micromol NADH/min/mg using Cibacron Blue 3GA Agarose and Oxamate-Agarose chromatographies. The enzyme is a tetramer, with molecular weight of 140 kDa for the native enzyme and 36 kDa for the subunit. The isoelectric point was at pH 5.7. The enzyme possesses high heat stability (T50 = 71.5 degrees C). The optimum pH for pyruvate reduction reaction was 6.5, while for lactate oxidation one, the maximum activity was at pH 9.1. The Km for pyruvate was minimal at 5 degrees C, the average environmental temperature of the isopod. The Km values determined at 30 degrees C and optimal pH for pyruvate reduction and lactate oxidation were 0.18 and 90.04 mM, respectively. Amino acid compositional analyses showed the strongest resemblance of the isopod isoenzyme to cod (Gadus morhua) LDH-C4.

[Factors influencing prophylaxis of caries and teeth treatment in children with mild mental retardation]. / Cynniki wplywajace na profilaktyke i leczenie zebów u dzieci upósledzonych umyslowo w stopniu lekkim.

The analysis indicated factors affecting caries prophylaxis and teeth treatment in children with mild mental retardation. The factors were sought in the personality of the child, its family environment and the work of the dentist. A significant effect on the cooperation of the dentist with the patient was exerted by such factors as the level of anxiety before dental procedure, motivation to treatment, intensity of teaching of oral hygiene, life period when teaching was started, frequency of teethbrushing, regular contacts with the dentist, and the mode of child reception by the dentist.

Monitoring protein cleavage and concurrent disulfide bond assignment using thermospray LC/MS.

The arrangement of the disulfide bridges of Cucurbita maxima trypsin inhibitor, CMTI I, has been confirmed by enzymatic and chemical cleavages of the native protein and analysis of the resulting disulfide-bridged fragments using thermospray liquid chromatography/mass spectrometry. Although the disulfide bridges of CMTI I have recently been assigned from the x-ray crystallographic structure, direct chemical analysis of the S-S bonds using classical techniques proved difficult. The CMTI I molecule is extremely resistant to enzymatic digestion, and only one site of the peptide chain (Met-8) can be used efficiently for chemical cleavage. A series of degradative conditions were employed in the studies reported here. The progress of protein modification was monitored directly by high-performance liquid chromatography/thermospray mass spectrometry. The disulfide pairings could be deduced directly from the mass spectra of the peptides produced by the fragmentation processes and resolved by high-performance liquid chromatography. In two instances, fragments involving a disulfide bond were isolated and further analyzed, and these confirmed the mass spectral assignments. The disulfide bridges identified, 3-20, 10-22 and 16-28, correspond to those of the x-ray structure and are consistent with those assigned for two other closely related trypsin inhibitors.

Peptide mapping using thermospray LC/MS detection: rapid identification of hemoglobin variants.

A rapid and easily interpreted method for peptide mapping is demonstrated with hemoglobin A and three variants. Digests of the globin chains are generated in columns containing immobilized trypsin. The resulting protein fragments are resolved by reverse phase HPLC and then analyzed by thermospray mass spectrometry. The entire process is carried out on-line. The result is a chromatographic trace which is two dimensional; in addition to the standard elution pattern, the individual mass spectra collected for each peak contain information about their identity and their purity. In two of the variants used, hemoglobin C and hemoglobin Baylor, the exact nature of the amino acid substitution could be determined unambiguously in a single analysis, from the masses of the new tryptic peptides observed. In the third case, hemoglobin S, the mass of the peptide containing the amino acid replacement is consistent with two separate sites of substitution. This ambiguity was resolved in a second analysis, using immobilized carboxypeptidase Y, prior to mass spectral analysis. The resulting partial digest permits reconstruction of the critical sequence region and correct assignment of the allelic site.

Synthesis of [alpha-methyltyrosine-4]angiotensin II: studies of its conformation, pressor activity, and mode of enzymatic degradation.

Modifications in angiotensin II and its antagonistic peptides that should have increased in vivo half-lives but not reduced biological activity were studied by determining the effect of alpha-methylation of the tyrosine in position 4. [alpha-Methyltyrosine-4]angiotensin II, synthesized by the solid-phase procedure, showed 92.6 +/- 5.3% pressor activity of angiotensin II. Incubation with alpha-chymotrypsin for 1 hr indicated absence of degradation although, under the same conditions, angiotensin II was completely degraded to two components. Comparison of the 1H NMR spectra in aqueous solution and the circular dichroism spectra in trifluoroethanol of angiotensin II and [alpha-methyltyrosine-4]angiotensin II suggested that alpha methylation of the tyrosine residue in angiotensin II does not lead to major changes in the overall solution conformation. These results are in contrast to those obtained with N-methylation in position 4, which drastically reduced the biological activity and produced remarkable changes in the peptide backbone and a severe limitation in rotational freedom of the side chains in tyrosine. Thus, it may be possible to synthesize potent angiotensin II analogs that have greater resistance to enzymatic degradation by alpha-methylation in position 4 (or 5) and simultaneous suitable modification at the NH2 and COOH termini.

Synthesis of angiotensin II analogues by incorporating beta-homotyrosine or beta-homoisoleucine residues.

[1-Sarcosine,4-beta-homotyrosine]-(I), [5-beta-homoisoleucine]-(II), and [1-sarcosine,5-beta-homoisoleucine]angiotensin II (III) were synthesized by Merrifield's solid-phase procedure to study the effect of pressor activity and duration of action. The analogues I--III possessed, respectively, 1.98, 2.82, and 29.2% pressor activity of angiotensin II (vagotomized, ganglion-blocked rats by single-injection procedure) and duration of action of 5.5, 6.7, and 4.7 min; the comparative duration of action of an equipressor dose of angiotensin II was 5.2, 6.3, and 5.3 min, respectively. When incubated with leucine aminopeptidase, degradation of II was as fast as that of angiotensin II; this degradation became considerably slower when position 1 was replaced with sarcosine. Incubation of all these analogues with chymotrypsin showed very little or no degradation up to 3 h. The results indicate that an increase in the chain length by one carbon atom in position 4 or 5 of angiotensin II increased resistance to degradation by chymotrypsin without any increase in in vivo duration of action. Further, all analogues showed low pressor activity.