Hemoglobin Deer Lodge (beta 2 His replaced by Arg). Consequences of altering the 2,3-diphosphoglycerate binding site.

Hemoglobin Deer Lodge is an abnormal human hemoglobin with arginine substituted for histidine at the beta 2 position. X-ray crystallography of normal human hemoglobin has shown that the beta 2 residue is normally part of the binding site for 2,3-diphosphoglycerate. The substitution of arginine for histidine at beta 2 affects both the kinetics and equilibria of ligand binding. When stripped of anions, Hb Deer Lodge has an increased oxygen affinity and a decreased degree of cooperativity relative to Hb A. The alkaline Bohr effect is slightly increased and there are marked increases in oxygen affinity below pH 6 and above pH 8. In the presence of 2,3-diphosphoglycerate the cooperativity in increases to nromal and the pH dependence of oxygen binding is reduced. This contrasts with the enhanced Bohr effect seen for Hb A in the presence of organic phosphates. Due to enhanced anion binding at high pH, Hb Deer Lodge has a slightly lower oxygen affinity than Hb A at pH 9 in the presence of 2,3-diphosphoglycerate or inositol hexaphosphate. Kinetic studies at neutral pH in the absence of organic phosphates revealed biphasicity in the rate of oxygen dissociation from Hb Deer Lodge, while approximately linear time courses were observed for Hb A. The fast phase of the oxygen dissociation kinetics shows great pH sensitivity, and organic phosphates increase the rate and percentage of the fast phase without greatly affecting the slow phase. The two phases are not resolvable at high pH. CO combination kinetics are much like those of Hb A except that "fast" and "slow" phases were apparent at wavelengths near the deoxy-CO isobestic point. We suggest that functional differences between the alpha and beta chains are enhanced in Hb Deer Lodge. After flash photolysis of the CO derivative, the percentage of quickly reacting material was slightly greater for Hb Deer Lodge than for Hb A. This may imply a somewhat greater tendency to dissociate into high affinity subunits. The substitution of arginine for histidine at beta 2 thus results in a macromolecule whose ligand-binding properties are significantly altered, the primary differences being expressed at high pH where Hb Deer Lodge binds anions more strongly than Hb A. The properties of Hb Deer Lodge are compared to those of other hemoglobin variants with substitutions at residues involved in binding of 2,3-diphosphoglycerate.     

The reactions of alpha-chymotrypsin and related proteins with ester substrates in non-aqueous solvents.

1. The reactivity of alpha-chymotrypsin toward p-nitrophenylacetate has been studied in dimethylformamide, dimethylsulfoxide, formamide and methylacetamide. p-Nitrophenol is liberated in dimethylsulfoxide only. 2. The reactions of alpha-chymotrypsin in dimethylsulfoxide are characterized by the same kinetic and equilibrium constants with either the p-nitrophenyl esters of straight chain carboxylic acids (from acetic to n-caprylic) or with the "specific substrate", N-carbobenzoxy-DL-phenylalanine p-nitrophenyl ester. This signifies that reactions of alpha-chymotrypsin in dimethylsulfoxide, unlike those in aqueous medium, have no specificity toward su-strate structure. 3. The stoichiometry of alpha-chymotrypsin reactions in dimethylsulfoxide was shown to be about five moles of substrate per mole of enzyme. After attaining this stoichiometry, the reaction is completed. 4. Optical rotatory dispersion spectra indicate that in non-aqueous media alpha-chymotrypsin undergoes a large conformational transition which results in a random coil. 5. Chymotrypsinogen, trypsin, trysinogen, lysozyme and serum albumin react with p-nitrophenylacetate in dimethylsulfoxide at rates which are approximately equal to those of alpha-chymotrypsin. Thus, the "activity" of alpha-chymotrypsin in dimethylsulfoxide toward p-nitrophenylacetate does not differ from the "activity" of other proteins, some of which are not even hydrolytic enzymes.     

A comparative kinetic study on the singlet molecular oxygen-mediated photoxidation of alpha- and beta-chymotrypsins.

Kinetic aspects of the sensitized photooxidation of alpha- and beta-chymotrypsins have been studied at pH 6 and 8. The sensitization, employing classical O2(1Deltag)-photogenerators, such as xanthene dyes, is a kinetically intricate process because of the presence of ground state dye-protein associations and to the simultaneous participation of superoxide ion and singlet molecular oxygen [O2(1Deltag)]. Both proteins, that possess the same distribution pattern of photooxidizable amino acids, suffer a pure O2(1Deltag)-mediated photodynamic attack, using the carbonylic sensitizer Perinaphthenone. Overall and reactive rate constants for the O2(1Deltag)-quenching (in the order of 108 and 107/M/s, respectively), and rates of oxygen consumption determined by time-resolved, spectroscopic and polarographic methods indicate that alpha- and beta-chymotrypsins are less photooxidizable at pH 6, as a result of an enhancement of the O2(1Deltag)-physical quenching component. In general terms, beta-chymotrypsin exhibits the greater overall proclivity to interact with O2(1Deltag), whereas structural factors, possibly evidenced by a higher exposure of the reactive tryptophan residues, impart an increased photooxidation degree to the proteins at pH 8, specially to the alpha-chymotrypsin.     

Peptide bond synthesis catalyzed by alpha-chymotrypsin.

alpha-Chymotrypsin [EC 3.4.21.1] catalyzed the syntheses of peptide bonds with various N-acylated amino acids or peptides having aromatic or hydrophobic amino acid residues at the C-terminal position as carboxyl components, and amino acid derivatives, peptides or their derivatives as amine components. A neutral pH was most efficient and quite high concentrations of alpha-chymotrypsin and starting materials were required for synthesis. Four amine components, hydrophobic or bulky amino acid residues were useful at the N-terminal position. Stereospecificity was also observed at the N-terminal position of amine components. Peptide synthesis was not usually seen when the products were soluble in the reaction mixture. This could be partly overcome by increasing the concentration of either the carboxyl or the amine component to more than ten times that of the other.     

Identification of a protein-binding surface by differential amide hydrogen-exchange measurements. Application to Bowman-Birk serine-protease inhibitor.

The binding surface of soybean trypsin/chymotrypsin Bowman-Birk inhibitor in contact with alpha-chymotrypsin has been identified by measurement of the change in amide hydrogen-exchange rates between free and chymotrypsin-bound inhibitor. Exchange measurements were made for the enzyme-bound form of the inhibitor at pH 7.3, 25 degrees C using fast-flow affinity chromatography and direct measurement of exchange rates in the protein complex from one-dimensional and two-dimensional nuclear magnetic resonance spectra. The interface is characterized by a broad surface of contact involving residues 39 through 48 of the anti-chymotryptic domain beta-hairpin as well as residues 32, 33 and 37 in the anti-chymotryptic domain loop of the inhibitor. A number of residues in the anti-tryptic domain of the protein also have an altered exchange rate, suggesting that there are changes in the protein conformation upon binding to chymotrypsin. These changes in amide exchange behavior are discussed in light of a model of the complex based on the X-ray crystallographic structure of turkey ovomucoid inhibitor third domain bound to a alpha-chymotrypsin, and the structure of free Bowman-Birk inhibitor determined in solution by two-dimensional nuclear magnetic resonance spectroscopy. The chymotrypsin-binding loop of Bowman-Birk inhibitor in the model is remarkably similar to the binding loop conformation in crystal structures of enzyme-bound polypeptide chymotrypsin inhibitor-I from potatoes, turkey ovomucoid inhibitor third domain, and chymotrypsin inhibitor-II from barley seeds.     

Active site residues of cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase from Comamonas testosteroni strain B-356

cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) from Comamonas testosteroni strain B-356 is the second enzyme of the biphenyl/polychlorinated biphenyl degradation pathway. Based on the crystal structure of a related BphB, three conserved residues, Ser142, Tyr155, and Lys159, have been suggested to function as a "catalytic triad" as for other members of the short-chain alcohol dehydrogenase/reductase (SDR) family. In this study, substitution of each triad residue was examined in BphB. At pH 9.0, turnover numbers relative to wild-type enzyme were as follows: Y155F, 0.1%; S142A, 1%; and K159A, 10%. Although the Michaelis constants of K159A and S142A for cis-2,3-dihydro-2,3-dihydroxybiphenyl increased about 20-fold, relatively little change was observed in the K(m) for dinucleotide. The K159A mutant, which showed little dehydrogenase activity at pH 7, was sharply activated by increasing the pH, reaching almost 25% of the activity of the wild-type enzyme at pH 9. 8. These three residues are therefore critical for BphB activity, as suggested by the crystal structure and similarity to other SDR family members. In addition, BphB showed a strong preference for NAD(+) over NADP(+), with a 260-fold higher specificity constant (k(cat)/K(m)). Evidence is presented that the inefficient use of NADP(+) by BphB might partly be due to the presence of an aspartate residue at position 36.     

How do calcium ions induce free radical oxidation of hydroxy-1,4-naphthoquinone? Ca2+ stabilizes the naphthosemiquinone anion-radical of echinochrome A

A surprising effect is the direct action of Ca(2+) on redox reactions of ortho-quinoid compounds. The effect of Ca(2+) on oxidation of the sea urchin pigment 6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone (echinochrome A) has been studied by electron paramagnetic resonance (EPR) spectroscopy, by UV/VIS absorbance spectroscopy, and by measurement of oxygen consumption. Echinochrome A per se reacted with dioxygen only in an alkaline solution; 2,3-semiquinone anion-radical of echinochrome A and superoxide anion-radical were the intermediates of the oxidation. Addition of calcium ions sharply increased the rate of echinochrome A autooxidation at alkaline pH and provoked oxidation at neutral pH. To explain this phenomenon we have focused on changes of the acid-base properties of echinochrome A in the presence of calcium and on stabilization of 2,3-semiquinone anion-radical of echinochrome A by Ca(2+). Dissociation constants (pK(a1), pK(a2), and pK(a3)) of echinochrome A determined by potentiometric titration were 5.20, 6.78, and >10 in calcium-free solution and 5.00, 6.10, and 7.15 in the presence of Ca(2+). We have found that Ca(2+) forms an insoluble adduct with the 2,3-semiquinone anion-radical. Thus, the effect of redox-inert calcium on the free radical reactions could be explained (i) by additional deprotonation of echinochrome A and (ii) by formation of a Ca(2+)-naphtho-2,3-semiquinone complex (calcium semiquinonate). Additionally, we have shown that the dried red spines from Strongylocentrotus intermedius possess paramagnetic properties; the EPR signal of the natural spines was similar to that of calcium semiquinonate obtained in our artificial chemical system.     

The chemoselective fluorescence labeling of alpha-chymotrypsin

The covalent fixation of the phosphinoyl residues in the active site of alpha-chymotrypsin is proved by the application of the fluorescent phosphinoyl fluorides 1 [( 5-(dimethylamino)-1-naphthyl]phenylphosphinoyl-fluoride) or 4 [(5-methoxy-1-naphthyl)phenyl-phosphinoylfluoride]. The differences in the rates of the phosphinoylation of alpha-chymotrypsin and "methyl-alpha-chymotrypsin" as compared to 1 agree with model reactions. In both enzymes the serine-OH in the active site is phosphinoylated. The non-fluorescent 4-nitrophenyl [5-(dimethylamino)-1-naphthyl]phenylphospinate (3) and the corresponding non-fluorescent 5-methoxynaphthyl derivative 5 inhibit alpha-chymotrypsin far more slowly than the corresponding fluorides 1 and 4. The phosphinoyl residues of the nitrophenyl esters 3 and 5 are covalently linked in a yield of 80% to the active site of the enzyme with evolution of fluorescence. 20% of the nitrophenyl ester inhibits the enzyme by adsorption.     

Oxidation of polyunsaturated fatty acids and lipids through thiyl and sulfonyl radicals: reaction kinetics, and influence of oxygen and structure of thiyl radicals.

Thiyl free radicals have been shown to react with polyunsaturated fatty acids via abstraction of bisallylic hydrogen, forming pentadienyl radicals, and via addition to the double bonds. In the absence of oxygen, the latter pathway leads to regeneration of thiyl radicals through beta-elimination or "repair" of the adduct radicals by thiols. In the presence of oxygen, fixation of thiyl-induced damage occurs through reaction of O2 with the pentadienyl radical (yielding conjugated dienyl peroxyl radicals) and also with the thiyl-to-double bond adduct radical. A quantitative reaction scheme evaluated from these data considers abstraction, addition, rearrangement, and repair reactions, and the evaluation of rate constants for the individual steps. Absolute rate constants have been measured, in particular, for reactions of thiyl free radicals from glutathione, cysteine, homocysteine, N-acetylcysteine, cysteine ethyl ester, penicillamine, captopril, mercaptoethanol, and dithiothreitol with polyunsaturated fatty acids (PUFAs) ranging from 18:2 to 22:6, and the lipids trilinolein and trilinolenin. The rate constants for hydrogen abstraction were found to be typically of the order of 10(7) mol-1 dm3 s-1 and to increase with increasing lipophilicity of the attacking thiyl radical. Thioperoxyl radicals, RSOO., were found to be rather unreactive toward PUFAs, in contrast to the isomer sulfonyl radicals, RSO2., which not only abstract hydrogen from the bisallylic methylene groups of the PUFAs (although only at relatively small yield) but also readily add to the PUFA double bonds (major pathway). Specific information was obtained on the optical properties of the thiyl radical derived from the ACE inhibitor captopril, CpS. (lambda max = 340 nm, epsilon = 460 +/- 50 mol-1 dm3 cm-1), and its conjugate disulfide radical anion (CpS:.SCp) (lambda max = 420 nm).     

Assessment of roles of surface histidyl residues in the molecular basis of the Bohr effect and of beta 143 histidine in the binding of 2,3-bisphosphoglycerate in human normal adult hemoglobin.

Site-directed mutagenesis has been used to construct two mutant recombinant hemoglobins (rHbs), rHb(betaH116Q) and rHb(betaH143S). Purified rHbs were used to assign the C2 proton resonances of beta116His and beta143His and to resolve the ambiguous assignments made over the past years. In the present work, we have identified the C2 proton resonances of two surface histidyl residues of the beta chain, beta116His and beta143His, in both the carbonmonoxy and deoxy forms, by comparing the proton nuclear magnetic resonance (NMR) spectra of human normal adult hemoglobin (Hb A) with those of rHbs. Current assignments plus other previous assignments complete the assignments for all 24 surface histidyl residues of human normal adult hemoglobin. The individual pK values of 24 histidyl residues of Hb A were also measured in deuterium oxide (D(2)O) in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer in the presence of 0.1 M chloride at 29 degrees C by monitoring the shifts of the C2 proton resonances of the histidyl residues as a function of pH. Among those surface histidyl residues, beta146His has the biggest contribution to the alkaline Bohr effect (63% at pH 7.4), and beta143His has the biggest contribution to the acid Bohr effect (71% at pH 5.1). alpha20His, alpha112His, and beta117His have essentially no contribution; alpha50His, alpha72His, alpha89His, beta97His, and beta116His have moderate positive contributions; and beta2His and beta77His have a moderate negative contribution to the Bohr effect. The sum of the contributions from 24 surface histidyl residues accounted for 86% of the alkaline Bohr effect at pH 7.4 and about 55% of the acid Bohr effect at pH 5.1. Although beta143His is located in the binding site for 2,3-bisphosphoglycerate (2,3-BPG) according to the crystal structure of deoxy-Hb A complexed with 2, 3-BPG, beta143His is not essential for the binding of 2,3-BPG in the neutral pH range according to the proton NMR and oxygen affinity studies presented here. With the accurately measured and assigned individual pK values for all surface histidyl residues, it is now possible to evaluate the Bohr effect microscopically for novel recombinant Hbs with important functional properties, such as low oxygen affinity and high cooperativity. The present study further confirms the importance of a global electrostatic network in regulating the Bohr effect of the hemoglobin molecule.     

Effect of irradiation and/or leucocyte filtration on RBC storage lesions

Red blood cell (RBC) storage lesions have been shown to be associated with some adverse reactions; numerous studies have focused on the lesions caused by storage, and few data on the RBC storage lesions caused by prestorage treatments of leucocyte filtration and irradiation. In this study, we examined the changes related with the RBC storage lesions, including 2,3-diphosphatidylglyceric acid (2,3-DPG), pH, free hemoglobin (Hb), supernatant free K+ and Na+ concentration, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH). Along with the increasing storage time, decreases in 2, 3-DPG levels, pH and Na+ concentration, increases in K+ and free Hb concentrations, and significant morphological changes in RBC in all groups were found. The changes in the groups of irradiation, leucocyte filtration and the combined irradiation and leucocyte filtration were more significant than those in the untreated group. Meanwhile, the MCV levels of the three treated groups were significantly lower than those in the untreated group, while the MCH variations were significantly higher. Our results suggest that irradiation and leucocyte filtration before storage may aggravate blood storage lesions.     

Effect of ligand binding and conformational changes in proteins on oxygen quenching and fluorescence depolarization of tryptophan residues

The rotational freedom of tryptophan residues in protein-ligand complexes was studied by measuring steady-state fluorescence anisotropies under conditions of oxygen quenching. There was a decrease in the oxygen bimolecular quenching constant upon complexation of trypsin and alpha-chymotrypsin with proteinaceous trypsin inhibitors, of lysozyme with N-acetylglucosamine (NAG) and di(N-acetyl-D-glucosamine) ((NAG)2) and of hexokinase with glucose. Binding of the bisubstrate analogue N-phosphonacetyl-L-aspartate (PALA) to aspartate transcarbamylase (ATCase) and binding of biotin to avidin resulted in increased oxygen quenching constants. The tryptophan of human serum albumin (HSA) in the F state was more accessible to oxygen quenching than that in the N state. With the exception of ATCase, the presence of subnanosecond motions of the tryptophan residues in all the proteins is suggested by the short apparent correlation times for fluorescence depolarization and by the low apparent anisotropies obtained by extrapolation to a lifetime of zero. Complex formation evidently resulted in more rigid structures in the case of trypsin, alpha-chymotrypsin and lysozyme. The effects of glucose binding on hexokinase were not significant. Binding of biotin to avidin resulted in a shorter correlation time for the tryptophan residues. The N --> F transition in HSA resulted in a more rigid environment for the tryptophan residue. Overall, these changes in the dynamics of the protein matrix and motional freedom of tryptophan residues due to complex formation and subsequent conformational changes are in the same direction as those observed by other techniques, especially hydrogen exchange. Significantly, the effects of complex formation on protein dynamics are variable. Among the limited number of cases we examined, the effects of complex formation were to increase, decrease or leave unchanged the apparent dynamics of the protein matrix.     

A new method for determining the absolute molarity of solutions of trypsin and chymotrypsin by using p-nitrophenyl N2-acetyl-N1-benzylcarbazate

1. p-Nitrophenyl N(2)-acetyl-N(1)-benzylcarbazate (NPABC) was synthesized and shown to acylate alpha-chymotrypsin stoicheiometrically; reaction at 25 degrees occurs almost instantaneously at pH7.04 and within 2min. at pH5.04 and there is no observable turnover during 10min. 2. The absolute molarity of solutions of alpha-chymotrypsin can be determined by spectrophotometric measurement of the p-nitrophenol liberated during the acylation step; the results obtained at pH5.04 and pH7.04 agree with one another and with those determined by the method of Erlanger & Edel (1964). 3. Trypsin reacts stoicheiometrically, but more slowly than alpha-chymotrypsin, with NPABC, and it, like chymotrypsin, can be spectrophotometrically titrated at pH7.04. At pH5.04, however, reaction between trypsin and NPABC is sufficiently slow for the reagent to be nearly specific for alpha-chymotrypsin. Specificity for one or other enzyme can be ensured by using soya-bean trypsin inhibitor or the chymotrypsin inhibitor l-1-chloro-3-toluene-p-sulphonamido-4-phenylbutan-2-one. Bovine thrombin does not react with NPABC. 4. Evidence is presented that indicates that acylation of alpha-chymotrypsin and trypsin by NPABC occurs at the active centres of the enzymes. 5. Evidence was obtained that indicates that one or more tryptophan residues move into a more hydrophobic environment when alpha-chymotrypsin and trypsin are acylated by NPABC.     

Optimization of hydroxylation of tyrosine and tyrosine-containing peptides by mushroom tyrosinase

Free tyrosine and tyrosine residues in various peptides and proteins are converted into dopa and dopa residues by tyrosinase (monophenol,L-dopa:oxygen oxidoreductase, EC 1.14.18.1) in the presence of reductants. The efficiency of the tyrosine-to-dopa conversion was examined under varied conditions, such as the substrate-to-tyrosine ratio, concentrations of reductant and oxygen in the reaction solution, pH, temperature and reaction time. The highest dopa yields were achieved with the following optimal conditions for hydroxylation: 0.1 M phosphate buffer at pH 7, 25 mM ascorbic acid, 1 mM tyrosine, 50 micrograms/ml tyrosinase and 20 degrees C. Using these conditions, up to 70% of free tyrosine was converted into dopa, and tyrosine residues in several synthetic peptides were also hydroxylated to dopa residues at ratios as high as free tyrosine. The preparation of hydroxylated analogues of the decapeptide (Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys), in particular, may contribute to a better understanding of adhesion in the dopa-containing mussel glue protein.     

Detection of native peptides as potent inhibitors of enzymes. Crystal structure of the complex formed between treated bovine alpha-chymotrypsin and an autocatalytically produced fragment, IIe-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp, at 2.2 angstroms resolution

Chymotrypsin is a prominent member of the family of serine proteases. The present studies demonstrate the presence of a native fragment containing 14 residues from Ile16 to Trp29 in alpha-chymotrypsin that binds to chymotrypsin at the active site with an exceptionally high affinity of 2.7 +/- 0.3 x 10(-11) M and thus works as a highly potent competitive inhibitor. The commercially available alpha-chymotrypsin was processed through a three phase partitioning system (TPP). The treated enzyme showed considerably enhanced activity. The 14 residue fragment was produced by autodigestion of a TPP-treated alpha-chymotrypsin during a long crystallization process that lasted more than four months. The treated enzyme was purified and kept for crystallization using vapour the diffusion method at 295 K. Twenty milligrams of lyophilized protein were dissolved in 1 mL of 25 mM sodium acetate buffer, pH 4.8. It was equilibrated against the same buffer containing 1.2 M ammonium sulfate. The rectangular crystals of small dimensions of 0.24 x 0.15 x 0.10 mm(3) were obtained. The X-ray intensity data were collected at 2.2 angstroms resolution and the structure was refined to an R-factor of 0.192. An extra electron density was observed at the binding site of alpha-chymotrypsin, which was readily interpreted as a 14 residue fragment of alpha-chymotrypsin corresponding to Ile-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp(16-29). The electron density for the eight residues of the C-terminus, i.e. Ala22-Trp29, which were completely buried in the binding cleft of the enzyme, was of excellent quality and all the side chains of these eight residues were clearly modeled into it. However, the remaining six residues from the N-terminus, Ile16-Glu21 were poorly defined although the backbone density was good. There was a continuous electron density at 3.0 sigma between the active site Ser195 Ogamma and the carbonyl carbon atom of Trp29 of the fragment. The final refined coordinates showed a distance of 1.35 angstroms between Ser195 Ogamma and Trp29 C indicating the presence of a covalent linkage between the enzyme and the native fragment. This meant that the enzyme formed an acyl intermediate with the autodigested fragment Ile16-Trp29. In addition to the O-C covalent bond, there were several hydrogen bonds and hydrophobic interactions between the enzyme and the native fragment. The fragment showed a high complementarity with the binding site of alpha-chymotrypsin and the buried part of the fragment matched excellently with the corresponding buried part of Turkey ovomucoid inhibitor of alpha-chymotrypsin.     

Influence of the pH on the photodynamic effect in lysozyme A comparative kinetic study with the sensitized photooxidation of isolated amino acids

Summary The kinetics of the eosin-sensitized photooxidation ([O₂(¹_g)]-mediated) of the protein lysozyme (Lyso) was investigated under two different pH conditions (pH 7 and pH 11). Rates of oxygen consumption and the fade in the protein fluorescence spectrum upon sensitized irradiation were monitored. Parallel studies on both denatured Lyso (absence of the four-S-S- bridges in the protein) and different mixtures of the photooxidizable amino acids of Lyso were also carried out. The mixtures maintained the same molar ratio as in the native protein, and were selected just in order to throw into relief the preferential amino acids that were being photooxidized at both pH values.Under work conditions Lyso was only photooxidizable at pH 7, whereas the opposite accounted for the denatured protein: only measurable oxygen consumption was detected at pH 11. Nevertheless, Lyso at pH 11, evidenced an important physical quenching of O₂(¹_g) due to the Tyr and Trp residues.The results for the native protein were interpreted on the basis of a previously described dark complex Eosin-Lyso, which selectively favours the photooxidation of the bounded protein. The Trp residues were the main reactive entities in the native protein. The photodinamic effect in denatured Lyso was characterized by the prevalence of Tyr residues as photooxidizable targets.In the discussion of the results, a comparisson with the photooxidation kinetics of the mixtures of free amino acids was made.Abbreviations O₂(³_g ^–) ground state triplet oxygen - O₂(¹_g) singlet molecular oxygen - Lyso lysozyme - LysoD denatured lysozyme - Eos eosin - FFA furfuryl alcohol - Trp tryptophan - Tyr tyrosine - Cys cysteine - Cis cystine - Met methionine - His histidine - AA amino acid - a.u. arbitrary units     

Differences in the photodestruction parameters of flavin fluorescence in normal and tumor cells at a decreased pH of the incubation medium

Further investigation of the peculiarities of flavin fluorescence photodestruction in malignant cells was made. In normal cells incubated in low pH (3.0-3.2) physiological solutions, the decrease in the oxidized flavoprotein fluorescence intensity under irradiation is the same as in normal pH condition, whereas in tumor cell in low pH solutions a significant increase in the photodestruction level was noticed. The cells isolated from foci of transformation, following treatment of 3T3 NIH fibroblasts with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine, displayed the photodestruction parameters similar to those in tumor cells. A two-step analysis of cells is proposed for distinguishing between the normal and malignant cells.     

alpha-Chymotryptic hydrolysis of derivatives of the specific substrates with substituents in the nucleus.

Steady state kinetic studies of alpha-chymotrypsin [EC 3.4.21.1]-catalyzed hydrolysis of nucleus-substituted derivatives of the specific substrates were made at pH 6.5 and 7.8. Ac-Trp(NCps)-OMe was hydrolyzed more readily than Ac-Trp-OMe owing to its smaller Km value. The kcat values of Ac-Trp(CHO)-OMe and Ac-Tyr(3-no2)-ome were higher than those of the corresponding unmodified substrates, suggesting that derivatives with a substituent as large as a formyl or nitro group at the epsilon-position are stereochemically favorable to the catalytic process. Derivatives of Ac-Phe-OMe with a chain of four atoms at the 3 or 4-position of the phenyl nucleus and 2,3-dihydropyrrolo[2,3-b]indoles derived from Ac-Trp-OMe were not hydrolyzed at all.     

Kinetic modeling of interesterification reactions catalyzed by immobilized lipase

Kinetic data for lipase-catalyzed interesterification reactions between free fatty acids and triglycerides were collected and the dynamics of the interesterification reactions were successfully modeled using tow rate experssions requiring a total of five adjustable parameters. One rate expression describes the disappearance of the free fatty acid (octanoic or linolenic acid), and the second describes the rate of release of fatty acid residues from the triglycerides (olive oil or milkfat). This model is able to account for the effects of the concentration of all chemical species participating in interesterification throughout the entire reaction. When the data for both milkfat and olive oil were subjected to nonlinear regression analyses using the same mathematical model, the parameter estimates for both systems were comparable. In addition to reproducing the tendencies observed experimentally, simulations of the interesterification system under a variety of initial conditions provided insight into the effects of several reaction variables which could not be examined experimentally. Among the most significant findings of the simulation work are (1) there is a limit beyond which increasing the initial concentration of water produces no further increase in the initial rate of the interesterification reaction; (2) an increase in the initial concentration of lower glycerides produces a concomitant increase in the rate of the interesterification reaction; (3) the free fatty acids inhibit the rate of hydrolysis of the fatty acid residues of the triglycerides; (4) there is a limit beyond which increasing the initial concentration of triglycerides produces no significant increase in the rate of either the hydrolysis reaction or the interesterification reaction. (c) 1994 John Wiley & Sons, Inc.     

The sensitivity of hemoglobin oxygen affinity to diphosphoglycerate and the characteristic pH of methemoglobin.

The sensitivity of the oxygen affinity of a hemoglobin to 2,3-diphosphoglyceric acid concentration has been defined as the change in log1/2O2 (deltalogp1/2O2) which results from saturating the hemoglobin with 2,3-diphosphoglyceric acid. The sensitivity varies from one hemoglobin species to another and is linearly rated to the difference in the logarithm of the binding constants of 2,3-diphosphoglyceric acid to deoxy- and oxyhemoglobin, the characteristic pH (pHch), and inversely proportional to the magnitude of the alkaline Bohr effect measured in a saturating amount of 2,3-diphosphoglyceric acid. Its magnitude is higher in large animals than in small animals and varies linearly with the charged amino acid composition of the hemoglobin. The charged amino acid residues must have been selected for in mammals with high metabolic needs and against in animals with low metabolic needs. Variability in the effect of 2,3-diphosphoglyceric acid on the oxygen transport in the different animal hemoglobins must therefore be the result of a positive Darwinian Selection of the charged amino acid residues in their hemoglobins. Furthermore, all the charged groups and not those at the binding site alone, affect the 2,3-diphosphoglyceric acid binding constant of a hemoglobin.