Hans G. Boman

Chromatography of rattlesnake venom A separation of three phosphodiesterases

1. 1. Venom from rattlesnake (Crotalus adamanteus) has been studied by chromatography on DEAE cellulose. Special interest has been devoted to phosphodiesterase and DNAase, while lecithinase and l-amino acid oxidase have been followed by semi-quantitative test only. 2. 2. Under optimal conditions three different forms of phosphodiesterases have been separated from lecithinase and l-amino acid oxidase. The heterogeneity has been confirmed by rechromatography and zone electrophoresis. 3. 3. All experiments have shown a parallelism between the phosphodiesterase and DNAase activities. 4. 4. A new test for l-amino acid oxidase activity is given, using l-amino as substrate and ninhydrin to determine remaining amino acid.

STUDIES ON MICROBIAL RNA. I. TRANSFER OF METHYL GROUPS FROM METHIONINE TO SOLUBLE RNA FROM ESCHERICHIA COLI.

Two partially purified enzyme preparations, one from yeast and one from Escherichia coli, have been used to study the transfer of methyl groups from methionine to soluble RNA (S-RNA) from E. coli. The reaction requires ATP and magnesium. The E. coli enzyme can incorporate methyl groups only into S-RNA from methionine-starved E. coli K12 W6 (a mutant with a deficient control of RNA synthesis). The yeast enzyme can methylate S-RNA from E. coli K12 in log phase as well as from methionine-starved cultures of W6. The methylation is not inhibited by periodate oxidation of the terminal adenosine residue in the S-RNA. The alkaline stability of the methylated S-RNA is clearly different from that of methionyl RNA. Methylated S-RNA has been degraded by snake venom enzymes as well as by alkaline hydrolysis. The digestion products have been examined by paper and anion exchange chromatography, respectively. In vitro, neither of the two enzymes can methylate purified ribosomal RNA (R-RNA). In vivo incorporation of methionine has, however, shown a significant methylation of both S-RNA and R-RNA. The composition of the methylated nucleosides obtained from R-RNA differs from that of S-RNA. Total RNA contains also smaller amounts of non-methylated RNA. Counter-current distribution of S-RNA has shown that the acceptors for the methyl groups can be separated from the acceptors for tyrosine.

Protein chromatography on an anion-exchange resin.

Abstract 1. 1. A technique for protein chromatography on the anion-exchange resin Dowex 2 is described. 2. 2. The stepwise elution of human serum albumin has been studied in some detail. 3. 3. A displacement technique has been used for the separation of the alkaline and acid phosphatases in human cancer serum. 4. 4. Human γ-globulin has been isolated directly from serum. The chromatographically purified γ-globulin has been examined in the ultracentrifuge and with paper electrophoresis. 5. 5. Human red-cell acid phosphatase has been separated from 99.9% of the hemoglobin. This means a 200-fold purification of the phosphatase. 6. 6. Horse-radish peroxidase and acid phosphatase (the latter enzyme not observed earlier in this material) have been separated and purified by means of stepwise elution. 7. 7. The displacement mechanism has been studied with the horse-radish enzymes and the results have been discussed in relation to the concepts of displacement chromatography. Heterogeneity was demonstrated for the horse-radish phosphatase. 8. 8. Approximate data for elution are given for 19 proteins.

Chromatography of prostatic phosphatase

Abstract 1. 1. A method for pH gradient elution chromatography of proteins is described. 2. 2. The method has been used for the chromatographic examination of the raw extracts of human semen and prostatic glands as well as for chromatographically purified samples of prostatic phosphatase. 3. 3. An enzyme preparation has been obtained which, judging from the chromatographic examination does not contain large amounts of impurities.

STUDIES ON MICROBIAL RNA.II. TRANSFER OF METHYL GROUPS FROM METHIONINE TO THE RNA OF A RIBONUCLEOPROTEIN PARTICLE.

When Escherichia coli W6 (a strain with a relaxed control of RNA synthesis) is starved for methionine, a ribonucleoprotein particle accumulates. These particles were partially purified, and the preparation was investigated by zone centrifuga-tion. It was found that the particles contained about 70% ribosomal RNA (R-RNA) and about 30% protein. The preparation was not homogeneous and contained additional protein and traces of S-RNA. These particles produced by methionine starvation of E. coli W6 were used as a substrate for the methylation of their own RNA. In the presence of ATP and a supernatant fraction, methyl groups from methionine were incorporated into a product obtained after phenol extraction and alcohol precipitation. Cold S-adenosylmethionine quenches this incorporation. [14C]methyl groups from S-adenosylmethionine were incorporated in the absence of supernatant liquid and ATP. The reaction product was hydrolysed by either 0·3 M -potassium hydroxide, pancreatic RNase or venom diesterase. Labelled RNA was prepared from a reaction mixture and fractionated by chromatography on Sephadex G200. Both R-RNA and S-RNA were methylated. The incorporation was proportional to the amount of methionine-starvation particles added. About one methylated base per 5000 bases was found as saturation value for R-RNA. The pattern of methylated nucleosides was found to be the same for R-RNA labelled in vivo and in vitro. This pattern differed from that for S-RNA. The dependence of the rate of methylation on the supernatant liquid was investigated with either methionine or S-adenosylmethionine as methyl donor. With the former, the supernatant liquid gave up to a tenfold stimulation; with the latter the stimulation was just detectable. For reactions with purified RNA from particles produced by methionine starvation of E. coli, the rate of methylation of R-RNA was strongly dependent on the amount of ribosomes present. These results suggest that R-RNA methylating enzymes are bound to particles and that the methylation of R-RNA is a step in the biosynthesis of ribosomes.

Fractionation of venom from the ringhals cobra.

Abstract 1. 1. Venom from the South African ringhals cobra ( Hemachatus haemachates ) has been studied by chromatography on DEAE cellulose and by column electrophoresis, with special regard to the isolation and purification of certain enzymes. 2. 2. In preparations of the venom obtained by commonly used procedures at least two chromatographically different phosphodiesterases have been found, one of them being partly transformed into the other on auto-incubation of the venom. 3. 3. Chromatographic analysis has shown that the method of collecting and drying the venom at room temperature does not give a well-defined material, but that such a material can be obtained by a rapid freeze-drying method. 4. 4. Chromatographic fractionations of 0.5 of freeze-dried venom have been made showing a complete separation of the phosphodiesterase from acetylcholine esterase and lecithinase A. Toxicity tests have revealed the existence of two well-separated toxins. 5. 5. By electrophoresis all the three enzymes mentioned have been separated from each other. 6. 6. The enzymic properties of the phosphodiesterase are discussed.

Penicillin Induced Lysis in Escherichia coli

SUMMARY: The paper is a survey of the action of α-aminobenzylpenicillin (ampicillin) on Escherichia coli. The rate at which lysis was induced in exponentially growing organisms was studied for different concentrations of ampicillin, using pure D- and L-forms as well as a 6:4 mixture. The interpolated concentration of penicillin which gives lysis in one generation, the ‘LIOG value’, has been used for characterization of penicillin derivatives with twelve different side chains. These LIOG values were also used for characterization of some penicillin-resistant mutants. The lysis rate has been recorded for cultures of concentrations between 4 x 105 and 108 rods/ml, and found to be independent of the population density. The use of five different media showed that for a given ampicillin concentration the time to lysis was proportional to the growth rate. The addition of penicillinase to a culture growing with penicillin rescued the organisms as late as a few minutes before lysis. Synergistic effects on the lysis rates were found with 6-amino-penicillanic acid and two amino acids, which as residues are side chains, in two of the penicillins tested. Different models for penicillin action in E, coli are discussed.

On the specificity of the snake venom phosphodiesterase.

The last two years have led to a great increase in our knowledge of the specificity of the venom phosphodiesterase; the present situation is reviewed elsewhere in this monograph by Hilmoe,’ Laskowski? and Tener.3 The purpose of this article is to describe and discuss a few kinetic experiments with diesterase degradation of desoxyribonucleic acid (DNA) in the presence of different metals and a chelating agent. The initial idea behind these experiments was that the formation constants for the complexes between divalent cations and the phosphatesof a polynucleotide chainmay vary because of the surrounding nucleotides. If so, a careful selection and control of the cation concentration might offer possibilities for a limited but specific digestion. The results obtained thus far do not seem to contradict this working hypothesis, but a t the same time they indicate that the description of the specificity of the diesterase has not yet been completed. The starting material for the enzyme preparation was freeze-dried venom from ringhals cobra* (Hemachatus haemachates, formerly named Sepedon haemachates). Our work with different venoms4* indicates that an evident autodigestion occurs during the “ordinary” collection procedure, and this fact may be the explanation for the difficulties many workers have experienced in reproducing enzyme preparations from snake venom. The freeze-dried ringhals venom, however, has proved to be reproducible; it is hoped to achieve i~ convenient and satisfactory preparation procedure for diesterase from this material. The sample used in the following experiments was purified by chromatography on DEAE ~ellulose.~ The enzyme was not definitely free from contaminating 5-nucleotidase, but the latter enzyme can hardly interfere with the hydrolysis of DNA when this is followed by the increase in the extinction of the lanthanum acid-soluble nucleotide material.4 The DNA used was from one preparation of calf thymus prepared according to Kay et aZ.6 The substrate solution was 0.1 per cent DNA in 0.1 M “Bis”HC1 buffer a t a pH of 9.1.1 Before the experiments, both enzyme and substrate were adjusted to 0.001 &I concentration of ethylenediamine tetraacetic acid (EDTA) and, afterward, to the different concentrations used of calcium and magncsium. After 20 min. of preincubation at 37” C., 100 pl. of enzyme (with Ezso = 0.23) was added to 1 ml. of DNA solution. At different times aliquots of 100 pl. were removed and added to cold samples of 1 ml. of 0.01 M La(N03)3 in 0.1 M HCl. At the end of the experiment, these solutions were centrifuged and the extinction a t 260 mp of the supernatant solution was measured. FIGURE 1 shows the results from 4 such experiments made with the

EFFECTS OF GAMMA RAYS ON SOLUTIONS OF HUMAN SERUM ALBUMIN. II. CHROMATOGRAPHIC STUDIES

Abstract It has been demonstrated that the effect of γ-rays on human serum albumin can be followed by chromatography on Dowex 2.

Chromatography of horse-radish enzymes: a separation of two acid phosphomonoesterases

Abstract 1. 1. Triethylaminoethylcellulose (TEAE-cellulose) has been used for a chromatographic investigation of a crude mixture of horse-radish peroxidase and acid phosphatase. 2. 2. Variations in the chromatographic schedule have been followed when increasing amounts of material have been applied to the same column. 3. 3. Under optimal conditions, peroxidase and two acid phosphatases could be separated in one experiment. 4. 4. A new way of rechromatographing, which permits the utilization of displacement effects, has been tried together with the usual rerunning of one single zone. 5. 5. The results are discussed in relation to various concepts of protein chromatography.