Batch purification of high-purity lysozyme from egg white and characterization of the enzyme modified by PEGylation

PEGylation is one of the most promising and extensively studied strategies for improving the pharmacological properties of proteins as well as their physical and thermal stability. Purified lysozyme obtained from hen egg white by batch mode was modified by PEGylation with methoxypolyethyleneglycol succinimidyl succinato (mPEG-SS, MW 5000). The conjugates produced retained full enzyme activity with the substrate glycol chitosan, independent of degree of enzyme modification, although lysozyme activity with the substrate Micrococcus lysodeikticus was altered according to the degree of modification. The conjugate with a low degree of modification by mPEG-SS retained 67% of its enzyme activity with the M. lysodeikticus substrate. The mPEG-SS was also shown to be a highly reactive polymer. The effects of pH and temperature on PEGylated lysozymes indicated that the conjugate was active over a wide pH range and was stable up to 50°C. This conjugate also showed resistance to proteolytic degradation, remained stable in human serum, and displayed greater antimicrobial activity than native lysozyme against Gram-negative bacteria.     

Tryptophan residues of phospholipase A2 from the venom of an Australian elapid snake (Pseudechis australis)

Tryptophan residues 31 and 69 (Trp-31 and Trp-69) in phospholipase A2 (Pa-11) from the venom of an Australian elapid snake, Pseudechis australis, were modified with N-bromosuccinimide (NBS) or with 2-nitrophenylsulphenylchloride (NPSC1). NBS oxidized only Trp-31, whereas NPSC1 reacted with both Trp-31 and Trp-69. Treatment of the enzyme with NBS at various pH values resulted in losses of enzymic and lethal activities. No protective effect on the oxidation with NBS was observed by the addition of calcium ion (20 mM) or lecithin (4 mM). The observations suggest that Trp-31 is exposed to the surface of the molecule, composes a part of the lipid-water interface recognition site around the active site and is essential for enzymic activity. Calcium ion addition to the solution caused a change in ultraviolet spectrum of the native enzyme Pa-11. The difference spectrum indicates that a charge effect caused a typical tryptophan blue shift in the Ca(2+)-enzyme complex. Pa-11 oxidized with NBS showed a smaller ultraviolet absorption difference on the addition of Ca2+ ion. The results show that the hypochromic effect induced upon the binding of Ca2+ is due to perturbation of the specific tryptophan residue (Trp-31) which is involved in the active site. Dissociation constant, Kd, of the Ca(2+)-enzyme complex was calculated to be 3.4 x 10(-4) M at pH 8.0.     

Reaction of carbodiimide for the introduction of p-bis(2-chloroethyl)amino-L-phenylalanine to lysozyme

In the synthesis of new prodrugs with inhibitoring action of tumour growth, a new nitrogen mustard derivative was obtained, proceeding of the coupling between an egg-white lysozyme with an antitumor amine nucleophile, the methyl ester of p-bis-(2-chloroethyl)amino-L-phenylalanine (Melphalan), catalyzed by 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide (EDC), at pH 5.0 and room temperature. In that case, the mechanism for the modification isn't selective of Asp101 in lysozyme. As in cases of histamine and D-glucosamine [3], it is evident that Melphalan is one type of amine who doesn't cause a selective modification of Asp101 but causes somewhat random reaction, because Asp101 is modified followed by modifications of other carboxyls. In this case, we suggest that the amine (Melphalan) may also bind to the substrate binding site in competition with EDC. With this type of amine, enzyme-nucleophile interactions predominate, and the selective activation of Asp101 by EDC is reduced to lead a more random reaction.     

Inhibition by a novel azole antifungal agent with a geranyl group on lanosterol 14 alpha-demethylase of yeast.

AFK-108 (1-[2-(2,4-dichlorophenyl)-2-((2E)-3,7-dimethylocta-2,6- dienyloxy)ethyl]-1H-imidazole) is a new imidazole derivative characterized by a geranyl substituent showing strong antifungal activity. Azole antifungal agents are known to be potent inhibitors of lanosterol 14 alpha-demethylase (P450(14)DM) of fungi. The role of the geranyl group of AFK-108 on interaction of AFK-108 with the target was studied by using Saccharomyces cerevisiae P450(14)DM as the model enzyme. AFK-108 and some of its derivatives bound to oxidized P450(14)DM with one-to-one stoichiometry and inhibited the demethylase activity. AFK-108 derivatives having the longer farnesyl or the shorter prenyl group showed lower affinity than AFK-108 for the enzyme. AFK-108 caused 100% inhibition at the equivalent concentration to P450(14)DM in the reaction mixture (0.07 microM), while the farnesyl derivative inhibited the activity by 60% at the same concentration. AFK-108 interfered with the binding of CO to the ferrous P450(14)DM. However, the interfering effect of the prenyl derivative was lower than that of AFK-108. Another AFK-108 derivative having the saturated 3,7-dimethyloctyl group was also a weaker inhibitor than AFK-108. These experiments suggest that the geranyl group of AFK-108 interacts with the substrate binding site of P450(14)DM that recognises the side chain of the substrate. AFK-108 is the first example of an azole derivative interacting with the side chain recognising region of the substrate binding site of P450(14)DM.     

Effect of carboxylate group modification on enzymatic and cardiotoxic properties of snake venom phospholipases A2

By treating Naja nigricollis and Naja naja atra phospholipase A₂ with carbodiimide and semicarbazide, we obtained derivatives having varied numbers of modified carboxylate groups. When tested on artificial and natural substrates, derivatives of both enzymes with a modified carboxylate group at the active site (Asp-49) retained little enzymatic activity ($\text{1}\text{4}$1 to 10%). However, the derivatives of N. nigricollis also lost most of their lethal potency (5% of native), while those of N. n. atra retained considerable lethality (29%). Caboxyl modification with protection of Asp-49 in N. n. atra enzyme resulted in a derivative with lethal potency equal to or greater than the native enzyme and enzymatic activity which was low on all substrates (12 – 17% of native). Similar protection of Asp-49 at the active site in N. nigricollis enzyme produced a derivative with decreased enzymatic activity on artificial substrate (22% of native) and decreased lethality (17 – 33% of native), but with full enzymatic activity on natural substrates. When tested on electrical and mechanical properties of the isolated perfused heart and the isolated ventricle muscle wall, the derivatives of both enzymes retained considerably more of the cardiotoxic activity than would have been expected based on their residual enzymatic activity. The one exception occurred with the least modified N. nigricollis derivative which had an unaltered Asp-49, this enzyme retained both cardiotoxic activity and full enzymatic activity on natural substrates. The extent of phospholipid hydrolysis following treatment was measured in the isolated heart preparation and in hearts removed from mice following i.v. injection of the phospholipases. Very low levels of phospholipid hydrolysis were observed and no correlation could be made between the extent of hydrolysis and the pharmacological potencies of these enzymes. Modification of the enzymatic active site, whether of Asp-49 in this study of His-48 in prior studies, leads to a large decrease in both enzymatic activity and lethal potency. Asp and Gluresidues outside of the enzymatic site contribute significantly to the lethal potency of the N. nigricollis enzyme and to the enzymatic activity of the N. n. atra enzyme. Based on these and previous data we conclude that changes in isoelectric points are not responsible for altered lethal potencies following chemical modification and that some pharmacological effects of snake venom phospholipases A₂ are due to a non-enzymatic action, suggesting two distinct but perhaps overlapping active sites.     

聚乙二醇对溶菌酶和粒细胞集落刺激因子的初步化学修饰

目的考察聚乙二醇 (PEG)修饰对溶菌酶和粒细胞集落刺激因子 (G CSF)活性的影响。方法以不同方法活化的PEG修饰溶菌酶 ,通过正交试验和单因素考察确定合适的修饰条件 ;溶菌酶活力测定采用溶壁小球菌法 ,抗原性测定采用试管沉淀法 ;G CSF活性测定以小鼠血浆中中性粒细胞数目增殖情况反映。结果当偶联上一个PEG分子时 ,溶菌酶的活性保留 13% ,抗原性显著减弱 ;G CSF经PEG修饰后对小鼠中性粒细胞数目的增加有显著促进作用 ,且作用时间明显延长。结论PEG修饰对G CSF血循环半衰期的延长有显著作用     

Synthesis and evaluation of lysozyme derivatives exhibiting an enhanced antimicrobial action

In order to generate novel preservatives exhibiting a broad antimicrobial spectrum against Gram-positive as well as Gram-negative bacteria, lysozyme was modified by the covalent attachment of caffeic acid and cinnamic acid, respectively. Linkage of these organic acids to lysozyme was achieved by the constitution of amide bindings between the carboxyl group of ligands and primary amino groups of the enzyme mediated by a carbodiimide. Compared to nonmodified lysozyme, the lytic activity of all resulting conjugates was reduced. In contrast, bacterial growth of Escherichia coli (ATCC 8739) could be strongly inhibited by lysozyme–caffeic acid conjugates and to a lower degree also by lysozyme–cinnamic acid conjugates. The minimal inhibitory concentration against E. coli was 0.05% for the lysozyme derivative of the highest antimicrobial activity. However, the efficacy of lysozyme derivatives against Staphylococcus aureus (ATCC 6538) was slightly reduced. As the antimicrobial spectrum of lysozyme altogether could be substantially widened, these derivatives represent promising candidates as novel preservatives for various pharmaceutical and cosmetic formulations.     

THE PH DEPENDENCE OF BINDING OF α, α‘-DIBROMO-P-XYLENESULFONIC ACID TO LYSOZYME

The chemical modification of lysozyme (I) has been accomplished with α,α'-dibromo-p-xylenesulfonic acid (DBX) at five different pH values. I was alkylated by DBX at room temperature (28°C) with decrease in enzyme activity. The rate of inactivation depended upon the pH at which alkylation was carried out. The highest rate was seen at alkaline pH values; the lowest at more acidic pH values. Amino acid analyses showed that two lysines and two tryptophan residues had been modified at pH 9; two lysines, one tryptophan and one methionine had reacted at pH 8. A histidine residue was bound at pH 6.5 together with a tryptophan residue. At the lower pH values (2.7, 4.5, 6.5), alkylation occurred with a single tryptophan residue each. Fluorescence and CD data both ruled out the participation of tryptophans 62 or 108. Labeling experiments showed that two residues of DBX-³⁵S were bound per molecule of I at both pH 9 and pH 8; one residue of DBX was bound per molecule of I at the other pH values. Sedimentation coefficients were characteristic of native lysozyme. The stoichiometry of binding and residue modification indicated that intra-molecular cross links were established. The pH dependence of the cross-linking provides means to measure several allowed intramolecular distances. The results presented here are consistent with the existence of side chain motion in lysozyme.     

The antiviral activity of naturally occurring proteins and their peptide fragments after chemical modification

Chemical modification of the proteins bovine serum albumin, alpha-lactalbumin, beta-lactoglobulin and chicken lysozyme by 3-hydroxyphthalic anhydride (3-HP) yielded compounds which exerted antiviral activity in vitro as compared with the native unmodified proteins. Of the three enveloped viruses tested, human herpes simplex virus type 1 (HSV-1), bovine parainfluenza virus type 3 and porcine respiratory corona virus, only HSV-1 proved sensitive to the 3-HP-proteins. All of the chemically modified proteins presented antiviral activity against HSV-1 when assayed before, during or after infection. However, to achieve HSV-1 inhibition, significantly higher concentrations of the modified proteins were required if present before infection as compared to during or after infection. Our results suggest that multiple mechanisms are involved in the inhibition of HSV-1 infection. Proteolytical digestion of albumin, alpha-lactalbumin, beta-lactoglobulin and lysozyme by trypsin, chymotrypsin and pepsin yielded several peptide fragments with antiherpetic activity. Chemical modification of these peptide fragments by 3-HP generated peptides with antiviral activity, however, this was almost always combined with a cytotoxic effect on the Vero cells. Overall, our results suggest that targeted chemical modification of some natural products might provide compounds effective against HSV-1 infection.     

Critical role of sulfenic acid formation of thiols in the inactivation of glyceraldehyde-3-phosphate dehydrogenase by nitric oxide.

The relationship between possible modifications of the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by nitric oxide (NO) and modified enzyme activity was examined. There are 16 free thiols, including 4 active site thiols, in a tetramer of GAPDH molecule. NO donors, sodium nitroprusside (SNP), and S-nitroso-N-acetyl-DL-penicillamine (SNAP) decreased the number of free thiols with a concomitant inhibition of GAPDH activity in a concentration- and time-dependent manner. After treatment for 30 min, free thiols were maximally decreased to 8-10 per GAPDH tetramer and enzyme activity was also inhibited to 5-10% of control activity. In the presence of 30 mM dithiothreitol (DTT), these effects were completely blocked. Since similar results were obtained in the case of hydrogen peroxide (H2O2) treatment, which is known to oxidize the thiols, these effects of nitric oxide donors were probably due to modification of thiol groups present in a GAPDH molecule. On the other hand, DTT posttreatment after the treatment of GAPDH with SNP, SNAP, or H2O2 did not completely restore the modified thiols and the inhibited enzyme activity. DTT posttreatment after the 30-min-treatment with these agents restored free thiols to 14 in all treatments. In the case of SNAP treatment, all 4 active sites were restored and enzyme activity reached more than 80% of the control activity, but in two other cases one active site remained modified and enzyme activity was restored to about only 20%. Therefore, all 4 free thiols in the active site seem to be very important for full enzyme activity. DTT posttreatment in the presence of sodium arsenite, which is known to reduce sulfenic acid to thiol, almost completely restored both thiol groups and enzyme activity. These findings suggest that nitric oxide inhibits GAPDH activity by modifications of the thiols which are essential for this activity, and that the modification includes formation of sulfenic acid, which is not restored by DTT. S-nitrosylation, which is one type of thiol modification by NO, occurred when GAPDH was treated with SNAP but not SNP. Analysis of thiol modification showed that SNAP preferentially nitrosylated the active site thiols, the nitrosylation of which fully disappeared by DTT posttreatment. It seems that SNAP nitrosylates the active site thiols of GAPDH to prevent these thiols from oxidizing to sulfenic acid.     

PREPARATION OF A TWO-DISULFIDE BONDED ENZYMICALLY ACTIVE DERIVATIVE FROM HEN EGG LYSOZYME

A method has been developed for preparation of an enzymically active two-disulfide bonded derivative from hen egg lysozyme. Lysozyme (0.15 mM) is incubated with 2 mM dithiothreitol at pH 7.8, 23° for 40 min. The products are reacted with [1-¹⁴C] iodoacetic acid and then purified by gel filtration and ion-exchange chromatography. An enzymically active derivative containing 4 mol of [1-¹⁴C] carboxymethyl groups and no free sulfhydryl groups is obtained in approximately 18% yield. Examinations of hydrodynamic volume, tryptophan fluorescence, CD and tryptic peptides containing [1-¹⁴C] carboxymethyl cysteine indicate that this derivative contains two presumably native disulfide bonds and two open disulfide bonds between Cys 6 and Cys 127 and between Cys 76 and Cys 94. The rest of the species in the incubation mixture are intact lysozyme. Thus, the species containing two presumably native disulfide bonds and four free sulfhydryl groups at Cys 6, Cys 76, Cys 94 and Cys 127 appears to be only the intermediate accumulating during reduction of lysozyme with dithiothreitol.     

Melted glyceryl palmitostearate (GPS) pellets for protein delivery

Lysozyme was incorporated into glyceryl palmitostearate (GPS) pellets by compression and melting at loadings of 2, 5 and 10% (w/w). Released lysozyme from both compressed and melted pellets showed good retention of enzymatic activity (>80% active). The percentage lysozyme recovered during in vitro release experiments, over 120 h, was significantly lower from the melted pellets (<15%) compared with compressed pellets (71-85%). Scanning electron microscopy suggested this difference in release was due to differences in porosity of the compressed and melted pellets. Inclusion of hydrophilic components, PEG 4000 and Gelucire 50/13, in the melted matrices increased the percentage of lysozyme released in vitro. Lysozyme released from GPS/PEG 4000 matrices showed good retention of enzymatic activity (>88% active) while that from GPS/Gelucire 50/13 showed reduced activity (68 and 51% active). PEG 4000 was not completely miscible with GPS at the concentrations studied and heterogenous systems resulted. At a loading of 20-35% (w/w) PEG 4000 in GPS greater than 80% of the incorporated lysozyme was released, indicating the likely achievement of interconnecting hydrophilic channels throughout the GPS matrix. In conclusion, melted GPS demonstrated potential as a matrix for the controlled release of proteins and release rates could be modified by inclusion of hydrophilic components.     

Chemical modification of arginine residues of Notechis scutatus scutatus notexin.

Notexin, a presynaptic phospholipase A2 (PLA2) neurotoxin isolated from Notechis scutatus scutatus venom, was inactivated by arginine-specific reagents, phenylglyoxal and 1,2-cyclohexanedione. Kinetic analyses of the modification reaction revealed that the inactivation of notexin followed pseudo-first order kinetics and the loss of PLA2 activity was correlated with the incorporation of one molecule of modification reagent per toxin molecule. However, the results of amino acid analysis and sequence determination revealed that two arginine residues at positions 43 and 79 of notexin were modified simultaneously. Modification of the arginine residues was accompanied with a decrease in the ability to inhibit the indirectly evoked contraction of chick biventer cervicis muscle and bind with synaptic membranes. The secondary structure of the toxin molecule did not significantly change after modification with phenylglyoxal as revealed by the CD spectra. The modified derivative retained its affinity for Ca2+, indicating that the modified arginine residues did not participate in Ca2+ -binding. Together with the notion that Arg-43 and Arg-79 of notexin are located in the proximity of its catalytic site and toxic site, respectively, our results suggest that modification of Arg-43 and Arg-79 should differently contribute to the observed decrease in the PLA2 activity and neurotoxic effect of notexin.     

Structure-activity relationships of new A,D-ring modified steroids as aromatase inhibitors: design, synthesis, and biological activity evaluation

Inhibition of aromatase is an efficient approach for the prevention and treatment of breast cancer. New A,D-ring modified steroid analogues of formestane and testolactone were designed and synthesized and their biochemical activity was investigated in vitro in an attempt to find new aromatase inhibitors and to gain insight into their structure-activity relationships (SAR). All compounds tested were less active than formestane. However, the 3-deoxy steroidal olefin 3a and its epoxide derivative 4a proved to be strong competitive aromatase inhibitors (K(i) = 50 and 38 nM and IC50 = 225 and 145 nM, respectively). According to our findings, the C-3 carbonyl group is not essential for anti-aromatase activity, but 5alpha-stereochemistry and some planarity in the steroidal framework is required. Furthermore, modification of the steroidal cyclopentanone D-ring, by construction of a delta-lactone six-membered ring, decreases the inhibitory potency. From the results obtained, it may be concluded that the binding pocket of the active site of aromatase requires planarity in the region of the steroid A,B-rings and the D-ring structure is critical for the binding.     

Chemical modification at subunit 1 of rat kidney Alpha class glutathione transferase with 2,3,5,6-tetrachloro-1,4-benzoquinone: close structural connectivity between glutathione conjugation activity and non-substrate ligand binding

2, 3, 5, 6-Tetrachloro-1, 4-benzoquinone (TCBQ) is a metabolite of pentachlorophenol known to react with cysteines of glutathione transferases (GSTs). TCBQ treatment of rat kidney rGSTA1-2 and rGSTA1-1 abolishes 70-80% conjugation of glutathione (GSH) to 1-chloro-2, 4-dinitrobenzene and results in strongly correlated quenching of intrinsic fluorescence of Trp-20 (R>0.96). rGSTA2-2 is only inhibited by 25%. Approximately 70% (rGSTA1-1) and 60% (rGSTA1-2) conjugation activity is abolished at TCBQ: GST stoichiometries near 1:1. The inactivation follows a Kitz/Wilson model with K(D) of 4.77+/-2.5microM for TCBQ and k(3) for inactivation of 0.036+/-0.01min(-1). A single tryptic peptide labelled with TCBQ was isolated from kidney rGSTA1-2 containing Cys-17 which we identify as the site of modification. Treatment with more than stoichiometric amounts of TCBQ modified other residues but resulted in only modest further inhibition of catalysis. We interpret these findings in terms of localised steric effects on the relatively rigid alpha-helix 1 adjacent to the catalytic site of subunit 1 possibly affecting the Alpha class-specific alpha-helix 9 which acts as a "lid" on the hydrophobic part of the active site. Homology modelling of rGSTA1-1 modified at Cys-17 of one subunit revealed only modest structural perturbations in the second subunit and tends to exclude global structural effects.     

2,4-Diamino-5-benzylpyrimidines as antibacterial agents. 13. Some alkenyl derivatives with high in vitro activity against anaerobic organisms

A series of 2,4-diamino-5-(3,5-dialkenyl-4-methoxy- or -4-hydroxybenzyl)pyrimidines was prepared from [(allyloxy)benzyl]pyrimidines by Claisen rearrangements, and the resulting allyl phenols were further modified by methylation and rearrangement to 1-propenyl analogues. Analogous 3,4-dimethoxy-5-alkenyl derivatives were prepared by similar techniques. High in vitro antibacterial activity was obtained against certain anaerobic organisms, such as Bacteroides species and Fusobacterium, which was equal to or better than the control, metronidazole, in several cases. The profile was similar against Neisseria gonorrhoeae and Staphylococcus aureus. The 3,5-bis(1-propenyl)-4-methoxy derivative 8 was 1 order of magnitude more active against Escherichia coli dihydrofolate reductase than its saturated counterpart, and it was also more active than trimethoprim, 1. However, it was considerably less active in vitro against the Gram-negative organisms. The 3,4-dimethoxy-5-alkenyl, -5-alkyl, and -5-alkoxy analogues had very high broad-spectrum antibacterial activity. However, pharmacokinetic studies of four of the compounds in dogs and rats and in vivo studies with an abdominal sepsis model in rats showed no advantages over trimethoprim.     

一种新四氢异喹啉类化合物H108体外抑制P-糖蛋白功能及对PC12细胞损伤的保护作用

目的 :观察新合成四氢异喹啉衍生物H10 8抑制P 糖蛋白 (P gp)功能及对PC12细胞损伤的保护作用。方法 :测定H10 8对K5 6 2 ADR细胞株及大鼠脑微血管内皮细胞 (RBMECs)内罗丹明 12 3(Rh12 3)积聚的影响 ,考察H10 8逆转P gp介导的多药耐药性及对血脑屏障上P gp药物外排功能的影响。以连二亚硫酸钠 (Na2 S2 O4)建立缺血缺氧损伤模型 ,过氧化氢 (H2 O2 )建立氧化应激损伤模型 ,硝普钠 (SNP)建立NO损伤模型 ,MTT法测定H10 8对三种PC12损伤细胞存活率的影响。结果 :H10 8浓度依赖性地增加K5 6 2 ADR及RBMECs细胞中Rh12 3的累积浓度。并可明显对抗Na2 S2 O4及SNP诱导的PC12细胞损伤 ,增加细胞存活率。结论 :H10 8具有一定的P gp逆转作 ,并可能具有一定的神经保护作用。其有可能成为一种新型、高效 ,特别是用于促进血脑屏障上药物转运的P gp逆转剂     

Hemoglobin S antigelation agents based on 5-bromotryptophan with potential for sickle cell anemia

5-Bromotryptophan (5-BrTrp) is the most potent amino acid derivative reported in the literature to inhibit the gelation of hemoglobin S (from sickle cell anemia patients). Trp-Trp is also more potent than Trp as an antigelation agent. Therefore, we have prepared a series of dipeptides containing 5-BrTrp and evaluated the antigelation activity. 5-BrTrp-5-BrTrp is the most potent, i.e., 5.9 times the activity of Trp, followed by 5-BrTrp-Trp and then Trp-5-BrTrp. This improved antigelation potency for 5-BrTrp-5-BrTrp and 5-BrTrp-Trp is very significant and will be pursued further as lead compounds with potential for sickle cell anemia.     

Histidine residues in human phenol sulfotransferases

Sulfotransferases are phase II drug-metabolizing enzymes that catalyze the sulfation of hydroxyl-containing compounds, leading to detoxification of xenobiotic toxicants. The universal sulfuryl donor is adenosine 3'-phosphate-5'-phosphosulfate. Human simple phenol sulfotransferase (P-PST) is one of the major human sulfotransferases that catalyze the sulfation of most phenols. Human monoamine phenol sulfotransferase (M-PST) has high affinity for monoamines and also catalyzes the sulfation of simple phenols at high substrate concentrations. In this report, the amino acid modification method was used for studies of His residues in the active site of P-PST and M-PST. The His specific modification reagent diethylpyrocarbonate was used for the modification of His residues in P-PST and M-PST. Diethylpyrocarbonate inactivation kinetic data suggest that there is one His residue in the active site that is critical for catalytic activity of both P-PST and M-PST. The modification has no effect on phenol or monoamine substrate binding for M-PST, but it does have an effect on adenosine 3'-phosphate-5'-phosphosulfate binding with M-PST. The experimental results agree with amino acid sequence alignment, mutation, and the crystal structures of P-PST and M-PST and suggest that His108 is the only critical His residue in both P-PST and M-PST. The differing roles His108 plays in P-PST and M-PST may explain the substrate specificity of the two isoforms.     

Surface modification of enzymes for therapeutic use: monomethoxypoly (ethylene glycol) derivatization of ribonuclease.

Bovine pancreatic ribonuclease A (RNase) was modified at various extent at the lysine residues by monomethoxypoly(ethylene glycol) (MPEG) activated as active ester. For pharmacokinetic experiments a radioactive adduct was also prepared with tritiated amino acid as spacer between polymer and protein. The modification reduced only slightly the RNase catalytic activity and Km towards the substrate cytidine-2',3'-cyclic monophosphate. On the other hand extensively modified MPEG-RNase samples, showed significant decrease in activity towards ribonucleic acid. The polymer modification did not change the pH activity profile, increased the stability to proteolytic digestion, while the behaviour towards denaturants and heat was not modified. The native and MPEG-RNase administered IV, IM and SC to rats, showed impressive differences in pharmacokinetics: the half-life of the modified enzyme, evaluated in blood by radioactivity, was increased of 40-50 folds with respect to the native form.