Analysis of streptokinase by validated liquid chromatography methods and correlation with an in vitro bioassay.

Reversed-phase and size-exclusion liquid chromatography methods were validated for the assessment of streptokinase. The reversed-phase method was carried out on a Jupiter C4 column (250 mm × 4.6 mm id) maintained at 25°C. The mobile phase consisted of 50 mM sodium sulfate solution pH 7.0 and methanol (90:10, v/v), run isocratically at a flow rate of 0.8 mL/min. The size-exclusion method was carried out on a Protein KW 802.5 column (300 mm × 8.0 mm id), at 25°C. The mobile phase consisted of 40 mM sodium acetate solution pH 7.0, run isocratically at a flow rate of 1.0 mL/min. Retention times were 19.3 min, and 14.1 min, and calibration curves were linear over the concentration range of 0.25-250 µg/mL (25.75-25 750 IU/mL) (r2 = 0.9997) and 5-80 µg/mL (515-8240 IU/mL) (r2 = 0.9996), respectively, for reversed-phase and size exclusion, with detection at 220 and 204 nm. Chromatographic methods were employed in conjunction with the in vitro bioassay for the content/potency assessment of Streptokinase, contributing to improve the quality control and ensure the efficacy of the biotherapeutic.

In-vitro clot lytic potential of Fagonia arabica: a comparative study of two methods.

The tube method developed in our laboratory is a simple, inexpensive and a classical whole blood clot lytic procedure through which clot lytic potential of Fagonia arabica was found to be significant. Microtiter plate clot lysis (MPCL) assay is a rapid and precise turbidimetric clot lysis method which includes measurements of maximum absorbance (Max Abs), area under the curve (AUC) along with the standard clot lysis time. In the present study we have compared and validated clot lytic potential of F. arabica extract by tube method and MPCL assay. Percentage of clot lysis was calculated by measuring the difference of the absorbance taken at 0 and 240 min in the case of MPCL assay, whereas with the tube method according to the weight difference. Fagonia arabica (50 ug/ml) was capable of clot lysis by MPCL assay and showed clot lysis pattern similar to 60 U/ml streptokinase (positive control). The clot lysis times were significantly different from one another (P value ≤0.001). When Max Abs and AUC were compared to the clot lysis time the correlation coefficient (r value) was significant too (P value ≤0.001). Moreover, we have also found that both the methods showed almost the same clot lysis percentage by streptokinase as well as F. arabica. The correlation coefficient between streptokinase, and F. arabica done by tube method and MPCL assay was found to be statistically significant (P < 0.05). Fagonia arabica had the clot lytic potential checked by in-vitro methods, namely MPCL assay and the method.

Electrochemical assay of plasmin activity and its kinetic analysis.

A sensitive and convenient electrochemical assay of plasmin activity and its kinetic analysis are described. Thus, a ferrocenyl peptide substrate (FcPS) having a plasmin-specific substrate sequence, Lys-Thr-Phe-Lys, and a Cys residue was prepared and immobilized on a gold electrode through the sulfur-gold linkage. The obtained electrode showed a redox signal based on the ferrocene moiety, suggesting the immobilization of FcPS on the electrode. After treatment of this electrode with plasmin, its electrochemical signal was decreased in proportion to an increase of the amount of plasmin. The detection limit for plasmin in this assay system was as low as 50 ng/ml or 0.15 mU/ml. Real-time monitoring of plasmin reaction on the electrode could also be achieved, and the kinetic parameters of this enzymatic reaction could be determined; for example, the k(cat)/K(m) value was 0.063 microM(-1) s(-1). Furthermore, a quantitative assay for streptokinase as a plasminogen activator was also demonstrated by using this system.

Acoustic determination of performance and equivalence of plasminogen activators.

A reliable method for the measurement of different plasminogen activators is of great interest for both manufacturing and clinical medicine. A one-step assay based on a thickness shear mode acoustic sensor has been developed for this purpose. Two separate mixtures of substrates (fibrinogen and plasminogen) and enzymes (thrombin and the plasminogen activator) were mixed, and placed on the acoustic sensor surface. During the assay, the resonant frequency of a quartz crystal oscillating in the thickness shear mode was measured and used to find a characteristic clot dissolution time, from the sample addition to the time at the maximum dissolution rate. Calibrations of the acoustic assay were done for tissue-type plasminogen activator (t-PA) as well as for the other plasminogen activators: urokinase (u-PA); streptokinase (SK) and staphylokinase (SAK). All gave relative standard deviations of about 12%. Since the same method was used for all of the activators, their activities were compared, resolving the differences between their unit definitions. Linear relationships were found between urokinase and streptokinase which activate plasminogen directly and between t-PA and staphylokinase which require fibrin as a cofactor. The relationship between the groups was found to curve, indicating the difference between the two mechanisms. The acoustic method, therefore, may be used as a rapid and cost-effective reference method for the standardization and comparison of different plasminogen activators.

Proteomics based on peptide fractionation by SDS-free PAGE.

Here we demonstrate the usefulness of peptide fractionation by SDS-free polyacrylamide gel electrophoresis and its applicability to proteomics studies. In the absence of SDS, the driving force for the electrophoretic migration toward the anode is supplied by negatively charged acidic amino acid residues and other residues as phosphate, sulfate and sialic acid, while the resulting mobility depends on both the charge and the molecular mass of the peptides. A straightforward method was achieved for SDS-PAGE of proteins, enzyme digestion, peptide transfer and fractionation by SDS-free PAGE, which was named dual-fractionation polyacrylamide gel electrophoresis (DF-PAGE). This method increases the number of identified proteins 2.5-fold with respect to the proteins identified after direct analysis, and more than 80% of assigned peptides were found in unique SDS-free gel slices. A vast majority of identified peptides (93%) have p I values below 7.0, and 7% have p I values between 7.0 and 7.35. Peptide digests that were derived from complex protein mixtures were in consequence simplified as peptides that are positively charged are not recovered in the present conditions. The analysis of a membrane protein extract from Neisseria meningitidis by this approach allowed the identification of 97 proteins, including low-abundance components.

International collaborative study to establish the 3rd International Standard for Streptokinase.

An international collaborative study was organized to calibrate a replacement for the current (2nd) International Standard (IS) for Streptokinase, stocks of which are almost exhausted. Two candidate preparations were assayed against the 2nd IS in a study involving 16 laboratories in 12 countries: preparation 88/824 (coded B), and preparation 00/464 (C and D, coded duplicates). Laboratories could use two methods provided, either a fibrin clot lysis assay or a solution chromogenic method, or an in-house method. Laboratories were encouraged to perform more than one method if possible. With the exception of one laboratory which gave outlying results for preparation 00/464, there was good agreement within and between laboratories and no significant differences between potencies using the different methods employed. This study demonstrates that a solution chromogenic assay is an acceptable format for potency determination of the streptokinase preparations in this study and fibrin is not necessary. It has now been agreed that a solution chromogenic plasminogen activation assay replace the current euglobulin reference method for streptokinase activity determination in the European Pharmacopoeia. Study participants, SSC of the International Society on Thrombosis and Haemostasis and the Expert Committee on Biological Standardization (ECBS) at the World Health Organization approved preparation 00/464 (C,D in the study) as the 3rd IS for Streptokinase with a potency of 1030 IU per ampoule.

A proposed reference method for plasminogen activators that enables calculation of enzyme activities in SI units.

A method has been developed for accurately and precisely measuring the activity of a range of plasminogen activators (PAs) used as thrombolytic agents, including streptokinase, tissue plasminogen activator (tPA) and variants, and urokinase (uPA), both single and two chain forms. Plasminogen activation is monitored in a transparent, solid fibrin matrix but uses chromogenic substrate hydrolysis, rather than changes in fibrin, to quantitate the activity of PAs. The method has been tested in two recent international collaborative studies involving tPA and streptokinase where it has been shown to perform very well. Furthermore, the method is based on sound enzymological principles and once correction for the competitive inhibition of fibrin(ogen) is made, the generation of plasmin can be determined in molar terms and hence the activity of PAs can be expressed and compared in SI units (rate of increase in molar concentration of plasmin) as well as International Units. The assay is also arranged in such a way to reflect the behavior of PAs in vivo during thrombolytic therapy and it is shown that the specific activity of streptokinase and tPA in this system reflects plasmin generation capacity of these thrombolytics for doses given in infusions for treatment of myocardial infarction. The method would make a suitable reference method for PAs and provides a rigorous means of studying and modeling the enzymology of fibrinolysis and will be helpful in the rational design of third generation thrombolytic agents.

Virulence of Streptococcus canis from canine streptococcal toxic shock syndrome and necrotizing fasciitis.

The recent recognition of streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) in dogs caused by Streptococcus canis highlights our lack of knowledge regarding the mechanisms of virulence of this organism. Fifteen isolates of S. canis from cases of canine STSS and/or NF were examined for the presence of 10 Streptococcus pyogenes-associated virulence genes by Southern hybridizations using gene probes generated by PCR. The isolates lacked DNA with homology to eight of the 10 gene probes (speA, speB, speC, mf, ssa, scp, hasA, ska) under low stringency conditions. Thirteen and 15 of 15 isolates hybridized with streptolysin O and M protein gene probes, respectively. Twelve of 15 S. canis isolates were resistant to phagocytosis in canine blood. Electron microscopy revealed the presence of proteinaceous cell surface fibrillae. These results suggest that S. canis possesses M proteins and encodes streptolysin O, but lacks some of the other recognized virulence genes with significant homology to those in S. pyogenes.

[Streptokinase: correlation between different methods of biological evaluation]. / Estreptoquinasa: correlación entre diferentes métodos de valoración biológica.

A study was carried out to establish an appropriate method for streptokinase (SK) potency determination (biological assay) in order to fulfil the main function of the Instituto Nacional de Medicamentos respecting products marketed in Argentina. The potency of different commercial samples of SK was determined against the International Standard, and three internationally accepted methods were used for this purpose: fibrin plate, clot lysis and chromogenic method. The analysis of results suggests that the fibrin plate method is the least precise and reproducible. The clot lysis and chromogenic methods demonstrated great precision and reproducibility, giving a correlation coefficient of 0.99. It is concluded that both of these methods are best suited to determine potency of SK commercial products.

Streptococcal pyrogenic exotoxin A, streptolysin O, exoenzymes, serotype and biotype profiles of Streptococcus pyogenes isolates from patients with toxic shock syndrome and other severe infections.

The determination of protein M and T serotypes, biotypes and pyrogenic (erythrogenic) exotoxin A (SPE A), streptolysin O (SLO), streptokinase (SK), hyaluronidase (HA) and cysteine proteinase release by 212 S. pyogenes isolates from patients with severe invasive group A streptococcal (GAS) infections, among them 74 cases of streptococcal toxic shock syndrome (STSS) has been investigated. M1 or M3 serotypes were expressed by 25% of the isolates (53/212), whereas 59% (125/212) belonged to 15 other different serotypes and 16% (34/212) were untypeable. Of the 74 isolates from STSS patients, 42% (31/74) expressed M1 and to a lesser extent M3 serotypes versus 19% of the non STSS isolates (26/138). Among the ten different biotypes known, biotypes 1 and 3 were prevalent, particularly the former in the case of STSS isolates. SPE A was detectably produced by about 25% (54/212) of the strains. However, as high as 40.5% of the STSS isolates (30/74) versus 17.4% of non STSS isolates (24/138) released SPE A. Moreover, 67% of the SPE A producing strains were of serotype M1 or M3. SK and HA were released by 71% and 10% of the isolates respectively. All strains released SLO (4 to 256 HU/ml) and 85% cysteine proteinase. No relationship between toxin or enzyme titer and the type of disease or clinical origin of the strains was found. Culture supernatants of all isolates showed moderate to high lymphocyte transforming activity with index values ranging from 14.5 to 50.3 including those strains which did not release detectable amounts of SPE A suggesting that SPE C and other mitogenic factor(s) are released by the isolates investigated.

Elevated IgG titer against the C region of streptococcal M protein and its immunodeterminants in patients with poststreptococcal acute glomerulonephritis.

OBJECTIVE: To determine the immune responses to the streptococcal M protein in patients with poststreptococcal acute glomerulonephritis (PSAGN). STUDY DESIGN: The gene coding type 12 M protein of group A streptococcus (M12), a known PSAGN-associated serotype, was cloned and expressed in Escherichia coli to investigate the specific immune responses to the M12 protein in patients with PSAGN. Recombinant M proteins for the variable N-terminal half (AB region) and conserved C-terminal half (C region) were produced separately. IgG titers against each region were measured by enzyme-linked immunosorbent assay in patients with PSAGN (n = 51), uncomplicated streptococcal pharyngitis (n = 26), chronic glomerulonephritis (n = 10), and in healthy control subjects (n = 51). Immunodominant domains within the M protein in PSAGN were further investigated by use of overlapping synthetic peptides. RESULTS: IgG titers against the C region, but not the AB region, were markedly higher in the PSAGN group than in other groups (p < 0.01), and these titers were maintained for at least several months after antistreptolysin O or antistreptokinase levels had returned to normal. Studies with overlapping synthetic peptides demonstrated that increased IgG reactivity was observed against the C repeat blocks. CONCLUSION: IgG titers against the C region are significantly elevated in patients with PSAGN, and it may be a diagnostic marker for PSAGN.

Partitioning of streptokinase in aqueous two-phase systems.

The partitioning of streptokinase in aqueous two-phase systems, containing poly(ethylene glycol) and dextran or poly(ethylene glycol) and salt was investigated. The protein partitions in favour of the upper, PEG-rich phase, if PEG of low molecular weight and potassium phosphate or ammonium sulphate were used. This property was found to be independent of the degree of purity and was exploited for the partial purification of streptokinase from crude material. The protein was shown to exhibit negligible affinity to diverse triazine dyes applied in affinity partitioning experiments.

Streptokinase activity among group A streptococci in relation to streptokinase genotype, plasminogen binding, and disease manifestations.

Certain genotypic variants of streptokinase (ska) of beta-hemolytic streptococci group A have been associated with acute post-streptococcal glomerulonephritis (APSGN). In our earlier studies on strains isolated from Ethiopian children with various streptococcal disease manifestation, we reported an even distribution of streptokinase genotypes with no association to disease patterns. Considering the possibility that strains could differ in their ability to secrete the protein, levels of streptokinase activity in culture supernatants of these strains were determined by a plasminogen activation assay using a synthetic tripeptide, H-D-valyl-leucyl-lysin-p-nitroaniline, as a substrate. Of the 53 streptococcal group A strains, ten (19%), which belonged to genotype ska4 and ska8, did not activate human plasminogen. These strains did not activate bovine, sheep, horse, rabbit or porcine plasminogens either. They represented at least five M protein and non-typeable serotypes, and were characterized by high human plasminogen binding activity. Six of the 53 strains (11%) harbouring genotype ska3 and ska7 showed low levels of human plasminogen activation. Strains of ska1 and ska2, 37/53, activated human plasminogen at a higher level (p < 0.005). Levels of plasminogen activation were not significantly different among the ska1 and ska2 strains associated with various streptococcal disease manifestations. Antibody levels against streptokinase were higher (p < 0.05) in convalescent sera from acute rheumatic fever and APSGN patients in comparison with sera from other patient categories and healthy controls. Streptokinase genotype and in vitro streptokinase production do not correlate directly to streptococcal disease manifestation, indicating a probable significance of additional streptococcal and/or host factors in the initiation of APSGN.

Variables influencing the clot lysis assay of streptokinase.

Clot lysis induced by streptokinase has been studied and the influences of pH and the concentrations of plasminogen, fibrinogen and buffer composition investigated. Variations in the concentrations of plasminogen strongly effect the SK apparent activity, maxima being obtained at different plasminogen concentrations linearly dependent on the concentration of fibrinogen. Buffer composition and pH also effect the apparent activity and the maxima obtained are dependent on the fibrinogen concentration. Analytical conditions are discussed and suitable sample and reagent volumes, as well as concentrations of the reactants and buffer environment, are suggested.

[The effect of sodium nucleinate on the population characteristics of Streptococcus groups A and C]. / Vliianie nukleinata natriia na populiatsionnye kharakteristiki streptokokkov grupp A i C.

The influence of sodium nucleinate on the growth kinetics, streptokinase activity and virulence of streptococcal populations, groups A and C, was studied. As revealed in these studies, the kinetics of the growth of the populations of both strains in the exponential phase did not depend on the concentration of sodium nucleinate in the culture medium. Measurements made on hours 15, 20 and 24 of growth showed the presence of close, direct and statistically significant correlation between the content of biomass, as well as streptokinase activity and specific streptokinase activity, and the concentration of sodium nucleinate in the culture medium. On the basis of the calculation of the coefficients of determination, the main part (70-96%) of the total dispersion of each of the above-mentioned characteristics dispersion of each of the above-mentioned characteristics could be attributed to variations in the concentration of sodium nucleinate. Five passages of faintly virulent streptococcal strains, groups A and C, were not accompanied by a rise in their virulence.

A collaborative study to establish the second international standard for streptokinase.

An International Standard for Streptokinase--Streptodornase (62/7) has been used to calibrate high purity clinical batches of SK since 1965. An international collaborative study, involving six laboratories, was undertaken to replace this standard with a high purity standard for SK. Two candidate preparations (88/826 and 88/824) were compared by a clot lysis assay with the current standard (62/7). Potencies of 671 i.u. and 461 i.u. were established for preparations A (88/826) and B (88/824), respectively. Either preparation appeared suitable to serve as a standard for SK. However, each ampoule of preparation A (88/826) contains a more appropriate amount of SK activity for potency testing, and is therefore preferred. Accelerated degradation tests indicate that preparation A (88/826) is very stable. The high purity streptokinase preparation, coded 88/826, has been established by the World Health Organisation as the 2nd International Standard for Streptokinase, with an assigned potency of 700 i.u. per ampoule.

[Study of the role of histidine residues in streptokinase molecule using modification with diethylpyrocarbonate]. / Issledovanie roli ostatkov gistidina v molekule streptokinazy s pomoshch'iu modifikatsii diétilpirokarbonatom.

The interaction of streptokinase with diethylpyrocarbonate resulting in partial inactivation of the protein was studied. Eight histidine residues are blocked per streptokinase molecule by this reagent. Ethoxyformylation of streptokinase histidyls is characterized by a rate constant corresponding to modification of free L-histidine. No reactivation of streptokinase was achieved by treatment of the modified protein with hydroxylamine. The CD spectroscopy data suggest that the residues modified by diethylpyrocarbonate are of no consequence for the stabilization of the protein secondary structure. The specificity of modification of streptokinase histidine residues by diethylpyrocarbonate is discussed. Based on the gel chromatography data, it was assumed that partial inactivation of streptokinase depends on the formation of protein oligomers with a decreased activatory function.

[Structural-dynamic and functional properties of native and modified streptokinase]. / Strukturno-dinamicheskie i funktsional'nye svoistva nativnoi i modifitsirovannoi streptokinazy.

The process of streptokinase modification by a polymer and the dynamics of protein molecules and the polymer in a protein-polymer (dialdehyde dextran, DAD) conjugate were studied with the aid of luminescent methods. Conclusions were drawn on the structure of the protein-DAD conjugate and the selection of optimum conditions for the preparation of modified streptokinase. It is shown that modified streptokinase is more stable to the action of denaturating agents. The interaction between streptokinase and plasminogen activated by it and changes in the streptokinase-plasminogen complex for modified streptokinase were detected.