Recognition of D-aspartyl residues in polypeptides by the erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase. Implications for the repair hypothesis.

We provide here the first direct evidence that D-aspartyl residues in peptides are substrates for the L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (EC 2.1.1.77). We do this by showing that D-aspartic acid beta-methyl ester can be isolated from carboxypeptidase Y digests of enzymatically methylated D-aspartyl-containing synthetic peptides. The specificity of this reaction is supported by the lack of methylation of L-aspartyl-containing peptides under similar conditions. Methylation of D-aspartyl residues in synthetic peptides was not observed previously because with Km values ranging from 2.5 to 4.8 mM, these peptides are recognized by the methyltransferase with 700-10,000-fold lower affinity than are their L-isoaspartyl-containing counterparts. The physiological significance of D-aspartyl methylation was investigated in two ways. First, analysis of in situ methylated human erythrocyte proteins showed that at least 22% of the methyl groups associated with the proteins ankyrin and band 4.1 are on D-aspartyl residues, suggesting that D-aspartyl methylation is an important function of the methyltransferase in vivo. Second, mathematical modeling of the protein aging and methylation reactions occurring in intact erythrocytes indicated that the accumulation of D-aspartyl residues can be reduced as much as 2-5-fold by the methyltransferase activity. Although this reduction is much less than that predicted for L-isoaspartyl residues, it may be significant in maintaining functional proteins throughout the 120-day life span of these cells.     

Distinct C-terminal sequences of isozymes I and II of the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase

We have purified the more acidic major isozyme (II) of the human erythrocyte L-isoaspartyl/D-aspartyl methyltransferase and compared its structure to that of the previously sequenced isozyme I. These isozymes are both monomers of 25,000 molecular weight polypeptides and have similar enzymatic properties, but have isoelectric points that differ by one pH unit. Analysis of 16 tryptic peptides of isozyme II accounting for 89% of the sequence of isozyme I revealed no differences between these enzyme forms. However, analysis of a Staphylococcal V8 protease C-terminal fragment revealed that the last two residues of these proteins differed. The Trp-Lys-COOH terminus of isozyme I is replaced by a Asp-Asp-COOH terminus in isozyme II. Southern blot analysis of genomic DNA suggests that the human genome [corrected] may contain only a single gene encoding the enzyme. We propose that the distinct C-termini of isozymes I and II can arise from the generation of multiple mRNA's by alternative splicing.     

Structural elements affecting the recognition of L-isoaspartyl residues by the L-isoaspartyl/D-aspartyl protein methyltransferase. Implications for the repair hypothesis.

We have synthesized a series of L-isoaspartyl-containing (isoD) peptides and characterized their interaction with the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase (EC 2.1.1.77). Our findings indicate that this enzyme interacts with 6 residues extending from the isoD-2 to isoD+3 positions in peptide substrates. Although peptides as simple as G-isoD-G are methylated with low affinity (Km = 17.8 mM), a wide variety of L-isoaspartyl-containing sequences in larger peptides are recognized with high affinity (Km less than 20 microM), the best yet discovered being VYP-isoD-HA, with a Km of 0.29 microM. Only two sequence elements have been found that can interfere with the high affinity binding of peptides of 4 or more residues, these being a prolyl residue in the isoD+1 position and negatively charged residues in the isoD+1, isoD+2, and/or isoD+3 positions. We investigated the effect of higher order structure on binding affinity using several L-isoaspartyl-containing proteins. Although conformation did, in some cases, lower the affinity of the methyltransferase for L-isoaspartyl residues, the range of kinetic constants for the methylation of these proteins was similar to that observed with the synthetic peptides. The L-isoaspartyl/D-aspartyl methyltransferase has been proposed to function in vivo to prevent the accumulation of L-isoaspartyl residues that arise spontaneously as proteins age. To examine whether such a mechanism is feasible given the wide range of substrate Km values observed in vitro, we set up a computer simulation to model the degradation and methylation reactions in aging human erythrocytes. Our results suggest that enough methyltransferase activity exists in these cells to significantly lower the expected number of L-isoaspartyl residues, even when these residues have millimolar Km values for methylation.     

Protein carboxyl methyltransferase facilitates conversion of atypical L-isoaspartyl peptides to normal L-aspartyl peptides

Prolonged incubation of L-isoaspartate-containing forms of lactate dehydrogenase (231-242), sperm activating peptide, and adrenocorticotropin (22-27) at 37 degrees C, pH 7.4, with S-adenosyl-L-methionine and protein carboxyl methyltransferase from bovine brain leads to extensive conversion of the atypical isopeptide bond to a normal peptide bond. For the lactate dehydrogenase-related peptide, conversion was 80% complete after 24 h. For the other two peptides, conversion reached a level of approximately 65% after 48 h. The mechanism of conversion involves (i) rapid enzymatic methylation of the alpha-carboxyl of the L-iso-Asp residue; (ii) nonenzymatic demethylation resulting in formation of an L-aspartyl cyclic imide; and (iii) a slow, nonenzymatic hydrolysis of the cyclic imide to form a mixture of 15-25% normal L-Asp peptide and 75-85% L-iso-Asp peptide. The regenerated L-iso-Asp peptide is remethylated and the cycle is repeated. The extent of conversion is limited by a competing side reaction wherein the L-imide slowly racemizes, leading to the formation of mainly D-iso-Asp peptide, which is not a substrate for the methyltransferase. The ability of protein carboxyl methyltransferase to initiate conversion of L-iso-Asp residues to normal L-Asp suggests a possible role for this enzyme in facilitating the repair or degradation of deamidated proteins in vivo.     

Protein repair methyltransferase from the hyperthermophilic archaeon Pyrococcus furiosus. Unusual methyl-accepting affinity for D-aspartyl and N-succinyl-containing peptides.

Protein l-isoaspartate-(d-aspartate) O-methyltransferases (EC ), present in a wide variety of prokaryotic and eukaryotic organisms, can initiate the conversion of abnormal l-isoaspartyl residues that arise spontaneously with age to normal l-aspartyl residues. In addition, the mammalian enzyme can recognize spontaneously racemized d-aspartyl residues for conversion to l-aspartyl residues, although no such activity has been seen to date for enzymes from lower animals or prokaryotes. In this work, we characterize the enzyme from the hyperthermophilic archaebacterium Pyrococcus furiosus. Remarkably, this methyltransferase catalyzes both l-isoaspartyl and d-aspartyl methylation reactions in synthetic peptides with affinities that can be significantly higher than those of the human enzyme, previously the most catalytically efficient species known. Analysis of the common features of l-isoaspartyl and d-aspartyl residues suggested that the basic substrate recognition element for this enzyme may be mimicked by an N-terminal succinyl peptide. We tested this hypothesis with a number of synthetic peptides using both the P. furiosus and the human enzyme. We found that peptides devoid of aspartyl residues but containing the N-succinyl group were in fact methyl esterified by both enzymes. The recent structure determined for the l-isoaspartyl methyltransferase from P. furiosus complexed with an l-isoaspartyl peptide supports this mode of methyl-acceptor recognition. The combination of the thermophilicity and the high affinity binding of methyl-accepting substrates makes the P. furiosus enzyme useful both as a reagent for detecting isomerized and racemized residues in damaged proteins and for possible human therapeutic use in repairing damaged proteins in extracellular environments where the cytosolic enzyme is not normally found.     

High-performance liquid chromatographic method to measure protein L-isoaspartyl/D-aspartyl o-methyltransferase activity in cell lysates

Protein l-isoaspartyl/d-aspartyl o-methyltransferase (PIMT) is a widely expressed protein repair enzyme that restores isomerized aspartyl residues to their normal configuration. Current methods for measuring PIMT activity have limited sensitivity or require radioactivity. We have developed a highly sensitive new assay method to measure PIMT activity in cell lysates. As a substrate, we used a fluorescently labeled delta sleep-inducing peptide (DSIP) that contains an isoaspartyl residue: 7-nitro-2,1,3-benzoxadiazole (NBD)-DSIP(isoAsp). The PIMT-catalyzed transfer of a methyl group onto this substrate can be detected with a simple high-performance liquid chromatography (HPLC) procedure. After the enzyme reaction, the methylated form of the peptide is stable and can be reproducibly separated from the unmethylated form in an acidic solvent and fluorometrically detected by HPLC. The limit of detection was estimated to be approximately 1 pmol of NBD-DSIP(isoAsp) (signal/noise ratio [S/N] = 3), and the quantitation limit of the activity was approximately 18 μg of total cell lysate from HEK293 cells (10.7 pmol/min/mg protein). This assay method is sensitive enough to detect PIMT activity in biological samples without the use of radioisotopes, offering significant advantages over previously reported methods.     

Sequence of the D-aspartyl/L-isoaspartyl protein methyltransferase from human erythrocytes. Common sequence motifs for protein, DNA, RNA, and small molecule S-adenosylmethionine-dependent methyltransferases

A widely distributed protein methyltransferase catalyzes the transfer of a methyl group from S-adenosyl-methionine to the free carboxyl groups of D-aspartyl and/or L-isoaspartyl derivatives of L-aspartyl and L-asparaginyl residues. This enzyme has been postulated to function in the repair or the catabolism of age-damaged proteins. We present here the complete amino acid sequence of the more basic isozyme I of this enzyme from human erythrocytes. The sequence was determined by Edman degradation and mass spectral analysis of overlapping trypsin, Staphylococcus aureus V8 protease, Pseudomonas fragi endoproteinase Asp-N, cyanogen bromide, and hydroxylamine-generated fragments. The NH2-terminus is modified by acetylation and the protein contains 226 amino acids for a calculated molecular weight of 24,575. This value is in good agreement with the molecular weight determined for the purified protein by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and by gel filtration chromatography under nondenaturing conditions. The identification of 2 different amino acid residues at both positions 22 and 119 may indicate the presence of allelic variants or of two or more closely related structural genes. Finally, comparison of this sequence with those of methyltransferases for RNA, DNA, and small molecules, as well as other S-adenosylmethionine-utilizing enzymes, shows that many of these proteins share elements of three regions of sequence similarity and may be structurally or evolutionarily related.     

Considerations in the identification of endogenous substrates for protein L-isoaspartyl methyltransferase: the case of synuclein

Protein L-isoaspartyl methyltransferase (PIMT) repairs abnormal isoaspartyl peptide bonds in age-damaged proteins. It has been reported that synuclein, a protein implicated in neurodegenerative diseases, is a major target of PIMT in mouse brain. To extend this finding and explore its possible relevance to neurodegenerative diseases, we attempted to determine the stoichiometry of isoaspartate accumulation in synuclein in vivo and in vitro. Brain proteins from PIMT knockout mice were separated by 2D electrophoresis followed by on-blot [(3)H]-methylation to label isoaspartyl proteins, and by immunoblotting to confirm the coincident presence of synuclein. On-blot (3)H-methylation revealed numerous isoaspartyl proteins, but no signal in the position of synuclein. This finding was corroborated by immunoprecipitation of synuclein followed by on-blot (3)H-methylation. To assess the propensity of synuclein to form isoaspartyl sites in vitro, samples of recombinant mouse and human α-synucleins were aged for two weeks by incubation at pH 7.5 and 37°C. The stoichiometries of isoaspartate accumulation were extremely low at 0.02 and 0.07 mol of isoaspartate per mol of protein respectively. Using a simple mathematical model based on the first order kinetics of isoaspartyl protein methyl ester hydrolysis, we ascribe the discrepancy between our results and the previous report to methodological limitations of the latter stemming from an inherent, and somewhat counterintuitive, relationship between the propensity of proteins to form isoaspartyl sites and the instability of the (3)H-methyl esters used to tag them. The results presented here indicate that synuclein is not a major target of PIMT in vivo, and emphasize the need to minimize methyl ester hydrolysis when using methylation to assess the abundance of isoaspartyl sites in proteins.     

Optimal conditions for the use of protein L-isoaspartyl methyltransferase in assessing the isoaspartate content of peptides and proteins.

Protein L-isoaspartyl methyltransferase provides a basis for enzymatic measurement of atypical, isoaspartyl linkages which make a major contribution to protein microheterogeneity. The low Vmax of the methyltransferase reaction and the instability of the methyl ester can hinder accurate determinations, and different laboratories using different conditions have achieved discrepant values for the isoaspartate content of the same proteins. To investigate the effects of these conditions, and to optimize the assay, isoaspartyl delta sleep-inducing peptide was methylated under a variety of conditions. We found that 1 microM methyltransferase was required to obtain stoichiometric modification of 2 microM peptide in 40-min reactions at pH 6.2 and 30 degrees C. A computer model utilizing kinetic constants obtained from studies on initial rates of methylation predicted the same requirement for enzyme concentration. Carrier protein was necessary for optimal methyltransferase activity at enzyme concentrations below 0.4 microM. Stoichiometric methylation required concentrations of S-adenosylmethionine to be in substantial excess over those of peptide; 50 microM S-adenosylmethionine is the minimum needed for complete modification of 10 microM peptide. Spontaneous demethylation was significant under all conditions tested, so that the methyl ester itself never reached a ratio of 1 mol/mol of total peptide. These results demonstrate that the most accurate measurements of isoaspartate are obtained when reactions are carried out at low peptide concentrations, high S-adenosylmethionine concentrations, and high enzyme concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein repair in the brain, proteomic analysis of endogenous substrates for protein L-isoaspartyl methyltransferase in mouse brain

Protein L-isoaspartyl methyltransferase (PIMT) catalyzes repair of L-isoaspartyl peptide bonds, a major source of protein damage under physiological conditions. PIMT knock-out (KO) mice exhibit brain enlargement and fatal epileptic seizures. All organs accumulate isoaspartyl proteins, but only the brain manifests an overt pathology. To further explore the role of PIMT in brain function, we undertook a global analysis of endogenous substrates for PIMT in mouse brain. Extracts from PIMT-KO mice were subjected to two-dimensional gel electrophoresis and blotted onto membranes. Isoaspartyl proteins were radiolabeled on-blot using [methyl-(3)H]S-adenosyl-L-methionine and recombinant PIMT. Fluorography of the blot revealed 30-35 (3)H-labeled proteins, 22 of which were identified by peptide mass fingerprinting. These isoaspartate-prone proteins represent a wide range of cellular functions, including neuronal development, synaptic transmission, cytoskeletal structure and dynamics, energy metabolism, nitrogen metabolism, pH homeostasis, and protein folding. The following five proteins, all of which are rich in neurons, accumulated exceptional levels of isoaspartate: collapsin response mediator protein 2 (CRMP2/ULIP2/DRP-2), dynamin 1, synapsin I, synapsin II, and tubulin. Several of the proteins identified here are prone to age-dependent oxidation in vivo, and many have been identified as autoimmune antigens, of particular interest because isoaspartate can greatly enhance the antigenicity of self-peptides. We propose that the PIMT-KO phenotype results from the cumulative effect of isoaspartate-related damage to a number of the neuron-rich proteins detected in this study. Further study of the isoaspartate-prone proteins identified here may help elucidate the molecular basis of one or more developmental and/or age-related neurological diseases.     

The L-isoaspartyl/D-aspartyl protein methyltransferase gene (PCMT1) maps to human chromosome 6q22.3-6q24 and the syntenic region of mouse chromosome 10.

We have mapped the genes for the human and mouse L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (EC 2.1.1.77) using cDNA probes. We determined that the human gene is present in chromosome 6 by Southern blot analysis of DNA from a panel of mouse-human somatic cell hybrids. In situ hybridization studies allowed us to confirm this identification and further localize the human gene (PCMT1) to the 6q22.3-6q24 region. By analyzing the presence of an EcoRI polymorphism in DNA from backcrosses of C57BL/6J and Mus spretus strains of mice, we localized the mouse gene (Pcmt-1) to chromosome 10, at a position 8.2 +/- 3.5 cM proximal to the Myb locus. This region of the mouse chromosome is homologous to the human 6q24 region.     

Synthetic peptide substrates for the erythrocyte protein carboxyl methyltransferase. Detection of a new site of methylation at isomerized L-aspartyl residues

Four hexapeptides of sequence L-Val-L-Tyr-L-Pro-(Asp)-Gly-L-Ala containing D- or L-aspartyl residues in normal or isopeptide linkages have been synthesized by the Merrifield solid-phase method as potential substrates of the erythrocyte protein carboxyl methyltransferase. This enzyme has been shown to catalyze the methylation of D-aspartyl residues in proteins in red blood cell membranes and cytosol. Using a new vapor-phase methanol diffusion assay, we have found that the normal hexapeptides containing either D- or L-aspartyl residues were not substrates for the human erythrocyte methyltransferase. On the other hand, the L-aspartyl isopeptide, in which the glycyl residue was linked in a peptide bond to the beta-carboxyl group of the aspartyl residue, was a substrate for the enzyme with a Km of 6.3 microM and was methylated with a maximal velocity equal to that observed when ovalbumin was used as a methyl acceptor. The enzyme catalyzed the transfer of up to 0.8 mol of methyl groups/mol of this peptide. Of the four synthetic peptides, only the L-isohexapeptide competitively inhibits the methylation of ovalbumin by the erythrocyte enzyme. This peptide also acts as a substrate for both of the purified protein carboxyl methyltransferases I and II which have been previously isolated from bovine brain (Aswad, D. W., and Deight, E. A. (1983) J. Neurochem. 40, 1718-1726). The L-isoaspartyl hexapeptide represents the first defined synthetic substrate for a eucaryotic protein carboxyl methyltransferase. These results demonstrate that these enzymes can not only catalyze the formation of methyl esters at the beta-carboxyl groups of D-aspartyl residues but can also form esters at the alpha-carboxyl groups of isomerized L-aspartyl residues. The implications of these findings for the metabolism of modified proteins are discussed.     

A new approach to the use of fluorogenic dinitrophenyl-containing substrates for determining the proteolytic activity of aspartyl proteinases

A method for the determination of proteolytic activity of aspartyl proteinases using known colored fluorogenic substrates was developed. The technique utilizes the chromophore properties of the dinitrophenyl (DNP) group. The approach proposed comprises separation of the initial peptide and subsequent measurement of absorption of the solution of the DNP-containing C-terminal fragment, produced by its enzymatic cleavage, at 360 nm. This method was used to determine the activity of calf chymosin, the pepsins from various sources, and the commercial preparations containing a mixture of enzymes without preliminary desalting. The method is simple and applicable under plant conditions.     

A new approach to the use of fluorogenic dinitrophenyl-containing substrates for determining the proteolytic activity of aspartyl proteinases

A method for the determination of proteolytic activity of aspartyl proteinases using known colored fluorogenic substrates was developed. The technique utilizes the chromophore properties of the dinitrophenyl (DNP) group. The approach proposed comprises separation of the initial peptide and subsequent measurement of absorption of the solution of the DNP-containing C-terminal fragment, produced by its enzymatic cleavage, at 360 nm. This method was used to determine the activity of calf chymosin, the pepsins from various sources, and the commercial preparations containing a mixture of enzymes without preliminary desalting. The method is simple and applicable under plant conditions.     

A new method for disulfide analysis of peptides.

A simple, sensitive, reliable method for determining disulfide groups in peptides is presented. The disulfides are cleaved in a brief treatment with strong alkali. Following neutralization with phosphoric acid, thiol resulting from the alkaline cleavage is estimated colorimetrically with 5,5′-dithio-bis(2-nitrobenzoic acid). In the presence of EDTA, the color yield is stable and is linear with the concentration of oxidized glutathione. The stoichiometry with other peptide disulfides appears to be somewhat variable but not so as to interfere with detection of peptide disulfides in chromatographic fractions. The present method compares favorably with two other proposed disulfide analytical methods. The cleavage assay is chromogenic with disulfides, thiols, and with certain blocked thiols but is not chromogenic with methionine and lanthionine.     

A new simple method for determination of erythrocyte filtrability.

A new simple filtration technique designed for measuring red cell filtrability in the routine laboratory use was developed. The suspension of the whole blood in saline (1:20,000 dilution) was processed on the Sartorius filter membranes, pore size 8 micron. The percentage of passed erythrocytes indicating red cell filtrability was determined. The suitability and perspective applicability of this method for studying various hematological disorders is proposed.     

Alternative splicing of the human isoaspartyl protein carboxyl methyltransferase RNA leads to the generation of a C-terminal -RDEL sequence in isozyme II.

We have isolated two cDNA clones that correspond to the mRNAs for two isozymes of the human L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (EC 2.1.1.77). The DNA sequence of one of these encodes the amino acid sequence of the C-terminal half of the human erythrocyte isozyme I. The other cDNA clone includes the complete coding region of the more acidic isozyme II. With the exception of potential polymorphic sites at amino acid residues 119 and 205, the deduced amino acid sequences differ only at the C-terminus, where the -RWK sequence of isozyme I is replaced by a -RDEL sequence in isozyme II. The latter sequence is identical to a mammalian endoplasmic reticulum retention signal. With the previous evidence for only a single gene for the L-isoaspartyl/D-aspartyl methyltransferase in humans, and with evidence for consensus sites for alternative splicing in corresponding mouse genomic clones, we suggest that alternative splicing reactions can generate the major isozymes previously identified in human erythrocytes. The presence of alternative splicing leads us to predict the existence of a third isozyme with a -R C-terminus. The calculated isoelectric point of this third form is similar to that of a previously detected but uncharacterized minor methyltransferase activity.     

A fast and efficient method for quantitative measurement of S-adenosyl-l-methionine-dependent methyltransferase activity with protein substrates

Modification of protein residues by S-adenosyl-l-methionine (AdoMet)-dependent methyltransferases impacts an array of cellular processes. Here we describe a new approach to quantitatively measure the rate of methyl transfer that is compatible with using protein substrates. The method relies on the ability of reverse-phase resin packed at the end of a pipette tip to quickly separate unreacted AdoMet from radiolabeled protein products. Bound radiolabeled protein products are eluted directly into scintillation vials and counted. In addition to decreasing analysis time, the sensitivity of this protocol allows the determination of initial rate data. The utility of this protocol was shown by generating a Michaelis-Menten curve for the methylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein by human protein arginine methyltransferase 1, variant 1 (hPRMT1v1), in just over 1 h. An additional advantage of this assay is the more than 3000-fold reduction in radioactive waste over existing protocols.     

Identification of isoaspartyl-containing sequences in peptides and proteins that are usually poor substrates for the class II protein carboxyl methyltransferase

We have found that a chicken egg lysozyme derivative (beta-101-lysozyme) containing an L-isoaspartyl residue at position 101 has a Km for methylation by the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase (EC 2.1.1.77) of 183 microM, about 30 times higher than that expected from previous studies with isoaspartyl-containing peptides. In the course of investigating the reasons for this poor enzyme recognition, we found that charged residues on the carboxyl side of isoaspartyl residues had a large effect on the affinity of the enzyme for synthetic peptides. This is best illustrated by the lysozyme-related peptide YVSisoDGDG, which has a Km for methylation of 469 microM. When the penultimate aspartyl residue is replaced by a cysteinyl residue, the Km drops to 4.6 microM, comparable to other peptides of similar size. Furthermore, replacing it with a cysteic acid residue results in a Km of 104 microM, suggesting that a negative charge at this position may lead to a weaker affinity of the peptide substrate for the methyltransferase. Assays with additional synthetic peptides indicate that moving the negative charge to the first or third residue on the carboxyl side of the isoaspartyl residue has a similar but less severe effect in reducing its affinity for the methyltransferase. Enzymatic methylation has recently been proposed to be the first step in the conversion of abnormal isoaspartyl residues to aspartyl residues. The results reported here, however, along with previous evidence that protein tertiary structure can inhibit isoaspartyl methylation, suggest that only a subclass of damaged sites are capable of efficiently entering a putative repair pathway; the sites not recognized by the methyltransferase may accumulate in vivo.     

Hydrolytic enzyme substrates. II. A new method for measuring periodate

This report describes an accurate and sensitive method for quantitatively measuring periodate concentration. The substances used to determine periodate are 4(p-nitrophenoxy)1,2-butanediol and 4(2,4-dinitrophenoxy)1,2-butanediol. These substances are readily oxidized by periodate yielding β-nitrophenoxy aldehydes which undergoes a facile β-elimination in base to yield the colored nitrophenolate ion. The concentration of the nitrophenolate ion is thus equivalent to the concentration of periodate. This report documents the validity of this reaction as an analytical method. The method was shown to be capable of accurately measuring periodate in concentrations as low as 10^?8M. Its value in biochemical analyses was demonstrated by quantitatively measuring the amount of periodate used to oxidize small quantities of adenosine 5′-phosphate, d-arabitol and d-glucose. Its accuracy, sensitivity and ease of use was shown by its utility in estimating the molecular weight of yeast transfer RNA using about 6 A₂₆₀ units of this material.