Specific host-guest interactions in a protein-based artificial transaminase.

Artificial enzymes can be created by covalent attachment of a catalytic active group to a protein scaffold. Recently, we assembled an artificial transaminase by conjugation of intestinal fatty acid binding protein (IFABP) with a pyridoxamine derivative via a disulfide bond; the resulting construct catalyzed a transamination reaction 200-fold faster than free pyridoxamine. To identify the origin of this increased catalytic efficiency computer modeling was first used to identify two putative residues, Y14 and R126, that were in close proximity to the gamma-carboxylate group of the substrate, alpha-ketoglutartate. These positions were mutated to phenylalanine and methionine, respectively, and used to prepare semisynthetic transaminases by conjugation to pyridoxamine (Px) or an N-methylated derivative (MPx). Kinetic analysis of the resulting constructs showed that the R126M mutation reduced substrate affinity 3- to 6-fold while the additional Y14F mutation had a negligible effect. These results are consistent with a model for substrate recognition that involves an electrostatic interaction between the cationic guanidinium group of R126 and the anionic carboxylate from the substrate. Interestingly, one of the conjugates that contains an N-methylated pyridoxamine catalyzes a transamination reaction with a k(cat)' value of 1.1h(-1) which is the fastest value for k(cat) we have thus far obtained and is 34-fold greater than that for the free cofactor in the absence of the protein.     

荧光染料R-藻红蛋白标记小鼠抗人CD4单克隆抗体

以R-藻红蛋白(R-PE)标记小鼠抗人CD4单克隆抗体,形成单色或和用其他荧光染料标记的CD系列单抗组成双色、多色的荧光试剂,应用于流式细胞仪检测分析。用异双功能交联试剂SPDP和SMCC分别活化R-PE和CD4单抗,用DTT使经SPDP活化后的R-PE巯基化,再与用SMCC活化的CD4单抗交联。使用NEM终止交联反应,经Sephacryl S-300柱在AKTA FPLC快速液相色谱系统(简称AKTA)监测下分离纯化。结果用R-PE标记的抗CD4单抗,检测正常人外周血淋巴细胞表面CD4抗原的表达,经流式细胞仪(简称FACS)分析表明,R-PE标记的CD4抗体特异性保持完好,荧光强度较高,还可与用FITC标记的其它CD系列单抗配伍成双标或多标试剂。使用SPDP,SMCC异双功能交联试剂和DTT还原剂,成功地偶联了R-藻红蛋白和CD4单克隆抗体,可应用于流式细胞仪检测分析。     

Synthesis of diketopiperazines with on-resin N-methylation and cyclative release.

Enantiomerically pure N-methylated diketopiperazines (DKP) can be obtained by treating a N-methylated resin-bound dipeptide with 20% piperidine in dimethylformamide via a process known as cyclative release. N-methylated resin-bound dipeptides can be formed from N-methylated precursors or N-methylation can be selectively performed on the resin. When on-resin N-methylation was performed on the C-terminal side of the dipeptide, diastereomers were formed. Yet the cyclative release is shown to be a stereoselective process, as seen using preformed N-methylated amino acids. The procedure was also applied to synthesize the pseudodiketopiperazine cyclo(Phepsi[CH2NH]Leu). When comparing nonmethylated, monomethylated and bismethylated derivatives, we find that N-methylation results in a dramatic increase in solubility.     

Enzymes by design: chemogenetic assembly of transamination active sites containing lysine residues for covalent catalysis

Artificial enzymes can be created by covalent conjugation of a catalytic active group to a protein scaffold. Here, two transamination catalysts were designed via computer modeling and assembled by chemically conjugating a pyridoxamine moiety within the large cavity of intestinal fatty acid binding protein. Each catalyst included a lysine residue, introduced via site-directed mutagenesis, that promotes catalysis by covalent interactions with the pyridoxamine group. Evidence for such interactions include the formation of a Schiff base with the pyridoxal form of the catalyst and a rate versus pH dependence that is bell shaped; both of these features are manifested in natural transaminases. The resulting constructs operate with high enantioselectivity (83-94% ee) and increase the rate of reaction as much as 4200-fold over the rate in the absence of the protein; this is a modest (12-fold) increase in catalytic efficiency (kcat/KM) compared to the conjugate lacking the lysine residue. Most importantly, these artificial aminotransferases are the first examples of designed bioconjugates capable of covalent catalysis, highlighting the potential of this chemogenetic approach.     

Preparation of an insulin conjugate with beta-galactosidase from E. coli for immunoenzyme assay

A modified procedure has been worked out for preparing a conjugate of porcine insulin with E. coli beta-galactosidase employing a heterobifunctional reagent, N-hydroxysuccinimidyl m-maleimidobenzoate. Optimal conditions for insulin acylation and subsequent coupling with beta-galactosidase were selected that afforded the conjugate in a high yield. The ability of the modified antigen to react with antibody was evaluated in the reaction of conjugate binding with immobilized monoclonal antibody to insulin. The conjugate almost completely retained the enzymatic activity and reacted with high specificity with the antibody to insulin. The conjugate can be used in competitive ELISA of insulin.     

应用异型双功能交联剂制备酶-抗体结合物

酶标免疫测定法(ELISA)中最关键的化合物是酶-抗体结合物,将酶和抗体交联起来需用交联剂。本文作者使用了N-琥珀酰亚胺基3-(2-吡啶基二硫)丙酸酯(简称SPDP)将辣根过氧化物酶(HRP)和兔抗小鼠IgG(兔IgG)交联起来。我们试验了SPDP/HRP,SPDP/IgG和HRP/IgG的不同比例,以期获得活性高的酶-抗体结合物。此外还研究了从结合物中去除自由HRP和自由IgG的方法。用SDS-PAGE及硝酸纤维膜电泳转移法证明本法制备的结合物不含HRP及IgG的自身聚合物。用ELISA法鉴定结合物制品时,一般稀释度可达到1:10,000以上,有的可达到1:20,000(当结合物浓度A_(280nm)=1.0,底物显色A_(492nm)=1.0时)。     

A novel biotinylated heterobifunctional cross-linking reagent bearing an aromatic diazirine

The synthesis of a p-[(3-trifluoromethyl)diazirine-3-yl]benzoic acid derivative is described as a new carbene generating heterobifunctional cross-linking reagent. The cross-linker carries a biotin moiety in order to make use of avidin—biotin technology for specific manipulation of cross-linked components. To evaluate the ability of this reagent, the inter-subunit cross-linking of egg-white avidin tetramer was investigated. As a typical application of avidin—biotin technology for cross-linking experiments, a chemiluminescent detection method was examined to identify photobiotinylated components. A cross-linked dimeric product with an apparent molecular mass of 38 kDa was clearly visualized by the combined use of a horseradish peroxidase—streptavidin conjugate and a luminol-based chemiluminescent system.     

Epitope mapping of the O-chain polysaccharide of Legionella pneumophila serogroup 1 lipopolysaccharide by saturation-transfer-difference NMR spectroscopy.

Two modifications of 5-acetimidoylamino-7-acetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (5-N-acetimidoyl-7-N-acetyllegionaminic acid) in the O-chain polysaccharide (OPS) of the Legionella pneumophila serogroup 1 lipopolysaccharide (LPS) concern N-methylation of the 5-N-acetimidoyl group in legionaminic acid. Both N-methylated substituents, the (N,N-dimethylacetimidoyl) amino and acetimidoyl(N-methyl)amino group, could be allocated to one single legionaminic acid residue in the long- and middle-chain OPS, respectively. Using mutants devoid of N-methylated legionaminic acid derivatives, it could be shown that N-methylation of legionaminic acid correlated with the expression of the mAb 2625 epitope. In the present study we investigated the binding of the LPS-specific monoclonal antibody mAb 2625 to isolated OPS with surface-plasmon-resonance biomolecular interaction analysis and saturation-transfer-difference (STD) NMR spectroscopy in order to map the mAb 2625 epitope on a molecular level. It could be demonstrated that the binding affinity of the N-methylated legionaminic acid derivatives was independent from the size of the isolated OPS molecular species. In addition, STD NMR spectroscopic studies with polysaccharide ligands with an average molecular mass of up to 14 kDa revealed that binding was mainly mediated via the N-methylated acetimidoylamino group and via the closely located 7-N-acetyl group of the respective legionaminic acid residue, thus indicating these derivatives to represent the major epitope of mAb 2625.     

The oxidation of Schiff bases of pyridoxamine with α-oxo acids by manganous ions and peroxidase

1. Oxygen was taken up rapidly when pyruvate was added to mixtures of pyridoxamine and Mn(2+) ions after lag periods that were shortened by peroxidase (donor-hydrogen peroxide oxidoreductase, EC 1.11.1.7). 2. The total oxygen uptake was proportional to the pyridoxamine added and was accompanied by the disappearance of pyridoxamine; the pyruvate acted catalytically and hydrogen peroxide was not formed. 3. At pH6 more than half the pyridoxamine that disappeared was accounted for as pyridoxal and ammonia; it is suggested that the primary reaction is the oxidative deamination of the pyridoxamine. 4. Results were similar when alpha-oxobutyrate or glyoxylate were substituted for pyruvate, except that the reactions were slower and the yield of pyridoxal less. 5. The oxidative decarboxylations of alpha-oxoglutarate and phenylpyruvate are catalysed by Mn(2+) ions and these reactions are activated by peroxidase; pyridoxamine increased both the rates and total oxygen uptakes in these reactions, and ammonia was produced. 6. The lag periods in the oxidation of mixtures of pyridoxamine and alpha-oxo acids, catalysed by Mn(2+) ions, were also shortened by traces of colloidal manganese dioxide. 7. It is suggested that the activating effect of peroxidase depends on its catalysis of manganese oxidation.     

Conformation of Leu-Arg-Arg-Ala-Ser-Leu-Gly bound in the active site of adenosine cyclic 3',5'-phosphate dependent protein kinase

Studies utilizing NMR spectroscopy have shown that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) probably binds Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) in one of two extended coil conformations (A or B). The relative reactivities of a series of N-methylated peptides based on the structure of peptide 1 might, therefore, be related to how well each can assume the A or B conformation. From estimates of the magnitude of steric interactions that would be induced by N-methylation of an amide in peptide 1 that is locked in either conformation, the ability of each peptide to form that conformation was predicted. The ability of A-kinase to catalyze phosphorylation of the N-methylated peptides correlated well with the ability of each peptide to form conformation A, but not conformation B. In accord with these findings, the reactivity of an unreactive N-methylated peptide was partially restored by a second change, which allowed the peptide to assume conformation A. These results suggest that, when bound in the enzymatic active site, peptide 1 has a conformation that resembles structure A much more closely than structure B.     

Dimethyl suberimidate as an effective crosslinker for antibody-enzyme conjugation.

Dimethyl suberimidate (DMS), a bifunctional reagent was used for the first time to crosslink the alpha-feto protein monoclonal antibodies (AFPMAb) to horse radish peroxidase (HRP). Three batches of conjugates were prepared, purified by Sephadex gel chromatography and evaluated for their immunological reactivity. The Rz values obtained for AFPMAb-HRP conjugate were 0.39 to 1.36. Under optimised conditions the ELISA results showed the optical density of 1.9. The iso-electric focusing for the conjugate revealed different degrees of crosslinking between antibodies and HRP. It was evident that isoperoxidase-C was involved in the crosslinking process. From the dot ELISA, as low as 25 pg of AFP in the test samples could be detected with AFPMab-HRP conjugate. The conjugate prepared by DMS was stable at 0 degrees C for more than 10 months.     

Preparation and in vitro photodynamic activities of novel axially substituted silicon (IV) phthalocyanines and their bovine serum albumin conjugates

Two novel axially substituted phthalocyanines, namely bis(4-(4-acetylpiperazine)phenoxy)phthalocyaninatosilicon (IV) (1) and its N-methylated derivative 2, have been synthesized. The dicationic phthalocyanine 2 is non-aggregated in water and exhibits good photophysical properties. The non-covalent BSA conjugates of these compounds have also been prepared. Compound 2 and the conjugate 2-BSA show extremely high photodynamic activities toward B16 melanoma cancer cell lines. The corresponding 50% growth-inhibitory (IC50) ratios are 33 and 38 nM, respectively.     

Synthesis of oligonucleotide derivatives with dansyl group at sugar fragment.

The method for introduction of dansyl group into the 2'-position of oligonucleotides is described. The dansyl group was introduced to 2'-position of uridine by the reaction of dansyl chloride with 2'-amino-2'-deoxyuridine, which was then converted to the 5'-dimethoxytrityl phosphorobisdiethylamidite derivative, (4). This reagent was used for the solid-phase synthesis of the oligonucleotide-dansyl conjugate, 5'-ACTCU(DNS)AGAGG. The conjugate could be purified by reversed-phase HPLC. The nucleoside composition of the conjugate was verified by the enzymatic digestion analysis.     

Preparation and characterization of a bovine serum albumin-catalase conjugate

A bovine serum albumin-catalase conjugate was prepared using a heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate. The purified conjugate had an M _r of 380 kDa. The half-life time of the conjugate was 6, 69 and 65 min after heat treatment, urea treatment and trypsin hydrolysis, respectively, whereas that of native catalase was 3, 22, 28 min, respectively.     

N-methylpyridoxamine: novel canine vitamin B6 urine metabolite

Cation-exchange HPLC analysis of urine from dogs given large daily doses of pyridoxamine revealed an unidentified metabolite hypothesized to be N-methylpyridoxamine. Identity was established by N-methylpyridoxamine synthesis and HPLC comparison to the canine metabolite. Compound synthesis was confirmed by IR, NMR, UV-vis and emission spectroscopy. It seems to have less fluorescent character than other routinely-measured vitamin B(6) metabolites. Upon administration of substantial pyridoxamine doses, N-methylpyridoxamine appears to be a quantifiable canine urine metabolite, although, at either pharmacological or dietary pyridoxamine intakes, its relevance to vitamin B(6) metabolism in other species, including humans, is not yet determined.     

Effect of pyridoxamine on chemical modification of proteins by carbonyls in diabetic rats: characterization of a major product from the reaction of pyridoxamine and methylglyoxal

Advanced glycation end products (AGEs) from the Maillard reaction contribute to protein aging and the pathogenesis of age- and diabetes-associated complications. The alpha-dicarbonyl compound methylglyoxal (MG) is an important intermediate in AGE synthesis. Recent studies suggest that pyridoxamine inhibits formation of advanced glycation and lipoxidation products. We wanted to determine if pyridoxamine could inhibit MG-mediated Maillard reactions and thereby prevent AGE formation. When lens proteins were incubated with MG at 37 degrees C, pH 7.4, we found that pyridoxamine inhibits formation of methylglyoxal-derived AGEs concentration dependently. Pyridoxamine reduces MG levels in red blood cells and plasma and blocks formation of methylglyoxal-lysine dimer in plasma proteins from diabetic rats and it prevents pentosidine (an AGE derived from sugars) from forming in plasma proteins. Pyridoxamine also decreases formation of protein carbonyls and thiobarbituric-acid-reactive substances in plasma proteins from diabetic rats. Pyridoxamine treatment did not restore erythrocyte glutathione (which was reduced by almost half) in diabetic animals, but it enhanced erythrocyte glyoxalase I activity. We isolated a major product of the reaction between MG and pyridoxamine and identified it as methylglyoxal-pyridoxamine dimer. Our studies show that pyridoxamine reduces oxidative stress and AGE formation. We suspect that a direct interaction of pyridoxamine with MG partly accounts for AGE inhibition.     

Design and study of peptide-based inhibitors of amylin cytotoxicity

The incidence of type II diabetes is on the increase each year and the World Health Organisation (WHO) predicts there to be over 360 million diabetic patients worldwide by the year 2030. Deposits consisting mainly of a small protein, called islet amyloid polypeptide (amylin), which aggregates into oligo-/polymeric beta sheet structures is responsible for cytoxicity to the pancreatic β-cells, thus inhibition of this process has been explored as a potential prevention or treatment. N-Methylated and non N-methylated peptides spanning the length of amylin_1–37 were synthesised and evaluated for their inhibition of full length amylin mediated cytoxicity to RIN-5F cells. The non N-methylated peptides were very effective in inhibiting the cytotoxicity while the N-methylated peptides were not. Both the N-methylated and non N-methylated versions of the 29-34 region were equally effective.     

Production and characterization of antibodies against the cross-reacting determinant of glycosyl-phosphatidylinositol-anchored acetylcholinesterase.

Dimeric acetylcholinesterase is anchored in the cell membrane by a glycosyl-phosphatidylinositol attached to the C-terminus of the protein. The complex glycan contains an antigenic epitope, the cross-reacting determinant (CRD), which is only revealed after removal of the diradylglycerol by phosphatidylinositol-specific phospholipase C (PI-PLC) but is cryptic in the amphiphilic form. Polyclonal antibodies were raised against the CRD of vertebrate acetylcholinesterase. The purified anti-CRD antibodies recognized only the PI-PLC treated hydrophilic forms of acetylcholinesterase from bovine erythrocytes and Torpedo, and of variant surface glycoprotein from trypanosomes but not the corresponding amphiphilic proteins. Competition experiments showed that inositol-1,2-cyclic phosphate and glucosamine inhibited the binding of the antibodies to the CRD. Furthermore, binding of the anti-CRD antibodies to acetylcholinesterase containing N-methylated glucosamine was markedly reduced. The amphiphilic N-methylated enzyme is less sensitive to digestion with PI-PLC than the non-methylated form. From our results we conclude that inositol-1,2-cyclic phosphate and glucosamine, especially the free amine group of this residue, contribute significantly to the epitope recognized by the anti-CRD antibodies.     

Targeted protein functionalization using His-tags

With the impressive growth in gene sequence data that has become available, recombinant proteins represent an increasingly vast source of molecular components, with unique functional and structural properties, for use in biotechnological applications and devices. To facilitate the use, manipulation, and integration of such molecules into devices, a controllable method for their chemical modification was developed. In this approach, a trifunctional labeling reagent first recognizes and binds a His-tag on the target protein's surface. After binding, a photoreactive group on the trifunctional molecule is triggered to create a covalent linkage between the reagent and the target protein. The third moiety on the labeling reagent can be varied to bring unique chemical functionality to the target protein. This approach provides: (1) specificity in that only His-tagged targets are modified, (2) regio-specific control in that the target is modified proximal to the His-tag, the position of which can be varied, and (3) stoichiometric control in that the number modifications is limited by the binding capacity of the His-tag. Two such labeling reagents were designed, synthesized, and used to modify both N- and C-terminally His-tagged versions of the enzyme murine dihydrofolate reductase (mDHFR). The first reagent biotinylated the enzyme,while the second served to attach an oligonucleotide to yield a protein-DNA conjugate. In all cases, modification in this manner brings new functionality to the protein while leaving the enzymatic activity intact. The protein-DNA conjugate was used to specifically immobilize the active enzyme through DNA hybridization onto polystyrene microspheres, a step toward creating a functional protein microarray.     

Activity of Pyridoxamine as a Substrate for Brain Pyridoxal Kinase

The substrate activity of pyridoxamine (PM) for brain pyridoxal (PL) kinase was examined in view of a recent report which indicated that PM was a poor substrate for this enzyme. Bovine brain PL kinase was shown by liquid chromatography to catalyze the phosphorylation of PM (Km = 65 microM). The identity of the reaction product, pyridoxamine 5'-phosphate, was confirmed by is ability to act as a substrate for liver pyridoxine (pyridoxamine) 5'-phosphate oxidase. The results, which indicate that PM is a good substrate for brain PL kinase, are consistent with the proposed role of intracellular phosphorylation in the uptake of vitamin B-6 brain tissue.