"Chitin Leash": a polysaccharide heterobifunctional cross-linking agent which can be cleaved by lysozyme.

6-O-[(2-Hydroxyethyl)poly(2-oxyethyl)]chitosan ("glycolchitosan") was oxidatively cleaved with nitrous acid and then partly acetylated with acetic anhydride, reacted with bromoacetyl-N-hydroxysuccinimide, and reacted further with acetic anhydride. Conditions were selected, including fractionation by size-exclusion chromatography, so that the resulting "Chitin Leash" had an estimated, average molecular weight of 10,000 (dextran standards), corresponding to a length of approximately 40 sugar residues. It possessed 0.9 terminal aldehyde and 2.6 random (presumably) side-chain bromoacetyl reactive groups per chain (average values). As a model system, the Chitin Leash was used to crosslink staphylococcal nuclease (SNase) to ribonuclease A (RNase) with retention of 75 and 78%, respectively, of the starting enzyme activities. For this coupling, the Nase was first converted to a sulfhydryl SNase derivative which retained 74% of the activity of starting enzyme. The yields in this synthesis were: 13% Chitin Leash from glycolchitosan, 24% Chitin Leash-RNase from Chitin Leash and 45% SNase-Chitin Leash-RNase from the latter conjugate. The ratio of SNase to RNase in this conjugate was 1.0:0.94. In a second preparation, in which [14C]acetic anhydride was used, a longer reaction time was employed for the coupling of Chitin Leash to RNase. This gave a 1.0:1.8:0.95 molar ratio of Nase: [14C]Chitin Leash: RNase, revealing multiple attachment of the [14C]Chitin Leash to RNase. The activity of the RNase in the final conjugate was 20%. The latter conjugate was approximately 70% hydrolyzed by diaminooctyl-succinyl-lysozyme, disconnecting the two enzymes while not affecting their activities.

Purification of DNA-derived deoxynucleotides from leukocytes involving nuclease elution of an ion-exchange column.

A method has been developed in which the DNA of leukocytes (as the buffy coat from blood) is isolated in the form of its constituent deoxynucleotides. The steps in this method are as follows: (1) lyse the leukocytes with sodium dodecyl sulfate (SDS) and enzymatically digest the proteins and RNA, (2) remove the SDS on a non-polar adsorbent (Bio-Beads SM-4) and then trap the DNA on a quaternary amine silica cartridge, (3) wash the column with 1 M NaCl-buffer, (4) digest the DNA on the column with staphylococcal nuclease and (5) elute the digested DNA with 0.5 M NaCl-buffer and digest it further with bovine spleen phosphodiesterase II to deoxynucleotide-3'-monophosphates. From a 40-microliters sample of butty coat was obtained 126 +/- 14 micrograms (two experiments, eight sample total) of deoxynucleotides. Reversed-phase high-performance liquid chromatography, which removed the added enzymes, showed only peaks for deoxynucleotides. For comparison, the amount of deoxynucleotides obtained from the leukocytes by an automated phenol extraction procedure was 101 +/- 5.4 micrograms (one experiment in triplicate).

Chromatographic enzyme immunoassay for T-2 toxin.

Both the active ester and maleimide moieties of the cross-linking reagent, N-[(gamma-maleimidobutyryl)oxy]succinimide (GMBS), were found to react with the primary amino groups on ribonuclease (RNase). This largely inactivated RNase towards a polymeric (but not monomeric) substrate. Citraconylating the RNase first, so that essentially only a single primary amino group remained to react with GMBS, overcame this problem. The subsequent maleimido-citraconyl-RNase was used to prepare a 1:1.1 M conjugate of anti-T-2 toxin Fab' and RNase (Fab'-RNase) in a 76% yield. The conjugate was used to detect as little as 0.1 microgram of T-2 toxin based on the ability of T-2 toxin to specifically displace Fab'-RNase complexed to a T-2 agarose affinity gel.

Staphylococcal nuclease high-performance liquid chromatographic characterization of diaminooctane-modified DNA and its biotin and fluorescein derivatives.

DNA was subjected to bisulfite-catalyzed transamination at the N4 sites of its cytosine residues with 1,8-diaminooctane (DAO). The product, DNA-DAO, was non-specifically degraded with a cloned staphylococcal nuclease (Nase). The products from the Nase digestion were determined by high-performance liquid chromatography (HPLC) to define the extent of reaction with DAO. Mostly, nucleoside 3'-monophosphates were obtained, along with four Nase-resistant dinucleotides: TpdGp, dApdGp, TpdCp-DAO and dApdCp-DAO. The addition of spleen phosphodiesterase II gave a faster hydrolysis and left no dinucleotides. A mixture of Nase, snake venom phosphodiesterase I and alkaline phosphatase gave a fast hydrolysis as well but two dinucleotides, apparently TpdC-DAO and dApdC-DAO, persisted. Further modification of the diaminooctyl side chains with fluorescein isothiocyanate or biotin N-hydroxysuccinimide ester was similarly investigated. Interestingly, derivatization of the DAO side chain with biotin eliminates the resistance of TpdCp-DAO and dApdCp-DAO to Nase digestion. This work provides some guidelines for using Nase, alone or with other nucleases, along with HPLC, for characterizing alkyldiamine DNA products, and should be similarly useful for studying other modifications of DNA.

Repetitive hit-and-run fluoroimmunoassay for T-2 toxin.

A monoclonal antibody for T-2 toxin is converted to a Fab'-fluorescein derivative. The latter is specifically complexed onto a T-2 agarose gel. Fifteen successive doses of T-2 ranging from 1 to 50 ng are then repetitively and linearly detected using a column packed with a small volume (0.2 ml) of this gel without recharging with Fab'-fluorescein. For these assays the effluent from the column is monitored with a spectrofluorometer.

Substrate-leash amplification with ribonuclease S-peptide and S-protein.

The S-peptide and S-protein fragments of ribonuclease S (RNase S, no EC no. assigned) have been immobilized onto separate Sepharose gels via a "leash" of polycytidylic acid substrate. Each of these gels releases its RNase fragment when treated with the complementary enzyme fragment or with RNase A (EC 3.1.27.5), and the released fragments recombine to give RNase S activity. Thus this system provides substrate-leash amplification (SLA), such that more enzymatic activity is eluted from the system than is applied. For example, 100 pg of RNase applied to the S-peptide gel is amplified by 1.9 X 10(4) to the equivalent of 1.9 micrograms of activity in 20 h, when followed by combination of the released S-peptide with excess S-protein. We also tested a three-stage amplification system, with a pair of S-peptide and S-protein gels at each stage. In this system the cumulative amplification of the initial 1-ng dose of RNase A is 4.9, 52, and 25-fold after each stage, respectively. Only 2 mg of each SLA gel is used per stage in these experiments, reflecting the magnitude of their production of RNase S activity.

Neonucleoproteins. Preparation and high-performance liquid chromatographic characterization of succinyl-lysozyme-diaminooctyl-polycytidylic acid and related polycytidylic acid conjugates.

Transamination conjugates of cytidine-3'-phosphate and polycytidylic acid (poly C) with diaminopropane, diaminohexane and diaminooctane (DAO) are formed both at 25 degrees C and 60 degrees C. The extent of reaction and formation of side products, with intermittent hydrolysis to mononucleotides in the case of aminoalkyl-poly C, is monitored by reversed-phase high-performance liquid chromatography. Both Dns-DAO-poly C and succinyl-lysozyme-DAO-poly C covalent conjugates are then prepared and similarly characterized, including separation on a size-exclusion diol high-performance liquid chromatography column. The retention of the latter on a wide-pore reversed-phase column seems to be controlled by the protein moiety.

The absence of a catechol estrogen effect on blood pressure in the male rat.

Since catechol estrogens are potent competitive inhibitors of catechol-O-methyl transferase (COMT), it has been suggested that they may prolong the half-life of catecholamines which in turn can cause hypertension. Thus, experiments were carried out to study the effect of catechol estrogens on blood pressure in the male rat following chronic administration. Results demonstrate that 2-hydroxyesterone (2,3-dihydroxyestra-1,3,5(10)-trien-17-one) and 2-hydroxy-estradiol (estra-1,3,5(10)-triene-2,3,17 beta-triol) even when administered in high doses do not alter blood pressure.

Urinary catechol estrogens in cycles stimulated by human menopausal gonadotropin.

Catechol estrogens, estrogen metabolites of potential physiologic significance, were measured in infertile women undergoing ovulation induction with human menopausal gonadotropins. Urinary 2-hydroxyestrone (2-OH-E1) specimens were obtained from 12 women in one or more stimulated cycles. The actual time for the administration of human chorionic gonadotropin to induce ovulation was based on serial plasma estradiol (E2) specimens. A significant correlation between plasma E2 and urinary 2-OH-E1 was demonstrated, similar but more pronounced than that seen in normal cycling women. This confirms previous work that showed that 2-OH-E1 is the major urinary estrogen metabolite in the nonpregnant state and further suggests that urinary catechol estrogens are a useful index of ovarian function.

Radioimmunoassay of 2-hydroxyestrone in plasma during the estrous cycle of the rat: interrelationships with estradiol, progesterone, and the gonadotropins.

A RIA for 2-hydroxyestrone (2-OHE1) in rat plasma has been developed. The assay employs an antiserum that is specific for catechol estrogens. Specificity is further ensured by purification of plasma extracts on Sephadex LH-20 columns before RIA. Blood was collected at 0 C in the presence of ascorbic acid to prevent oxidation. Under these conditions, the conversion of 2-OHE1 to methylated derivatives was found to be negligible. Plasma 2-OHE1, LH, FSH, PRL, estradiol, and progesterone were measured at 3-h intervals throughout the 4-day estrous cycle of the rat. The 2-OHE1 concentration varied from undetectable to 11 pg/ml plasma. No clearly defined relationship with the other hormones analyzed was observed. Thus, it is unlikely that changes in circulating 2-OHE1 levels are involved in the regulation of the gonadotropin surge and ovulation.