Inactivation and removal of human immunodeficiency virus in monoclonal purified antihemophilic factor (human) (Hemofil M).

Cold supernatant which was prepared from factor VIII:C containing cryoprecipitate was seeded with HIV-1, then treated with a mixture of tri (n-butyl) phosphate (TNBP) and triton X-100. A greater than 10(11)-fold reduction of HIV-1 infectivity was observed. In a separate experiment, cold supernatant which had been seeded with HIV-1 was chromatographed on an immunoaffinity column, resulting in a 10(4)-fold reduction of infectivity. None of the 17 patients treated with the purified product and followed for at least three months has shown serologic evidence of HIV-1 or other viral infections.

Immunoglobulin A concentrations in commercial immune globulins.

Commercial immune globulins contain varying concentrations of IgA as a minor constituent. For patients who are both IgA deficient and IgA hypersensitive, the administration of most immune globulins is contraindicated due to potentially lethal anaphylactic reactions. We have measured the IgA levels in a broad range of commercial immune globulin preparations by means of a competitive-binding solid-phase radioimmunoassay. Of the products tested, all immune globulins intended for intramuscular administration and most immune globulins for intravenous use had IgA levels of over 150 micrograms/ml. Only two of the intravenous products had IgA concentrations in the range of 10 micrograms/ml or less.

The incidence of HTLV-III/LAV seroconversion and non-A, non-B hepatitis in recipients of plasma products.

Several groups including ours at Hyland have studied the incidence of HTLV-III/LAV seroconversion and non-A, non-B hepatitis (NANBH) in recipients of products made from unscreened plasma. Of 18 previously untreated patients who received heat-treated factor VIII concentrate (HEMOFIL T), none seroconverted for HTLV-III/LAV. However, 11 of 13 similarly treated patients showed aminotransferase elevations indicative of NANBH. None of 21 patients receiving anti-inhibitor coagulant complex (Autoplex) seroconverted for HTLV-III/LAV, while 28 of 50 patients receiving other forms of treatment did seroconvert. Of 30 recipients of AUTOPLEX, 9 had elevated aminotransferase levels. Since all patients had hepatitis B markers when the study began, a diagnosis of NANBH cannot be made. Of 16 patients who received large doses of immune globulin for intravenous administration (IGIV, GAMMAGARD), none seroconverted for HTLV-III/LAV. Retrospective and prospective studies of 157 recipients of GAMMAGARD did not reveal a single case of NANBH attributable to receipt of this product. In contrast, NANBH has occurred following administration of HEMOFIL T, indicating that heating in the dried state does not eliminate the NANBH agent(s). In spite of lingering concerns about NANBH infectivity, the available data strongly suggest that heat treatment and/or cold ethanol fractionation render these plasma products safe from HTLV-III infectivity.

Safety of an intravenous immunoglobulin preparation: lack of seroconversion for human immunodeficiency virus antibodies.

A retrospective analysis of 18 patients who had received a human intravenous immunoglobulin (IGIV) preparation was undertaken to ascertain the safety of this preparation with respect to transmission of human immunodeficiency virus (HIV), the virus that causes the acquired immune deficiency syndrome (AIDS). Patients were followed up by means of periodic enzyme-linked immunosorbent assay (ELISA) for the presence of circulating antibodies against HIV; a negative ELISA was evidence that HIV had not been transmitted to the recipients of IGIV. Results in 16 patients were negative, and two patients were determined to have had false-positive ELISAs because the Western blot test was negative for seroconversion. It is thus concluded that the IGIV product tested has little or no potential for transmitting HIV.

Amino acid sequence of a peptide containing an essential cysteine residue of pig heart aconitase.

Pig heart aconitase reacts with one mole of phenacyl bromide per molecule to give complete inactivation due to the alkylation of a cysteine reside at the active site. A tryptic peptide containing this essential residue has been isolated and its amino acid sequence determined at Ile-Gln-Leu-Leu-Cys *-Pro-Leu-Leu-Asn-Gln-Phe-Asp-Lys by manual methods and by the use of an automated solid phase sequencer. There is a limited similarity in amino acid sequence between this peptide and other peptides containing the cysteine residues involved in the binding of the iron-sulfur clusters of high-potential iron-sulfur protein of Rhodopseudomonas gelatinosa and rubredoxins from various bacteria.

Analysis of the iron-sulfur cluster of aconitase by natural and magnetic circular dichroism.

We have examined the iron-sulfur cluster of aconitase, a high-potential iron-sulfur protein, by absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectroscopy. The MCD spectrum of unactivated aconitase, which is presumably oxidized, is similar to those of reduced two iron-two sulfide ferredoxins but distinct from the MCD of known four iron-four sulfide proteins. The magnitude of the natural CD of unactivated aconitase also suggests the absence of four iron-four sulfur clusters. Reduction of the enzyme with dithionite and activation with the cysteine-ascorbate-ferrous ion activation mixture generate spectra which are significantly different from those of any iron-sulfur protein seen to date. We interpret these results as indicating that aconitase does not contain a four iron-four sulfur cluster generally thought to be characteristic of high-potential iron-sulfur proteins. It could contain a two iron-two sulfur center or some other center such as a cyclic three iron-three sulfur center.

Isoleucyl transfer ribonucleic acid synthetase. Steady-state kinetic analysis.

A steady-state kinetic analysis was conducted of the overall aminoacylation reaction catalyzed by isoleucyl-tRNA synthetase. The patterns of Lineweaver-Burk plots obtained indicated that tRNA adds to the enzyme only after isoleucyl adenylate formation and pyrophosphate release. These kinetic patterns were consistent with the bi-uni-uni-bi Ping Pong mechanism generally accepted for this aminoacyl-tRNA synthetase, but they could also be accommodated by a mechanism in which a second molecule of L-isoleucine added to the enzyme between isoleucyl adenylate formation and aminoacylation of tRNA [Fersht, A.R., & Kaethner, M.M. (1976) Biochemistry 15, 818]. The values of the kinetic parameters favor the latter mechanism. The results of this kinetic analysis indicated that the affinity of isoleucyl-tRNA synthetase for Mg.ATP was enhanced upon binding of L-isoleucine and vice versa. It also indicated that the affinity of the enzyme for L-isoleucine is decreased upon binding tRNA and vice versa. The values of dissociation constants calculated for each of the substrates by this study generally compared well with those determined by other authors using a variety of kinetic and equilibrium methods.

Isoleucyl transfer ribonucleic acid synthetase. Competitive inhibition with respect to transfer ribonucleic acid by blue dextran.

The inhibitory effects of blue dextran and a small dye molecule derived from it (F3GA-OH) on the steady-state reaction catalyzed by Escherichia coli isoleucy-tRNA synthetase have been studied. Blue dextran gave uncompetitive inhibition with respect to Mg.ATP, mixed inhibition with respect to L-isoleucine, and competitive inhibition with respect to tRNA. The small dye molecule (F3GA-OH) was also competitive with respect to tRNA. These inhibition patterns were not consistent with the bi-uni-uni-bi Ping Pong mechanism generally accepted for aminoacyl-tRNA synthetases. They were consistent with a mechanism in which a second L-isoleucine is bound after isoleucyl-AMP synthesis and before transfer of the isoleucyl moiety to tRNA. Enzyme-bound L-isoleucine lowered the affinity of the enzyme for blue dextran approximately fivefold, a value comparable to the ninefold lowering of the enzyme's affinity for tRNA upon binding L-isoleucine. The affinity of the synthetase for F3GA-OH (K1 = 1.0 X 10(-7) M) is approximately fivefold higher than its affinity for blue dextran (K1 = 5.3 X 10(-7) M). These results indicate that blue dextran and its derivatives may be useful for kinetic and physical studies of polynucleotide binding sites on proteins as well as NAD and ATP sites.

Amino acid sequence of ATP phosphoribosyltransferase of Salmonella typhimurium.

The amino acid sequence of ATP phosphoribosyltransferase [1-(5'-phosphoribosyl)-ATP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.17] of Salmonella typhimurium has been determined. The amino acid sequence analysis was carried out with a combination of manual and automated methods. It was complemented by DNA sequence analysis (done in another laboratory) of the hisG gene, which codes for it. The subunit polypeptide chain contains 299 amino acid residues and has a molecular weight of 33,216. The amino-terminal segment of the protein is relatively basic in character and has limited sequence homologies with the lac repressor and histidinol dehydrogenase. In addition, the protein contains a 40-residue segment that has 13 residues identical with the sequence surrounding the active-site cysteine of glyceraldehyde-3-phosphate dehydrogenase.