BNP Human, His

What is BNP Human, His and what is its biological significance?

BNP Human, His refers to recombinant human B-type Natriuretic Peptide with an N-terminal histidine tag. It is a single, non-glycosylated polypeptide chain containing 83 amino acids (positions 27-102 of the full sequence) including a 7-amino acid N-terminal His tag, with a total molecular mass of 9.4 kDa .

BNP acts as a cardiac hormone with diverse biological actions including natriuresis, diuresis, vasorelaxation, and inhibition of renin and aldosterone secretion. It plays a crucial role in cardiovascular homeostasis by helping restore the body's salt and water balance and improving heart function . Originally discovered in porcine brain tissue in 1988 (thus initially named "brain natriuretic peptide"), research has since established that BNP is primarily produced and secreted by the ventricular myocardium in response to increased ventricular blood volume and stretching .

Methodologically, researchers should note that while studying BNP's functions, its physiologic actions resemble those of Atrial Natriuretic Peptide (ANP), including decreased systemic vascular resistance and central venous pressure along with increased natriuresis. The combined effect is reduced blood pressure through decreased systemic vascular resistance (afterload) and decreased cardiac output from reduced central venous pressure (preload) .

How do the different forms of BNP relate to each other in research applications?

The BNP system consists of several interrelated molecular forms that researchers must distinguish between:

• BNP-32: The mature, biologically active 32-amino acid polypeptide form, which is the principal effector molecule .

• proBNP-108: The precursor molecule from which BNP-32 is cleaved. Research indicates that proBNP is actually the predominant BNP-immunoreactive form circulating in human blood, particularly in heart failure patients .

• NT-proBNP-76: The biologically inactive 76-amino acid N-terminal fragment released when proBNP is cleaved to form BNP-32 .

The relationship between these forms is critical for experimental design as recent research has revealed that in patient plasma, the molar concentration of NT-proBNP exceeded that of proBNP by almost tenfold, and the mean proBNP:BNP ratio was 6.3 (range 1.8-10.8) . This has significant implications for assay selection and data interpretation.

Methodologically, researchers should be aware that currently available BNP immunoassays react with both mature BNP and proBNP; therefore, measured plasma BNP represents the sum of mature BNP + proBNP . For differentiation between these forms, specialized assays targeting specific epitopes must be employed.

What are the optimal storage and handling conditions for BNP Human, His in laboratory settings?

For optimal experimental outcomes with BNP Human, His, researchers should adhere to the following protocol:

• Storage: The lyophilized product should be stored at -20°C for long-term stability. After reconstitution, aliquot and store at -80°C to prevent freeze-thaw cycles that can degrade the peptide.

• Reconstitution: Add deionized water to prepare a working stock solution of approximately 0.5 mg/ml and allow the lyophilized pellet to dissolve completely . For cell culture applications, the solution must be sterile-filtered as the product is not provided in sterile form.

• Handling: BNP Human, His is typically formulated in 20 mM Tris buffer with 50 mM NaCl at pH 7.5 . When preparing working dilutions, use buffers with similar composition to maintain stability.

• Quality verification: Before experimental use, verify integrity by SDS-PAGE, which should show greater than 95% purity .

It's essential to note that BNP is intended for laboratory research use only and should not be used for therapeutic applications, as drugs, agricultural or pesticidal products, food additives, or household chemicals .

How can researchers develop specific assays to distinguish between different BNP forms?

Distinguishing between BNP forms requires carefully designed immunoassays with specific antibodies. Based on recent research methodologies:

• Antibody Selection Strategy:

  • Use monoclonal antibodies specifically designed for recognition of unique epitopes on BNP-32, proBNP, and NT-proBNP

  • Test antibodies in dual-site combinations using time-resolved immunoassays for optimal specificity

• Assay Development Protocol:

  • Employ synthetic and recombinant antigens for calibration curves

  • Validate with plasma samples from heart failure patients

  • Establish detection limits (research has shown specific detection limits for proBNP, BNP, and NT-proBNP assays at 0.4, 3, and 10 ng/L, respectively)

• Chromatographic Separation Technique:

  • Implement gel filtration fast protein liquid chromatography (FPLC) to analyze proBNP and related molecules

  • Use protein fractionation with Sep-Pak C18 cartridges for improved separation

  • Analyze distinct molecular weight peaks (NT-proBNP at 25 kDa, proBNP at 37 kDa, and two peaks for BNP immunoreactivity)

This methodological approach allows researchers to accurately quantify the relative proportions of each BNP form, crucial for experimental work examining the pathophysiology of heart failure and other cardiovascular conditions.

What methodological approaches are best for studying BNP expression regulation in cellular models?

To effectively study BNP expression regulation in cellular models, researchers should implement these methodological approaches:

• Transcriptional Regulation Analysis:

  • Target the conserved AUUUA unit in the 3′-untranslated region of BNP mRNA, which promotes degradation and differs from ANP mRNA regulation

  • Examine shear stress response elements (SSRE) located in the proximal promotor at positions -161-bp, -641-bp, and -652-bp

  • Implement site-directed mutations of SSRE elements or co-transfection with NF-kB activity suppressors to assess strain-activated human BNP promotor activity

• Cellular Stress Response Protocol:

  • Study the transcriptional repressor element NRSE (located at -552 of the human BNP promoter) which represses basal BNP promoter activity

  • Examine cardiac hypertrophic signaling induced by fibronectin through this pathway

  • Compare ventricular and atrial expression patterns, as ventricular BNP mRNA increases more significantly under pathological conditions

• O-Glycosylation Impact Assessment:

  • Implement a protocol to examine O-glycosylation at multiple sites during transport to the Golgi apparatus

  • Analyze how these post-translational modifications affect cleavage efficiency and bioactivity

This comprehensive approach provides insights into the complex regulation of BNP gene expression, which is dynamically altered by both physiological and pathological conditions, unlike ANP gene expression.

How should researchers interpret BNP measurements in patients receiving ARNI therapy?

Interpreting BNP measurements in patients receiving Angiotensin Receptor-Neprilysin Inhibitor (ARNI) therapy requires special methodological considerations:

• Temporal Dynamics Analysis Protocol:

  • Implement a longitudinal measurement approach as BNP levels initially increase after ARNI administration due to reduced degradation by neprilysin

  • Later measurements typically show decreased levels as reduced myocardial wall stress leads to lower BNP production, which offsets the initial increase

• Differential Form Assessment:

  • Note that circulating protease neprilysin has lower substrate specificity for BNP than ANP

  • Recognize that proBNP and NT-proBNP are not degraded by neprilysin

  • Consider that measured plasma BNP represents mature BNP + proBNP

• Assay Selection Guidance:

  • For patients on ARNI therapy, NT-proBNP measurements may provide more consistent results as they are not affected by neprilysin inhibition

  • When using BNP assays, implement a correction factor or temporal trend analysis rather than single measurements

Despite these complexities, BNP remains a useful biomarker for heart failure in the ARNI era, though mild increases early during ARNI administration should be considered in result interpretation .

What are the optimal experimental controls when using BNP Human, His in receptor binding studies?

When conducting receptor binding studies with BNP Human, His, implement these methodological controls:

• Positive Control Protocol:

  • Include native BNP-32 without His-tag to assess potential interference of the His-tag with receptor binding

  • Use ANP as a comparative control since it binds to the same receptors but with 10-fold higher affinity

• Negative Control Strategy:

  • Employ a scrambled peptide with similar amino acid composition but different sequence

  • Use heat-denatured BNP to confirm binding specificity requires proper tertiary structure

• Receptor Specificity Verification:

  • Design experiments to distinguish between NPRA and NPRB binding, as BNP binds primarily to NPRA and to a lesser extent to NPRB

  • Implement competition assays with receptor-specific antagonists

• Binding Quantification Method:

  • Use radiolabeled peptides or fluorescence-based techniques to determine binding kinetics

  • Calculate affinity constants (Kd) and maximum binding capacity (Bmax) through Scatchard analysis

This comprehensive control framework ensures valid interpretation of receptor binding data and enables differentiation between specific biological effects and artifacts related to the His-tag or experimental conditions.

How can researchers effectively compare BNP and NT-proBNP as biomarkers in experimental models?

To effectively compare BNP and NT-proBNP as biomarkers in experimental models, researchers should implement this methodological framework:

• Paired Measurement Protocol:

  • Simultaneously measure BNP, NT-proBNP, and proBNP in the same samples to establish relative ratios

  • Account for the fact that NT-proBNP values are typically 5-10 times higher than BNP values

• Half-life Considerations:

  • Design sampling timepoints based on the different biological half-lives (BNP's half-life is approximately twice as long as ANP's, and NT-proBNP's is even longer)

  • Implement area-under-curve analyses rather than single timepoints

• Clearance Pathway Analysis:

  • Include renal function parameters in experimental design, as BNP is cleared by the kidneys

  • Stratify data analysis based on renal function status

• Assay Cross-reactivity Assessment:

  • Validate that BNP assays detect both mature BNP and proBNP

  • Confirm NT-proBNP assays specifically detect the N-terminal fragment

  • Document proBNP:BNP ratios, which research has shown to average 6.3 in patient plasma (range 1.8-10.8)

This methodological approach provides a more comprehensive understanding of the natriuretic peptide system dynamics and improves interpretation of biomarker data in cardiovascular research studies.

What experimental approaches best reveal the functional differences between glycosylated and non-glycosylated BNP forms?

To investigate functional differences between glycosylated and non-glycosylated BNP forms, implement these experimental approaches:

• Comparative Bioactivity Protocol:

  • Measure natriuretic, diuretic, and vasodilatory responses in cellular and animal models

  • Quantify receptor activation using cGMP production as a second messenger

  • Compare ED50 values (effective dose for 50% response) for each form

• Structural Analysis Methodology:

  • Employ circular dichroism spectroscopy to assess secondary structure differences

  • Use nuclear magnetic resonance (NMR) to examine tertiary structural changes

  • Implement molecular dynamics simulations to predict binding interface alterations

• Degradation Kinetics Approach:

  • Measure neprilysin-mediated degradation rates for each form

  • Assess plasma half-life in ex vivo systems

  • Quantify clearance rates through receptor-mediated endocytosis

• O-Glycosylation Site Mapping:

  • Analyze O-glycosylation at multiple sites during transport through the Golgi apparatus

  • Create site-directed mutants with modified glycosylation sites

  • Assess impact on proBNP processing efficiency to BNP-32

This systematic approach provides critical insights into how post-translational modifications affect BNP bioavailability and activity, helping researchers understand the complex processing of this important cardiac hormone in both normal and pathological conditions.

What are the most common pitfalls in BNP research and how can they be methodologically addressed?

Researchers working with BNP Human, His should anticipate and address these common experimental challenges:

• Immunoassay Cross-reactivity Resolution:

  • Challenge: Standard BNP assays detect both mature BNP and proBNP , leading to overestimation of active BNP-32.

  • Solution: Implement sandwich immunoassays using antibodies targeting unique epitopes specific to each form. Research has demonstrated success with detection limits of 0.4, 3, and 10 ng/L for proBNP, BNP, and NT-proBNP assays, respectively .

• Sample Stability Protocol:

  • Challenge: BNP degradation during storage affects measurement accuracy.

  • Solution: Process samples immediately and store at -80°C. For longer-term storage, add protease inhibitors. Quantify degradation rates under various conditions for your specific experimental setup.

• His-tag Interference Management:

  • Challenge: The His-tag may alter protein folding or receptor binding.

  • Solution: Include parallel experiments with tagged and non-tagged versions. If interference is detected, consider enzymatic tag removal using specific proteases before functional studies.

• Solubility Issues Resolution:

  • Challenge: Improper reconstitution leading to aggregation or precipitation.

  • Solution: Follow recommended reconstitution protocol using deionized water to prepare a working stock solution of approximately 0.5 mg/ml, allowing complete dissolution . Filter solutions before cell culture applications.

• Experimental Model Selection:

  • Challenge: Choosing models that accurately reflect human BNP biology.

  • Solution: Consider species differences in BNP structure (human BNP-32 vs. porcine BNP-26 vs. rat BNP-45) when selecting experimental models. Use human cell lines for receptor studies when possible.

Addressing these methodological challenges systematically improves experimental reproducibility and data quality in BNP research.

How can researchers validate that their recombinant BNP Human, His maintains proper biological activity?

To ensure recombinant BNP Human, His maintains proper biological activity, implement this validation protocol:

• Structural Integrity Assessment:

  • Perform SDS-PAGE under reducing and non-reducing conditions to verify proper molecular weight (9.4kDa) and disulfide bond formation

  • Implement circular dichroism spectroscopy to confirm secondary structure elements

  • Verify amino acid sequence using mass spectrometry to confirm the presence of all 83 amino acids including the 7-amino acid His-tag

• Functional Bioassay Panel:

  • Measure cGMP production in cells expressing natriuretic peptide receptors

  • Quantify calcium mobilization in response to BNP binding

  • Assess vasodilatory effects on isolated vessel rings

• Receptor Binding Verification:

  • Determine binding affinity to NPRA and NPRB receptors using competitive binding assays

  • Compare binding constants with literature values for native BNP

  • Confirm that binding occurs with 10-fold lower affinity than ANP

• Physiological Response Measurement:

  • In cellular models, verify natriuretic and diuretic signaling pathways

  • Compare EC50 values with native BNP-32 to ensure similar potency

  • Confirm ability to inhibit renin and aldosterone secretion pathways

This comprehensive validation approach ensures that experimental outcomes reflect true biological properties rather than artifacts from recombinant production.

What methodological considerations are important when developing a BNP standard curve for quantitative assays?

Developing reliable BNP standard curves for quantitative assays requires these methodological considerations:

• Reference Material Selection Protocol:

  • Use certified reference materials with defined purity (>95%) and confirmed sequence

  • Consider whether BNP Human, His or non-tagged BNP is most appropriate for your specific assay

  • Include proBNP standards if your assay detects both forms, as research shows proBNP is the principal BNP-immunoreactive form in circulation

• Matrix Effect Minimization Strategy:

  • Prepare standards in the same matrix as samples (e.g., serum-free media for cell culture, buffer matching plasma composition for clinical samples)

  • Implement standard addition methods to verify recovery and matrix effects

  • Consider depleted matrix approaches for highly complex biological samples

• Dynamic Range Optimization:

  • Design a standard curve covering the physiological range (0-20 pg/mL for normal subjects) and pathological range (increasing with disease severity)

  • Use a minimum of 6-8 concentration points with duplicate or triplicate measurements

  • Implement log-transformation or 4-parameter logistic regression rather than linear fitting

• Quality Control Implementation:

  • Include low, medium, and high QC samples within and across batches

  • Calculate intra- and inter-assay coefficients of variation (target <10% and <15%, respectively)

  • Determine lower limit of quantification (LLOQ) based on precision profile (CV <20%)

This methodological framework ensures accurate quantification across the relevant concentration range and facilitates comparison of results between different experimental conditions and research groups.

What are the current gaps in BNP research methodology that researchers should address?

Current BNP research methodology presents several gaps that require innovative approaches:

• Improved Form-Specific Detection Methods:

  • Challenge: Current BNP immunoassays react with both mature BNP and proBNP , complicating interpretation.

  • Research Need: Develop highly specific assays that can selectively quantify BNP-32, proBNP-108, and NT-proBNP-76 in complex biological samples without cross-reactivity.

  • Methodological Approach: Implement epitope mapping followed by monoclonal antibody development targeting unique regions of each form.

• Post-Translational Modification Characterization:

  • Challenge: O-glycosylation affects proBNP processing and bioactivity , but comprehensive modification mapping is incomplete.

  • Research Need: Establish the complete profile of post-translational modifications and their impact on bioactivity.

  • Methodological Approach: Combine mass spectrometry glycoprofiling with site-directed mutagenesis of modification sites.

• Standardization of BNP Measurements:

  • Challenge: Different assays show poor concordance between laboratories.

  • Research Need: Develop universally applicable reference materials and standardization protocols.

  • Methodological Approach: Coordinate international efforts to establish common calibrators and measurement procedures.

• Receptor Subtype Specificity Research:

  • Challenge: Differential signaling through NPRA versus NPRB receptors is poorly characterized.

  • Research Need: Clarify the physiological significance of BNP binding to different receptor subtypes.

  • Methodological Approach: Develop receptor subtype-specific antagonists and conditional knockout models.

Addressing these methodological gaps will significantly advance our understanding of BNP biology and improve its utility in both research and clinical applications.

How might novel analytical techniques enhance BNP research in the coming years?

Emerging analytical techniques offer promising enhancements to BNP research methodology:

• Single-Molecule Detection Applications:

  • Technology: Total internal reflection fluorescence (TIRF) microscopy and nanopore sensing

  • Research Advantage: Enables detection of BNP at physiological concentrations without amplification

  • Methodological Implementation: Develop fluorescently labeled antibodies targeting different epitopes of BNP forms for co-localization studies

• Advanced Chromatography Integration:

  • Technology: Ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry

  • Research Advantage: Builds upon current gel filtration FPLC approaches to achieve enhanced separation of BNP forms

  • Methodological Implementation: Develop multi-dimensional chromatography protocols specific for natriuretic peptide separation

• Digital Biomarker Approaches:

  • Technology: Microfluidic-based digital ELISA systems

  • Research Advantage: Improves sensitivity by orders of magnitude compared to conventional immunoassays

  • Methodological Implementation: Design BNP-specific capture antibodies compatible with single-molecule array technology

• Structural Biology Techniques:

  • Technology: Cryo-electron microscopy and advanced NMR methods

  • Research Advantage: Provides atomic-level structural information about BNP-receptor complexes

  • Methodological Implementation: Express stabilized forms of natriuretic peptide receptors for structural studies in complex with BNP

These emerging methodologies have the potential to revolutionize our understanding of BNP biology by providing unprecedented sensitivity, specificity, and structural insights into this important cardiac hormone system.