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We analyzed seminal plasma of 88 normozoospermic, 40 oligozoospermic and 32 azoospermic donors. During this study, we focus to record the protamine concentration in the seminal plasma of azoospermic donors. The seminal protamine concentrations were found to be 19.6–62.8 IU/ml in normozoospermic donors; 25.4–100.8 IU/ml in oligozoospermic donors; and, most notably, 23.7–219.4 IU/ml in azoospermic donors. These results indicate that, based on seminal plasma protamine concentrations, even azoospermic donors were able to produce as much sperm as normo- and/or oligozoospermic donors. Using statistical analyses, significant differences were found between azoospermic and normozoospermic donors (p = 0.0018). Protamine content was found to be a direct marker for the presence of sperm. The data from this study provided evidence for a new therapeutic approach for testicular varicose veins, which are found in obstructive or non-obstructive azoospermia. High seminal protamine concentrations indicated the future possibility of acquiring childbearing sperm for insemination sperm injection (ICSI) and testicular sperm extraction (TESE), even with azoospermic donors. Given these results, we also suggest a new cut-off value for acquisition of childbearing sperm in selection for ICSI.

A novel label-free fluorescence nanosensor was developed for ultrasensitive detection of protamine and heparin based on fluorescence resonance energy transfer (FRET) between NaYF4:Yb,Er upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs). The FRET system was formed by the electrostatic adsorption of AuNPs on UCNPs, and the fluorescence of UCNPs was significantly quenched. When protamine was added to the mixture of UCNPs-AuNPs, the AuNPs interacted with protamine and then desorbed from the surface of UCNPs and aggregated, resulting in the recovery of the fluorescence of UCNPs. On the addition of both protamine and heparin, the FRET system formed owing to the stronger interaction between heparin and protamine than that with AuNPs, leading to a marked fluorescence quenching of UCNPs. The concentrations of protamine and heparin were proportional to the changes of the fluorescence of UCNPs. The linear response range was obtained over the concentration ranges of 0.02 to 1.2μg/ml and 0.002 to 2.0μg/ml with low detection limits of 6.7 and 0.7ng/ml for protamine and heparin, respectively. Simultaneous measurement of protamine and heparin in human serum can be achieved, suggesting that the nanosensor can be used in a complex biological sample matrix.

Interferences in human plasma immunoassay are severe challenge that affects the sensitivity and reproducibility of the assay. The clotting factor fibrinogen is a negatively charged protein and is one of the most common sources of interference in immunoassays, and its removal increases the sensitivity and reproducibility. Here, we present a highly sensitive and reproducible method for the detection of prostate specific antigen (PSA) in human plasma immunoassays. Protamine sulfate, a highly positively charged protein, was used to precipitate fibrinogen via ionic interaction to improve the sensitivity and reproducibility of human plasma immunoassay. In a sandwich ELISA for PSA using plasma and protamine-treated plasma samples, the limit of detection was improved from 413 pg/mL in plasma to 235 pg/mL in protamine-treated plasma samples, and the coefficient of variation known as a measure of reproducibility was significantly lowered by protamine treatment. The use of protamine sulfate in human plasma immunoassays for detection of PSA using quartz crystal microbalance (QCM) biosensors resulted in increased sensitivity and reproducibility by about 2-fold and 3-fold, respectively, relative to when not using protamine sulfate. Based on these results, protamine sulfate was the best choice to increase the sensitivity and reproducibility in immunoassays using plasma samples.

We report here on a highly sensitive and rapid detection technique, multipulse flash chronopotentiometry, for the anticoagulant polyion heparin and its antidote protamine. The technique is based on a localized titration of the polyions at the surface of an appropriately formulated polymeric ion-selective membrane devoid of ion exchange properties to prohibit spontaneous extraction processes. A defined ion flux from the sample side to the membrane is induced electrochemically by applying a current pulse of appropriate amplitude and sign. The resulting depletion of the measured ions at the membrane surface gives rise to a characteristic limiting current or transition time and is observed as an inflection point in the resulting chronopotentiogram. The limiting current and the square root of the transition time are linear functions of the concentration of the polyion and yield sensitive and rapid analytical information attractive for clinical diagnostics applications. The polyion protamine is detected in 10-fold diluted blood samples in a matter of seconds via a cathodic current pulse. The utility of the technique for monitoring heparin/protamine titrations in physiological saline solutions is demonstrated.

Quantum dot (QD) nanohybrids provide an effective route to explore the new properties of materials and are increasingly used as highly valuable sensitive (bio) chemical probes. Interestingly, the room-temperature phosphorescence (RTP) of 3-mercaptopropionic acid (MPA)-capped Mn-doped ZnS QDs could be remarkably enhanced by the addition of protamine. Based on the above finding, a simple, sensitive, and selective method for rapid detection of protamine was successfully designed. With this method, protamine as a cationic peptide interacts electrostatically with MPA-capped Mn-doped ZnS QDs to form MPA-capped Mn-doped ZnS QD/protamine complexes, which leads to the aggregation of QDs and enhances the RTP intensity. Under the optimized conditions, the RTP intensity change was linearly proportional to the concentration of protamine in the range 0.2-3.0 μg ml(-1), and the limit of detection was 0.14 μg ml(-1). The proposed method was successfully applied to detect protamine in protamine sulfate injection and human serum samples with satisfactory results, and the recovery ranged from 96.5 to 105.6%.

Two relatively simple electrochemical assay methods suitable for the measurement of plasminogen activators (including urokinase (u-PA), streptokinase (SK), and tissue plasminogen activator (t-PA)) in plasma samples are described. In one approach, the initial rate of decrease in the potentiometric response of a polycation-sensitive membrane electrode toward protamine is monitored after addition of a preincubated reaction mixture containing the sample and exogenous plasminogen. The plasmin formed from plasminogen by the activators catalyzes the decomposition of the arginine-rich protamine substrate, yielding smaller polycationic fragments that are not sensed by the electrode. Alternately, the sample, plasminogen, and protamine can be incubated together, and the remaining protamine in this reaction mixture can be measured at a fixed point in time by placing the electrode into the mixture and recording the electromotive force response. Working curves found with both methods for plasma samples spiked with varying levels of the activators cover the expected therapeutic activity ranges found in the plasma of patients treated with these “clot-busting” drugs.

A polycation-sensitive membrane electrode based on the ion-exchanger dinonylnaphthalene sulfonate has previously been developed and used as an end-point detector for the determination of unfractionated heparin in whole blood samples via simple potentiometric titration with protamine. Herein, we report the application of the same methodology for the quantitation of a commercial low-molecular-weight heparin (LMWH) preparation (Fragmin) in whole blood samples at concentrations up to 2 U/ml. Further, an analogous polyanion (heparin)-sensitive electrode is used to estimate the binding constants between protamine and various LMWH preparations. The equilibrium constants (Keq) and the number of binding sites per mole of heparin (n) are determined by recasting the data in the form of a Scatchard plot. Results show that the average molecular weight and molecular weight distribution of the LMWH preparation are important parameters affecting their binding with protamine. Comparable binding constants are obtained for the same LMWH preparations titrated with a synthetic protamine analog, [+18RGD] [acetyl-EA(R2A2R2A)4R2GRGDSPA-NH2].

A simple and sensitive electrochemical method suitable for real time detection of trypsin-like proteinase inhibitors is described. The method is based on utilizing a protamine (a polycationic substrate for trypsin-like proteinases)-sensitive membrane electrode to monitor, potentiometrically, the initial rate of protamine decomposition upon the addition of a proteinase–antiproteinase test solution. In the presence of proteinase inhibitors, the initial rate of change in electromotive force is dependent on the concentration of proteinase inhibitor in the sample solution. The feasibility of this new assay method is demonstrated by detecting four inhibitors of trypsin-like proteinases: α 1 -antiproteinase inhibitor, α 2 -macroglobulin, aprotinin, and soybean inhibitor, using trypsin as the indicator proteinase. The efficacy of inhibition by each species, as expressed by I 50 values (concentration of the inhibitor that induces 50% of the maximum proteinase inhibition), is shown to correlate well with literature values for the association constant of the proteinase–antiproteinase complex ( k assoc ). The proposed electrochemical assay for aprotinin is examined further using trypsin, plasmin, and kallikrein as the proteinase indicator reagents. It is found that the trypsin–aprotinin system offers the highest sensitivity and lowest detection limit for aprotinin detection. Application of the proposed method for measuring aprotinin in pretreated plasma samples is also reported.

A simple and rapid homogeneous enzyme-based binding assay is described to study the degree of interaction between glycosaminoglycans and various macromolecules/peptides. The method is based on the homogeneous inhibition of a highly positively charged enzyme, acid deoxyribonuclease II (EC 3.1.22.1), by glycosaminoglycan polyanions, such as heparin, chondroitin 4-sulfate, and dermatan sulfate. Catalytic activity of DNase II is inhibited to nearly 100% by relatively small amounts of these glycosaminoglycan molecules. In the presence of species that bind these polyanions, the activity of the enzyme is regained in an amount proportional to the concentration of the species present. Thus, the relative binding affinities of various species with a given GAG can be assessed rapidly by comparing the concentration of the compound required to reverse the enzyme inhibition to 50% of the maximum value (ED 50 values). The feasibility of this binding assay principle is demonstrated by measuring the ED 50 values of five macromolecules: polylysine, polyarginine, protamine, low-density lipoprotein (LDL), and high-density lipoprotein (HDL), using heparins of different size, as well as chondroitin 4-sulfate and dermatan sulfate as the GAG polyanions. The applicability of the assay method is further extended to study GAG–peptide interactions. A variety of small synthetic peptides (8–13 amino acid residues) derived from the heparin-binding domains of protamine and type IV collagen are used as model peptide species. Relative GAG-binding affinities of these macromolecules/peptides are compared to previous literature values, and data obtained via a new electrode-based titration method.

Affinity coelectrophoresis (ACE) is a technique for characterizing ligand/nucleic acid binding interactions under equilibrium conditions. It is used here to characterize the protamine clupeine Z binding to several DNA fragments in order to define the use and limitations of ACE for ligands that bind cooperatively and nonspecifically to nucleic acids. The results demonstrate that the ACE data for cooperative, nonspecific ligands can be analyzed using the McGhee-von Hippel model and that binding-site sizes can be accurately determined using lattices containing as few as one site. However, binding constants can be greatly underestimated for some cooperative ligands with large-site sizes if small lattices are used. The salt dependence of the binding constant can also be determined but is limited to salt concentrations less than approximately 300 mM. Given the simplicity and reproducibility of the ACE assay, it should find many applications for studying binding interactions for a variety of cooperative and noncooperative nucleic acid binding peptides and proteins.

O-Desulfated heparin (ODSH) is a promising new anti-inflammatory agent for the prevention of reperfusion injury following myocardial infarction or stroke. This partially desulfated heparin derivative has less anticoagulant activity than unfractionated heparin but retains the inherent anti-inflammatory properties of heparin. Thus, ODSH could be administered at the high doses needed to achieve desired anti-inflammatory function without risk of hemorrhage. However, given the very low anticoagulant activity of this species, traditional methods for heparin determination in clinical samples might not be well suited for ODSH measurements. In this article, a novel titrimetric method for detection of ODSH in buffer and plasma is described using a protamine-sensitive polymer membrane electrode as the detector. Titrations of ODSH with the heparin antagonist protamine yield sharp endpoints with sensitivity to ODSH in the micrograms per milliliter range for plasma samples. The stoichiometry for protamine interaction with ODSH is determined to average 1.39 microg protamine/microg ODSH in plasma. This technology is further applied to a toxicokinetic study of ODSH in an animal model, demonstrating the ability to detect the changes in ODSH concentrations in biological samples.

A polymeric membrane electrode that exhibits significant and analytically useful potentiometric response to submicromolar levels of the heparin antagonist, protamine, is reported. The sensor is prepared by incorporating a lipophilic cation exchanger, potassium tetrakis(4-chlorophenyl)borate (KTpClPB) (at 1 wt%), within a specially formulated polymer membrane composed of 33 wt% 2-nitrophenyl octyl ether (2-NPOE), and 66 wt% poly(vinyl chloride) (PVC). When the polymer film is mounted in an appropriate electrode body, the resulting membrane electrode responds reproducibly to protamine via a nonequilibrium quasi-steady-state change in the phase boundary potential at the membrane/sample interface. Such response can be used to directly monitor, via classical potentiometric titrations, the binding between protamine and a variety of native (porcine and beef) as well as low-molecular-weight heparins. Scatchard analysis of the EMF titration data provides binding constants and stoichiometries for protamine-heparin interactions. The electrode can be further used to follow the enzymatic digestion of protamine by trypsin. In the presence of a given level of protamine, initial rates of potential decrease (-dE/dt) are shown to be linearly related to trypsin activity in solution over the range of 0-130 units/ml. The speed and simplicity of the protamine sensor make it an attractive alternative to classical methods for studying the interaction of protamine with other biologically important macromolecules as well as the proteolytic activity and reaction kinetics of trypsin.

Heparin was immobilized in a defined orientation on Sepharose, agarose, and polyacrylamide supports by coupling through its reducing end. This is expected to mimic the attachment of heparin to the protein core in the naturally occurring proteoglycan and impart better ligand binding efficiency by exposing all the binding sites available in the naturally occurring heparin. The coupling chemistry was accomplished by modifying heparin at its reducing end to introduce reactive functionality that can react with appropriately functionalized supports. Three reducing end modified heparins were synthesized and characterized: 2,6-diaminopyridinyl heparin, containing a reactive amino group at the reducing end; omega-hydrazido-adipyl-azo heparin, containing a hydrazido group at the reducing end; and heparin lactone, containing a reactive ester functionality at the reducing end. These heparin derivatives were then reacted with the supports to give directionally immobilized heparin using different coupling chemistries: coupling of reducing end modified heparins to amine-containing supports (i.e., omega-aminohexyl Sepharose and omega-aminobutyl agarose), hydrazide-containing supports (i.e., Emphaze hydrazide), and activated carboxy-containing supports (i.e., activated 6-aminohexanoic acid Sepharose, Emphaze azlactone). The heparinized matrices were prepared and analyzed for their heparin content and protamine binding capacity.

Heparin plays an important role in anticoagulation and several other biological processes. Cleavage of heparin by nitrous acid results in a reactive 2,5-anhydromannose (Am) which can be used to selectively insert primary and secondary amines by reductive amination. Low-molecular-mass heparin (LMMH) was bound to 4-(2-aminoethylphenol) as shown by nuclear magnetic resonance spectroscopy (NMR), high-performance size-exclusion chromatography (HPSEC), polyacrylamide gel electrophoresis (PAGE), and ultraviolet/visible (uv/vis) spectroscopy. 1H NMR spectra revealed an average sequence of (IdoA2SO3-GlcNSO36SO3)9-IdoAaSO3-Am-tyramine and a 50% binding rate of tyramine to LMMH. LMMH-Tyr had an anticoagulant activity of 108 antifactor Xa activity (aXa) U/mg and 42 antifactor IIa activity (aIIa) U/mg. The compound was neutralized by protamine. The N-alkylamine derivative was adopted to label LMMH with iodine-125 by oxidation with chloramine T. Fluorescein-5-isothiocyanate (Fitc) was used to label LMMH-Tyr with fluorescence. NMR, HPSEC, PAGE, and uv/vis spectroscopy demonstrated the binding of Fitc to LMMH-Tyr. 1H NMR spectra indicated that about 80% of the LMMH-Tyr was labeled at the secondary amino group. The fluorescent compound exhibited 70 aXa and 5 aIIa U/mg and was neutralized by protamine. The selectively bound labeled heparin derivatives are "endpoint attached" and have intact anticoagulant activity.

Extensive purification of DNA polymerase alpha-primase resulted in a marked loss of the DNA polymerase alpha activity. This loss is due partly to the elimination of some basic proteins from the enzyme preparation since the activity of purified enzyme was stimulated 10- to 15-fold by the addition of various basic proteins, including all five classes of histones, protamine, poly-L-lysine, and poly-L-arginine, at a concentration of 2 micrograms/0.2 ml in the presence of 20 micrograms/0.2 ml of activated DNA. The optimum concentration of the basic proteins and the maximum activity attained at that concentration varied with varying concentrations of the template primer used, indicating that the observed stimulation is caused by an interaction between these basic proteins and activated DNA. The enzyme activity with an optimal concentration of activated DNA was markedly inhibited by the addition of denatured DNA. The suppressed enzyme activity could be restored by an appropriate concentration of histone H1. These results suggest that histone H1 and other basic proteins protect the enzyme from forming an abortive complex with single-stranded DNA or with a long stretch of the single-stranded part of activated DNA as single-stranded DNA-specific binding proteins do (M. Sapp, H. König, H. D. Riedel, A. Richter, and R. Knippers (1985) J. Biol. Chem. 260, 1550-1556). Spermine also showed a similar stimulatory effect. All acidic proteins tested were ineffective.

Protamine sulfate is used to release histones and other basic proteins from the DNA of chromatin. This phenomenon becomes the basis of a versatile method for analysis of the nucleic acid and protein composition of nucleoprotein samples, which is termed here in situ protamine release. When protamine is added to a nucleoprotein sample in 5% acetic acid and 8 m urea, at a concentration of 1.0%, ≥94% of the histones are released from the DNA of chromatin, comparable to the release of histones using sodium dodecyl sulfate. This makes in situ protamine release the method of choice for the analysis of acid-soluble proteins on acid/urea-based gels, where the DNA must be removed from the protein prior to electrophoresis. Compared to DNase I release or acid extraction, protamine release is found to be the simplest, most reliable, and most effective method for removing the acid-soluble proteins from DNA. Protamine is either added to the sample (very much like the detergent, sodium dodecyl sulfate), or electrophoresed through a gel containing nucleoprotein, thus displacing proteins in its path. A serendipitous advantage of protamine is that it can also serve as a carrier for the precipitation of dilute nucleoprotein samples with ethanol, and 3 m m Mg 2+ , to concentrate the nucleoprotein in preparation for analysis. A unique feature of the in situ protamine-release method is that the DNA is not lost or destroyed and can therefore be used for subsequent analysis.

Fluorescamine is a useful reagent in monitoring protein-DNA interactions only if a convenient method of separating the complex from free protein is available. Sedimentation of the complex provides such a method at least in the case of histone-like proteins capable of extensive interaction with DNA. This approach is therefore complementary to the filter binding assay. When the interaction of protein and DNA is compared by both methods, a clear-cut distinction between two steps is obtained: (i) a nucleation step that can be measured by the filter binding assay: and (ii) the cooperative growth of the complex that can only be measured by the sedimentation assay. The method is also useful to detect small amounts of protease contamination in DNA preparations.

α- and e-amino groups of basic chromosomal proteins (protamines and histones) are available for the reaction with fluorescamine. The extent of the reaction depends on the respective pKa values but linear relationships between the protein concentration and fluorescence were found at all pH values tested (5.5–10.5). Upon interaction with DNA, the pKa values of protein amino groups are raised to an extent which excludes them from the fluorescamine reaction under quasiphysiological conditions. Protein displacements caused by dissociating agents may conveniently be followed kinetically and can be quantified by appropriate applications of fluorescamine.

The conditions of the Hirsch-Rosen assay (1974, Anal. Biochem.60, 389–394) for protein kinase activity oncomplete isoelectric focusing gels have been analyzed and further developed with the consequence that the test can be easily adapted to other protein kinases in a quantitative manner. Special attention was given (i) to the breakdown of the pH gradient in relation to gel size and Ampholine concentration in order to achieve optimal pH ranges for the assay, (ii) to the introduction of histone as a substrate besides protamine, and (iii) to the distribution of the particular substrates and products throughout the gel. The results of the protein kinase assay on gels were shown to be linear for at least 1 h, and to be dependent on the amount of ATP and on the amount of protein kinase applied, thereby fulfilling the requirements necessary to yield quantitative data.

A quantitative colorimetric assay for serum carboxypeptidase B (SCPB, anaphylatoxin inactivator, kininase I) is described. SCPB is known to possess an enzymatic specificity for cleaving COOH-terminallysyl and arginyl residues which is similar to the specificity of bovine pancreatic carboxypeptidase B. One function of SCPB involves the inactivation of C3a and C5a, the two complement derived anaphylatoxins. Since cobalt markedly enhances the activity of the enzyme, serum is treated with CoCl 2 before the SCPB assay is performed. Salmine, a protamine from salmon sperm, was selected as the substrate because it contains multiple COOH-terminal arginyl residues and is digested more rapidly by SCPB than other common substrates of carboxypeptidase B, including hippuryl-arginine and benzyl-glycylarginine. The kinetics for arginine release from salmine were first-order throughout the course of the assay and the colorimetric values obtained were related to micromols of arginine released. A unit of SCPB is defined as one nanomol of arginine released per minute per milliliter of serum. The range of SCPB activity in serum from healthy individuals was found to be 318 to 466 units. The medians of SCPB activity in sera obtained from patients with Dengue shock syndrome and with shock following intravenous dextran infusion were both lower than the mean SCPB activity of healthy individuals. SCPB levels in patients homozygous and heterozygous for cystic fibrosis were within the normal range.

The ability of protamine sulfate to effect the quantitative precipitation of 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD):Ah receptor complexes from rat liver cytosol has been developed into a new assay for the identification, quantitation, and characterization of the Ah receptor. The method is reliable, uncomplicated, and rapid, and can be applied to large numbers of samples. The major advantage of the assay is that protamine sulfate appears to selectively precipitate the Ah receptor protein and does not precipitate a number of other proteins that bind [3H]TCDD nonspecifically.

Only 30% of commercial heparin reacts with antithrombin III (ATIII). This study shows that the interaction is pH dependent: 100% of the heparin binds to ATIII at pH 3.0, 30% at physiological pH. Binding of ATIII, platelet factor 4, and protamine to heparin was studied using a new rocket precipitin electrophoresis procedure, adapted from the Laurell rocket immunoelectrophoresis procedure. Protamine is incorporated into agarose gel, and heparin mixtures with protamine, ATIII, or platelet factor 4 electrophoresed into the gel from a series of wells. The residual free heparin is precipitated by the protamine in a rocket-shaped arc, the height of which is proportional to the amount of free heparin. No antibody is employed. This procedure is useful for quantitation of heparin and for studying the binding of heparin to proteins.

Directions are given for the use of Reinecke's salt as a precipitating agent in the preparation, purification and concentration of histones. The histones as well as other basic compounds of an acid tissue extract are quantitatively precipitated as reineckate complexes. Only predominantly basic proteins such as histones, protamines, lysozyme, and cytochrome c (reduced form) remain as insoluble reineckates when the pH is raised to 8.3. Precipitation with Reinecke's salt and subsequent extraction of the precipitate at pH 8.3 do not affect aggregation of the histone molecules. After extraction of the histone-reineckate with acid acetone, the histones can be dissolved in the concentration wanted.

Chromatographic resolution of protein mixtures: egg albumin and lysozyme, and another containing insulin and protamine (Mugil cephalus) was achieved on paper strips using a several step elution with neutral salt solutions of increasing concentrations. Experimental data concerning the influence of cations and anions on chromatographic separation of proteins on paper strips are presented.

Techniques for rapidly aggregating suspensions of small vesicles made either of natural biological membrane or of phospholipid or phospholipid-protein mixtures by addition of one of the basic polypeptides, polylysine or protamine, have been investigated. Either filtration or centrifugation may be used to rapidly (15 to 30 s) and completely (over 98%) separate the vesicular aggregate from the suspending medium. At low values of the polylysine-to-vesicle weight ratio, aggregation is observed to increase with increasing polylysine concentration. At high values of this same ratio, aggregation decreases with increasing polylysine concentration. With protamine, like polylysine, aggregation increases with protamine concentration at low values of the weight ratio. At high values of the ratio, aggregation induced by protamine does not rapidly decrease with increasing protamine concentration as it does with polylysine. Explanations are given for these observations. While leakage induced by polylysine may be troublesome under some conditions with phospholipid vesicles or reconstituted systems, protamine aggregation has been found to induce much less leakage than polylysine aggregation. The leakage rate induced by protamine was not found to be significantly different from the control leakage rate for the first few minutes after addition of protamine under any of the conditions tested. Since this provides ample time for filtration, the protamine aggregation-filtration technique seems to be the method of choice for separation of many types of small vesicles from the suspending medium. It combines the advantages of rapid separation, complete separation, low rate of vesicle leakage, and versatility in being able to separate the vesicles from uncharged molecular components such as sugars where ion-exchange techniques will not work (1), as well as from ionic components. Low cost and simplicity are further advantages of the technique.

Uricase was found to be stabilized by protamine from salmon testis. Protamine was then bound to controlled-pore glass beads aminohexyl CPG 500 using glutaraldehyde. Microbial uricase was readily immobilized on the protamine bound to glass beads. The immobilized uricase proved to be stable even at 70 degrees C, whereas free uricase was inactivated at 45 degrees C and showed activity over a broader pH range than free uricase. Automated analysis of uric acid was facilitated using the immobilized uricase. The standard curve for uric acid was linear in the range of 2 to 10 micrograms/sample and passed through the origin. This automated procedure was also applicable to the determination of uric acid in human serum. Protamine bound to glass beads is expected to be useful for the simple immobilization and stabilization of enzymes.

The technique of separating small vesicles from suspension by aggregation with protamine or polylysine, followed by either filtration or centrifugation has proved a very useful one. This is because the minimum times necessary to separate vesicles from the suspending medium are quite short (15 to 30 s) and the separation is almost complete (over 98% in the cases tested). The primary limitation of this technique, until now, has been that it could only be used for a terminal assay, because the aggregates produced could not be easily disaggregated. It has been found that after aggregation of vesicles by protamine and their separation from suspension by low-speed centrifugation, the addition of an excess of heparin over protamine, followed by vortexing or homogenizing the pellet in fresh medium, satisfactorily disaggregates the vesicles. This process has been tested with phospholipid vesicles and proteoliposomes and with vesicles from inner mitochondrial membrane, sarcoplasmic reticulum, and red blood cell membrane, producing satisfactory resuspensions in all cases tested. Leakage of [14C]sucrose or 86Rb+ from phospholipid vesicles was not found to be significantly more rapid from protamine-heparin-treated vesicles, than from similar vesicles treated with protamine alone or from untreated control vesicles. Good separation of the resuspended vesicles from the remaining protamine and heparin was obtained with phospholipid vesicles and proteoliposomes using a gel filtration technique. Most of the protamine and heparin could be removed from the vesicles made of natural biological membrane using a combination of ion exchange chromatography and gel filtration. It was found possible to use this aggregation-centrifugation-disaggregation technique to increase the concentration of cytochrome oxidase vesicles without significantly decreasing the respiratory control ratio for these vesicles below that of a control which was only vortexed.

A procedure is described which resolves histones and other very basic cellular polypeptides from solubilized whole cells by two-dimensional gel electrophoresis. The entry of histones into the gel was apparently quantitative when salt and protamine were added to solubilization buffers containing urea and detergents. Histones and basic polypeptides in the histone region of the gels were identified and characterized by comparison with purified histones and by determining lysine to arginine ratios of individual spots. Phosphorylated derivatives of the nucleosome histones were clearly resolved from stained spots in the charge dimension. Some phosphorylated H1's comgrated with the stained spots and some were retarded in the charge dimension. Acetylated nucleosome histones were nearly coincident with stained spots. The usefulness of this technique for evaluating changes in post-translational modification of histones was illustrated by showing that one H1-like protein increased in 32 P content when C-6 cells were treated with a β-adrenergic agonist.

It is usually necessary to remove nucleic acids from microbial extracts in order to avoid their interference with the isolation of enzymes from the extract. This may be particularly important where the purification procedure includes chromatography on an anion-exchange column such as DEAE-Sephadex (1). Methods that have been used have included precipitation with Mn, destruction of the nucleic acids with nucleases, and precipitation with basic substances, usually protamine sulfate or streptomycin sulfate. It is likely that there may be advantages, in some cases at least, in using other basic proteins for this purpose, since the results obtained with the previous methods have not always been satisfactory. The procedure described in this paper utilizes high concentrations of lysozyme to precipitate nucleic acids from a bacterial extract. The separation obtained with lysozyme was efficient, reproducible, and superior to the separation obtained with protamine sulfate.

Optimal assay conditions for analyses of the catalytic subunit activity of the cyclic AMP-dependent protein kinase using a well-defined, commercially available synthetic peptide as the phosphate acceptor are defined. Activity of purified catalytic subunit toward the synthetic peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (PK-1; Kemptide) was 1.5- to 45-fold greater than activity toward other commonly used substrates such as histone fractions, casein, and protamine. The effects of buffer, pH, Mg2+, and protein kinase concentration on activity toward PK-1 were investigated. The optimal assay conditions determined were as follows: 20 m m Hepes or phosphate buffer, pH 7.5, 100 μ m PK-1, 100 μ m [γ-32P]ATP, 3 m m MgCl2, 12 m m KCl, and 20–200 ng of catalytic subunit assayed at 30°C. Since PK-1 is the only commercially available, well-defined substrate for this enzyme, adaption of the proposed standard assay conditions for the analyses of purified catalytic subunit activity will permit direct comparison of kinetic parameters and purity of enzyme preparations from multiple preparations.